Bacteria as Human Pathogens

Chapter 4

Bacteria as Human Pathogens

Staphylococcus

• Staphylococci are Gram-positive cocci occurring in clusters. They can be cultured on normal nutrient mediums both aerobically and anaerobically. The most important species from the viewpoint of human medicine is S. aureus. A number of extracellular enzymes and exotoxins such as coagulase, alpha toxin, leucocidin, exfoliating, enterotoxins, and toxic shock toxin are responsible for the clinical symptoms of infections by this pathogen, which are observed in the three types of invasive infections, pure toxicoses, and mixed forms. The antibiotics of choice for therapy of these infections are penicillinase-resistant penicillin. Laboratory diagnosis involves identification of the pathogen by means of microscopy and culturing. S. aureus is a frequent pathogen in nosocomial infections and limited outbreaks in hospitals. Hand washing by medical staff is the most important prophylactic measure in hospitals. Coagulase-negative staphylococci are classic opportunists. S. epidermidis and other species are frequent agents in foreign body infections due to their ability to form biofilms on the surfaces of inert objects. S. saprophyticus is responsible for between 10 and 20% of acute urinary tract infections in young women.

Staphylococcus Aureus

• Morphology and culturing. Fig. 4.1a shows the appearance of Gram-stained S. aureus. This is a facultative anaerobe that is readily cultured on normal nutrient mediums at 37 8C. Colonies as in Fig. 4.1b develop after 24 hours of incubation. Hemolytic zones are frequently observed around the colonies.

• Fine structure. The cell wall consists of a thick layer of murein. Linear teichoic acids and polysaccharides are covalently coupled to the murein polysaccharide (Fig. 3.10, p. 154). The lipoteichoic acids permeating the entire murein layer are anchored in the cell membrane. Teichoic and lipoteichoic acids can trigger activation of complement by the alternative pathway and stimulate macrophages to secrete cytokines. Cell wall-associated proteins are bound to the peptide components of the murein. Clumping factor, fibronectin-binding protein, and collagen-binding protein bind specifically to fibrinogen, fibronectin, and collagen, respectively, and are instrumental in adhesion to tissues and foreign bodies covered with the appropriate matrix protein. Protein A binds to the Fc portion of immunoglobulins (IgG). It is assumed that “false” binding of immunoglobulins by protein A prevents “correct” binding of opsonizing antibodies, thus hindering phagocytosis.

• Extracellular toxins and enzymes. S. aureus secretes numerous enzymes and toxins that determine, together with the fine structures described above, the pathogenesis of the attendant infections. The most important are:

• Plasma coagulase is an enzyme that functions like thrombin to convert fibrinogen into fibrin. Tissue microcolonies surrounded by fibrin walls are difficult to phagocytose.

• a-toxin can have lethal CNS effects, damages membranes (resulting in, among other things, hemolysis), and is responsible for a form of dermonecrotic.

• Leucocidins damages microphages and macrophages by degranulation.

• Exfoliating is responsible for a form of epidermolysis.

• Food poisoning symptoms can be caused by eight serologically differentiated enterotoxins (A-E, H, G, and I). These proteins (MW: 35 Kad) are not inactivated by heating to 100 8C for 15–30 minutes. Staphylococcus enterotoxins are superantigens (see p. 72).

• Toxic shock syndrome toxin-1 (TSST-1) is produced by about 1% of Staphylococcus strains. TSST-1 is a superantigen that induces clonal expansion of many T lymphocyte types (about 10%), leading to massive production of cytokines, which then give rise to the clinical symptoms of toxic shock.

• Pathogenesis and clinical pictures. The pathogenesis and symptoms of S. aureus infections take one of three distinct courses:

• Invasive infections. In this type of infection, the pathogens tend to remain in situ after penetrating through the derma or mucosa and to cause local infections characterized by purulence. Examples include furuncles (Fig. 4.2), carbuncles, wound infections, sinusitis, otitis media, and mastitis puerperal is.

• Inert foreign bodies (see p. 158 for examples) can be colonized by S. aureus. Colonization begins with specific binding of the staphylococci, by means of cell wall-associated adhesion proteins, to fibrinogen or fibronectin covering the foreign body, resulting in a biofilm that may function as a focus of infection.

• Toxicoses. Food poisoning results from ingestion of food contaminated with enterotoxins. The onset a few hours after ingestion takes the form of nausea, vomiting, and massive diarrhea.

• Mixed forms. Dermatitis exfoliative (staphylococcal scalded skin syndrome, Ritter disease), pemphigus neonatorum, and bullous impetigo are caused by exfoliating-producing strains that infect the skin surface. Toxic shock syndrome (TSS) is caused by strains that produce TSST-1. These strains can cause invasive infections but may also only colonize mucosa. The main symptoms are hypotension, fever, and a scarlatiniform rash.

• Diagnosis. This requires microscopic and culture-based pathogen identification. Differentiating S. aureus from the coagulase-negative species is achieved by detection of the plasma coagulase and/or the clumping factor. The enterotoxins and TSST-1 can be detected by means immunological and molecular biological methods (special laboratories).

• Therapy. Aside from surgical measures, therapy is based on administration of antibiotics. The agents of choice for severe infections are penicillinase resistant penicillin, since 70–80% of all strains produce penicillinase. These penicillin are, however, ineffective against methicillin-resistant strains, and this resistance applies to all beta lactams.

• Epidemiology and prevention. S. aureus is a frequent colonizer of skin and mucosa. High carrier rates (up to 80%) are the rules among hospital patients and staff. The principal localization of colonization in these persons is the anterior nasal mucosa area, from where the bacteria can spread to hands or with dust into the air and be transmitted to susceptible persons.

Coagulase-Negative Staphylococci (CNS)

• CNS are an element in the normal flora of human skin and mucosa. They are classic opportunists that only cause infections given a certain host disposition.

• S. epidermidis. This is the pathogen most frequently encountered in CNS infections (70–80% of cases). CNS cause mainly foreign body infections. Examples of the foreign bodies involved are intravasal catheters, continuous ambulant peritoneal dialysis (CAPD) catheters, endoprostheses, metal plates and screws in osteosynthesis, cardiac pacemakers, artificial heart valves, and shunt valves. These infections frequently develop when foreign bodies in the macroorganism are covered by matrix proteins (e.g., fibrinogen, fibronectin) to which the staphylococci can bind using specific cell wall proteins. They then proliferate on the surface and produce a polymeric substance—the basis of the developing biofilm. The staphylococci within the biofilm are protected from antibiotics and the immune system to a great extent. Such biofilms can become infection foci from which the CNS enter the bloodstream and cause sepsislike illnesses. Removal of the foreign body is often necessary.

• S. saprophyticus is responsible for 10–20% of acute urinary tract infections, in particular dysuria in young women, and for a small proportion of cases of nonspecific urethritis in sexually active men. Antibiotic treatment of CNS infections is often problematic due to the multiple resistance often encountered in these staphylococci, especially S. hemolyticus.

Streptococcus and Enterococcus

• Streptococci are Gram-positive, nonmotile, catalase-negative, facultatively anaerobic cocci that occur in chains or pairs. They are classified based on their hemolytic capacity (a-, b-, c-hemolysis) and the antigenicity of a carbohydrate occurring in their cell walls (Lancefield antigen). b-hemolytic group A streptococci (S. pyogenes) cause infections of the upper respiratory tract and invasive infections of the skin and subcutaneous connective tissue. Depending on the status of the immune defenses and the genetic disposition, this may lead to scarlet fever and severe infections such as necrotizing fasciitis, sepsis, or septic shock. Sequelae such as acute rheumatic fever and glomerulonephritis have an autoimmune pathogenesis. The a-hemolytic pneumococci (S. pneumoniae) cause infections of the respiratory tract. Penicillins are the antibiotics of choice. Resistance to penicillins is known among pneumococci, and is increasing. Laboratory diagnosis involves pathogen detection in the appropriate material. Persons at high risk can be protected from pneumococcal infections with an active prophylactic vaccine containing purified capsular polysaccharides. Certain oral streptococci are responsible for dental caries. Oral streptococci also cause half of all cases of endocarditis.

• Classification. The genera Streptococcus and Enterococcus comprise a large number of species. Table 4.2 lists the most important.

• a-, b-, c-hemolysis. a-hemolysis. Colonies on blood agar are surrounded by a green zone. This “greening” is caused by H2O2, which converts hemoglobin into methemoglobin. b-hemolysis. Colonies on blood agar are surrounded by a large, yellowish hemolytic zone in which no more intact erythrocytes are present and the hemoglobin is decomposed. c-hemolysis. This (illogical) term indicates the absence of macroscopically visible hemolytic zones.

• Lancefield groups Many streptococci and enterococci have a polymeric carbohydrate (C substance) in their cell walls called the Lancefield antigen. They are classified in Lancefield groups A-V based on variations in the antigenicity of this antigen. Specific characteristics of enterococci that differentiate them from streptococci include their ability to proliferate in the presence of 6.5% NaCl, at 45 8C and at a pH level of 9.6.

Streptococcus pyogenes (A Streptococci)

• Morphology and culturing. Gram-positive cocci with a diameter of 1 lm that form chains (Fig. 4.3a). Colonies on blood agar (Fig. 4.3b) show b-hemolysis caused by streptolysins (see below).

• Fine structure The murein layer of the cell wall is followed by the serogroup A carbohydrate layer, which consists of C substance and is covalently bound to the murein. Long, twisted protein threads that extend outward are anchored in the cell wall murein: the M protein. A streptococci are classified in serovars with characteristic M protein chemistry. Like the hyaluronic acid capsules seen in some strains, the M protein has an antiphagocytic effect.

• Extracellular toxins and enzymes. The most important in the context of pathogenicity are:

• Streptolysin O, streptolysin S. Destroy the membranes of erythrocytes and other cells. Streptolysin O acts as an antigen. Past infections can be detected by measuring the antibodies to this toxin (ant streptolysin titer.

• Pyrogenic streptococcal exotoxins (PSE) A, B, C. Responsible for fever, scarlet fever exanthem and enanthem, sepsis, and septic shock. The pyrogenic exotoxins are superantigens and therefore induce production of large amounts of cytokines (p. 77).

• Streptokinase. Dissolves fibrin: facilitates spread of streptococci in tissues.

• Hyaluronidase. Breaks down a substance that cements tissues together.

• DNases. Breakdown of DNA, producing runny pus.

• Pathogenesis and clinical pictures. Streptococcal diseases can be classified as either acute, invasive infections or sequelae to them.

• Invasive infections. The pathogens enter through traumas or microtraumas in the skin or mucosa and cause invasive local or generalized infections (Fig. 4.4). The rare cases of severe septic infection and necrotizing fasciitis occur in persons with a high-risk MHC II allotype. In these patients, the PSE superantigens (especially PSEA) induce large amounts of cytokine by binding at the same time to the MHC II complex and the b chain of the T cell receptor. The excess cytokines thus produced are the cause of the symptoms.

• Sequelae. Glomerulonephritis is an immune complex disease (p. 113) and acute rheumatic fever may be a type II immune disease (p. 109).

• Diagnosis. What is involved in diagnosis is detection of the pathogen by means of microscopy and culturing. Group A antigen can be detected using particles coated with antibodies that precipitate agglutination (latex agglutination, conglutinations). Using these methods, direct detection of A streptococci in tonsillitis is feasible in the medical practice. However, this direct detection method is not as sensitive as the culture. Differentiation of A streptococci from other b-hemolytic streptococci can be realized in the laboratory with the bacitracin disk test, because A streptococci are more sensitive to bacitracin than the other types.

• Therapy. The agents of choice are penicillin G or V. Resistance is unknown. Alternatives are oral cephalosporins or macrolide antibiotics, although resistance to the latter can be expected. In treatment of septic shock, a polyvalent immunoglobulin is used to inactivate the PSE.

• Epidemiology and prophylaxis. Infection frequency varies according to geographical area, season, and age. Humans are the only pathogen reservoir for S. pyogenes. Transmission is by direct contact (smear infection) or droplets. The incubation period is one to three days. The incidence of carriers among children is 10–20% but can be much higher depending on the epidemiological situation. Carriers and infected persons are no longer contagious 24 hours after the start of antibiotic therapy. Microbiological follow-up checks of patients and first-degree contacts are not necessary (exception: rheumatic history). In persons with recurring infections or with acute rheumatic fever in their medical histories, continuous penicillin prophylaxis with a long-term penicillin is appropriate (e.g., 1.2 million IU benzathine penicillin per month)

Streptococcus pneumoniae (Pneumococci)

• Morphology and culturing. Pneumococci are Gram-positive, oval to lancet shaped cocci that usually occur in pairs or short chains (Fig. 4.5a). The cells are surrounded by a thick capsule. When cultured on blood agar, S. pneumoniae develop a-hemolytic colonies with a mucoid (smooth, shiny) appearance (hence “S” form, Fig. 4.5b). Mutants without capsules produce colonies with a rough surface (“R” form).

• Morphology and culturing. Pneumococci are Gram-positive, oval to lancet shaped cocci that usually occur in pairs or short chains (Fig. 4.5a). The cells are surrounded by a thick capsule. When cultured on blood agar, S. pneumoniae develop a-hemolytic colonies with a mucoid (smooth, shiny) appearance (hence “S” form, Fig. 4.5b). Mutants without capsules produce colonies with a rough surface (“R” form).

• Pathogenesis and clinical pictures. The capsule protects the pathogens from phagocytosis and is the most important determinant of pneumococcal virulence. Unencapsulated variants are not capable of causing disease. Other potential virulence factors include neurolysin with its effects on membranes and an IgA1 protease. The natural habitat of pneumococci is provided by the mucosa of the upper respiratory tract. About 40–70% of healthy adults are carriers. Pneumococcal infections usually arise from this normal flora (endogenous infections). Predisposing factors include primary cardiopulmonary diseases, previous infections (e.g., influenza), and extirpation of the spleen or complement system defects. The most important pneumococcal infections are lobar pneumonia and bronchopneumonia. Other infections include acute exacerbation of chronic bronchitis, otitis media, sinusitis, meningitis, and corneal ulcer. Severe pneumococcal infections frequently involve sepsis.

• Diagnosis. The laboratory diagnosis includes detection of the pathogen in appropriate test samples by means of microscopy and culturing. Pneumococci can be differentiated from other a-hemolytic streptococci based on their greater sensitivity to optochin (ethyl hydrocupration hydrochloride) in the disk test or their bile solubility. Bile salts increase autolysis in pneumococci.

• Therapy. Penicillin is still the antibiotic of choice. There have been reports of high-frequency occurrence of strains resistant to penicillin (South Africa, Spain, Hungary, USA). These strains are still relatively rare in Germany, Switzerland, and Austria (5–10%). Macrolide antibiotics are an alternative to penicillin, but resistance to them is also possible.

• Epidemiology and prophylaxis. Pneumococcal infections are endemic and occur in all seasons, more frequently in the elderly. Humans are the natural pathogen reservoir. The vaccine product Pneumovax! is available for immunization purposes. It contains 25 mg of the purified capsule polysaccharides of each of 23 of the most frequent serovars. Eighty to ninety percent of all isolated pneumococci have antigens contained in this vaccine, which is primarily indicated in persons with predisposing primary diseases. There is also a seven-valent conjugate vaccine that is effective in children under two years of age (p. 33). Exposure prophylaxis is not necessary

Streptococcus agalactiae (B Streptococci)

• B streptococci occasionally cause infections of the skin and connective tissues, sepsis, urinary tract infections, pneumonia, and peritonitis in immunocompromised individuals. About one in 1000 neonates suffers from a sepsis with or without meningitis. These infections manifest in the first days of life (early onset type) or in the first weeks of life (late onset type). In the early onset form, the infection is caused intra partum by B streptococci colonizing the vagina. Potential predisposing factors include birth complications, premature birth, and a lack of antibodies to the capsule in mother and neonate.

Oral Streptococci

• Most of the oral streptococci of the type often known as the viridines group have no group antigen. They usually cause a-hemolysis, some c-hemolysis as well. Oral streptococci are responsible for 50–70% of all cases of bacterial endocarditis, overall incidence of which is one to two cases per 100 000 annually. The origins of endocarditis lie in invasion of the vascular system through lesions in the oral mucosa. A transitory bacteremia results. The heart valves are colonized, and a biofilm is formed by the organism. Predisposing factors include congenital heart defects, acute rheumatic fever, cardiac surgery, and scarred heart valves. Laboratory diagnosis of endocarditis involves isolation of the pathogen from blood cultures. Drug therapy of endocarditis is carried out with either penicillin G alone or combined with an aminoglycoside (mostly gentamicin). Bactericidal activity is the decisive parameter.

Enterococcus (Enterococci)

• Enterococci are a widespread bacterial genus (p. 220) normally found in the intestines of humans and other animals. They are nonmotile, catalase-negative, and characterized by group antigen D. They are able to proliferate at 45 8C, in the presence of 6.5% NaCl and at pH 9, qualities that differentiate them from streptococci. As classic opportunists, enterococci show only low levels of pathogenicity. However, they are frequently isolated as components of a mixed flora in nosocomial infections (p. 343). Ninety percent of such isolates are identified as E. faecalis, 5–10% as E. faecium. Among the most dangerous enterococcal infections is endocarditis, which must be treated with a combination of an aminopenicillin and streptomycin or gentamicin. Therapeutic success depends on the bactericidal efficacy of the combination used. The efficacy level will be insufficient in the presence of high levels of resistance to either streptomycin (MIC >1000 mg/l) or gentamicin (MIC >500 mg/l) or resistance to the aminopenicillin. Enterococci frequently develop resistance to antibiotics. Strains manifesting multiple resistance are found mainly in hospitals, in keeping with the classic opportunistic character of these pathogens. Recently observed epidemics on intensive care wards involved strains that were resistant to all standard anti-infective agents including the glycopeptides vancomycin and teicoplanin.

Gram-Positive, Anaerobic Cocci

• Gram-positive, strictly anaerobic cocci are included in the genera Peptococcus and Peptostreptococcus. The only species in the first genus is Peptococcus niger, whereas the latter comprises a number of species. The anaerobic cocci are commonly observed in normal human flora. In a pathogenic context they are usually only encountered as components of mixed florae together with other anaerobes or facultative anaerobes. These bacteria invade tissues through dermal or mucosal injuries and cause subacute purulent infections. Such infections are either localized in the head area (cerebral abscess, otitis media, mastoiditis, sinusitis) or lower respiratory tract (necrotizing pneumonia, pulmonary abscess, empyema). They are also known to occur in the abdomen (appendicitis, peritonitis, hepatic abscess) and female genitals (salpingitis, endometriosis, Tubo-ovarian abscess). Gram-positive anaerobic cocci may also contribute to soft-tissue infections and postoperative wound infections. See p. 317ff. for clinical details of anaerobe infections.

Bacillus

• The natural habitat of Bacillus anthracis, a Gram-positive, sporing, obligate aerobic rod bacterium, is the soil. The organism causes anthrax infections in animals. Human infections result from contact with sick animals or animal products contaminated with the spores. Infections are classified according to the portal of entry as dermal anthrax (95% of cases), primary inhalational anthrax, and intestinal anthrax. Sepsis can develop from the primary infection focus. Laboratory diagnosis includes microscopic and cultural detection of the pathogen in relevant materials and blood cultures. The therapeutic agent of choice is penicillin G.

• Occurrence. Anthrax occurs primarily in animals, especially herbivores. The pathogens are ingested with feed and cause a severe clinical sepsis that is often lethal.

• Morphology and culturing. The rods are 1 lm wide and 2–4 lm long, no flagellated, with a capsule made of a glutamic acid polypeptide. The bacterium is readily grown in an aerobic milieu.

• Pathogenesis and clinical picture. The pathogenicity of B. anthracis results from its antiphagocytic capsule as well as from a toxin that causes edemas and tissue necrosis. Human infections are contracted from diseased animals or contaminated animal products. Anthrax is recognized as an occupational disease.

• Dermal, primary inhalational, and intestinal anthrax are differentiated based on the pathogen’s portal of entry. In dermal anthrax, which accounts for 90–95% of human B. anthracis infections) the pathogens enter through injuries in the skin. A local infection focus similar to a carbuncle develops within two to three days. A sepsis with a foudroyant (highly acute) course may then develop from this primary focus. Inhalational anthrax (bioterrorist anthrax), with its unfavorable prognosis, results from inhalation of dust containing the pathogen. Ingestion of contaminated foods can result in intestinal anthrax with vomiting and bloody diarrheas.

• Diagnosis. The diagnostic procedure involves detection of the pathogen in dermal lesions, sputum, and/or blood cultures using microscopic and culturing methods.

• Therapy. The antimicrobial agent of choice is penicillin G. Doxycycline (a tetracycline) or ciprofloxacin (a fluoroquinolone) are possible alternatives. Surgery is contraindicated in cases of dermal anthrax.

• Epidemiology and prophylaxis. Anthrax occurs mainly in southern Europe and South America, where economic damage due to farm animal infections is considerable. Humans catch the disease from infected animals or contaminated animal products. Anthrax is a classic zoonosis.

Clostridium

• Clostridia are 3–8 lm long, thick, Gram-positive, sporing rod bacteria that can only be cultured anaerobically. Their natural habitat is the soil. The pathogenicity of the disease-causing species in this genus is due to production of exotoxins and/or exoenzymes. The most frequent causative organism in anaerobic cellulitis and gas gangrene (clostridial myonecrosis) is C. perfringens. Tetanus is caused by C. tetani. This pathogen produces the exotoxin tetanospasmin, which blocks transmission of inhibitory CNS impulses to motor neurons. Botulism is a type of food poisoning caused by the neurotoxins of C. botulinum. These substances inhibit stimulus transmission to the motor end plates. Pseudomembranous colitis is caused by C. difficile, which produces an enterotoxin (A) and a cytotoxin (B). Diagnosis of clostridial infections requires identification of the pathogen (gas gangrene) and/or the toxins (tetanus, botulism, colitis). All clostridia are readily sensitive to penicillin G. Antitoxins are used in therapy of tetanus and botulism and hyperbaric O2 is used to treat gas gangrene. The most important preventive measure against tetanus is active vaccination with tetanus toxoid.

The Pathogens That Cause Gas Gangrene (Clostridial Myonecrosis) and Anaerobic Cellulitis

• Pathogen spectrum. The pathogens that cause these clinical pictures include Clostridium perfringens, C. Novy, C. septicemia, and C. histolyticum. Species observed less frequently include C. sporogeneses', C. Sardelli, and C. liferenters. The most frequent causative pathogen in gas gangrene is C. perfringens.

• Toxins, enzymes. The toxins produced by invasive clostridia show necrotizing, hemolytic, and/or lethal activity. They also produce collagens proteinases, DNases, pectinases, and hyaluronidase, all of which destroy tissue structures, resulting in accumulations of toxic metabolites.

• Pathogenesis and clinical picture. Due to the ubiquitous presence of clostridia, they frequently contaminate open wounds, often together with other microorganisms. Detection of clostridia in a wound is therefore no indication of a clostridial infection. These infections develop when a low tissue redox potential makes anaerobe reproduction possible, resulting in tissue necrosis. Two such infections of differing severity are described below:

• Anaerobic cellulitis. Infection restricted to the fascial spaces that does not affect musculature. Gas formation in tissues causes a cracking, popping sensation under the skin known as crepitus. There is no toxemia.

• Gas gangrene (clostridial myonecrosis). An aggressive infection of the musculature with myonecrosis and toxemia. The incubation period varies from hours to a few days.

• Diagnosis. The diagnostic procedure includes identification of the pathogens in relevant materials by means of microscopy and culturing. Identification of anaerobically grown cultures is based on morphological and physiological characteristics.

• Therapy. Primary treatment is surgical, accompanied by antibiosis (penicillin, cephalosporins). Treatment with hyperbaric O2 in special centers has proved effective: patients breathe pure O2 through a tube or mask in a pressure chamber (3 atm = 303 kPa) several times during two-hour periods.

• Epidemiology and prevention. True gas gangrene is now a rare condition. Timely operation of contaminated wounds is the main preventive measure.

Clostridium tetani (Tetanus)

• Tetanus (lockjaw) is an acute clostridial disease, its clinical manifestations do not result directly from the invasive infection but are rather caused by a strong neurotoxin.

• Toxin. Tetanospasmin (an AB toxin, p. 16) consists of two polypeptide chains linked by a disulfide bridge. The heavy chain binds specifically to neuron receptors. The light chain is a zinc-metalloprotease that is responsible for proteolysis of components of the neuroexocytosis apparatus in the synapses of the anterior horns of the spinal cord. This stops transmission of inhibitory efferent impulses from the cerebellum to the motor end plates.

• Pathogenesis and clinical picture. These ubiquitous pathogens invade tissues following injuries (Fig. 4.8a). Given anaerobic conditions, they proliferate and produce the toxin (see above), which reaches the anterior horns of the spinal cord or brain stem via retrograde axonal transport. The clinical picture resulting from the effects of the toxin is characterized by increased muscle tone and spasms induced by visual or acoustic stimuli. The cramps often begin in the facial musculature (rises sardonicus, Fig. 4.8b), then spread to neck and back muscles (opisthotonos). The patient remains lucid.

• Diagnosis. The preferred method is toxin detection in wound material in an animal test (mouse) based either on neutralization or detection of the toxin gene with PCR. The pathogen is difficult to culture.

• Therapy. Antitoxic therapy with immune sera is applied following a meticulous wound cleaning. The patient’s musculature must also be relaxed with curare or similar agents.

• Epidemiology and prophylaxis. Tetanus is now rare in developed countries due to widespread vaccination practice with incidence rates of approximately one case per million inhabitants per year. The frequency of occurrence is much higher in developing or underdeveloped countries. Worldwide, about 300 000 persons contract tetanus every year, with a lethality rate of approximately 50%. Thus, the importance of the active vaccination as a protective measure can hardly be overstated (see p. 33 for vaccination schedule). A dose of Td should be administered once every 10 years to sustain protection (p. 33). A booster shot is also required in case of severe injury if the patient’s last inoculation was administered longer than five years before, and in case of minor injury longer than 10 years. Human tetanus immunoglobulin (250 IU).

Clostridium botulinum (Botulism)

• Foodborne botulism is not an infection, but rather an intoxication, that is, the toxin is ingested with food. Infant botulism involves ingestion of spores and wound botulism results from infection of a wound.

• Toxin. The very strong botulinum neurotoxin is a heat-labile protein. Seven toxigenic types are differentiated, each of which produces an immunologically distinct form of botulinum toxin. Types A, B, and E cause poisoning in humans. The toxin is a metalloprotease that catalyzes the proteolysis of components of the neuroexocytosis apparatus in the motor end plates, resulting in flaccid paralysis of the muscular.

• Pathogenesis and clinical picture. Classic botulism results from eating spoiled foods in which the toxin has been produced under anaerobic conditions by C. botulinum. The toxin is absorbed in the gastrointestinal tract, and then transported to the peripheral nervous system in the bloodstream. Within a matter of hours or days paralysis symptoms occur, especially in the nerves of the head. Frequent symptoms include seeing double, difficulty swallowing and speaking, constipation, and dry mucosa. Lethality rates range from 25–70%, depending on the amount of toxin ingested. Death usually results from respiratory paralysis. Wound botulism results from wound infection by C. botulinum and is very rare. Infant botulism, first described in 1976, results from ingestion of spores with food (e.g., honey). Probably due to the conditions prevailing in the intestines of infants up to the age of six months, the spores are able to proliferate there and produce the toxin. The lethality of infant botulism is low (<1%).

• Diagnosis. Based on toxin detection by means of the mouse neutralization test.

• Therapy. Urgent administration of a polyvalent antitoxin.

• Epidemiology and prevention. Botulism is a rare disease. Exposure to the toxin is a food hygiene problem that can be avoided by taking appropriate precautions during food production. Aerosolized botulinum toxin has been used experimentally as a bioweapon.

Clostridium difficile (Pseudomembranous Colitis)

• C. difficile occurs in the fecal flora of 1–4% of healthy adults and in 30–50% of children during the first year of life. The factors that lead to development of the disease are not known with certainty. Cases of pseudomembranous colitis are observed frequently under treatment with clindamycin, aminopenicillins, and cephalosporins (hence the designation antibiotic-associated colitis), but also occur in persons not taking antibiotics. Occasional outbreaks are seen in hospitals. The pathological mechanism is based on formation of two toxins. Toxin A is an enterotoxin that causes a dysfunction characterized by increased secretion of electrolytes and fluids. Toxin B is a cytotoxin that damages the mucosa of the colon. The clinical course includes fever, diarrhea, and spasmodic abdominal pains. Coloscopy reveals edematous changes in the colon mucosa, which is also covered with yellowish-whitish matter. Laboratory diagnosis involves culturing the pathogen from patient stool and detection of the cytotoxin in bacteria-free stool filtrates on the basis of a cytopathic effect (CPE) observed in cell cultures, which CPE is then no longer observed after neutralization with an antiserum. Toxins A and B can also be detected with immunological test kits (ELISA tests, see p. 127f.). A specific therapy is not required in many cases. Antibiotic treatment is indicated in severe cases. The agent of choice is currently metronidazole.

Listeria, Erysipelothrix, and Gardnerella

• Listeria monocytogenes are diminutive Gram-positive rods with peritrichous flagellation that are quite motile at 20 8C and can be cultured aerobically on blood agar. They occur ubiquitously in nature. Human infections may resultif 106–109 pathogens enter the gastrointestinal tract with food. Listeriae are classic opportunists. In immunocompetent persons, an infection will either be clinically silent or present the picture of a mild flu. In immunocompromised patients, the disease manifests as a primary sepsis and/or meningoencephalitis. More rarely, listeriae cause endocarditis. Listeriosis during pregnancy may result in spontaneous abortion or connatal listeriosis (granulomatosis infantiseptica). Penicillins (amoxicillin) and cotrimoxazole, sometimes in combination with aminoglycosides, are used in therapy. Listeriosis is a rare infection characterized by sporadic occurrence. Occasional gastrointestinal epidemics due to contaminated food may result from the coincidence of unfortunate circumstances.

Listeria monocytogenes

• he only listeriae that cause human disease are L. monocytogenes and the rare species L. Ivanovo. The designation L. monocytogenes results from the observation that infections of rodents, which are much more susceptible than humans, are accompanied by a Mono cytosis.

• Morphology and culturing. The small Gram-positive rods feature peritrichous flagellation. They show greater motility at 20 8C than at 37 8C. Culturing is most successful under aerobic conditions on blood agar. Following incubation for 18 hours, small gray colonies surrounded by inconspicuous hemolytic zones appear. The zones are caused by listeriolysin O. Listeriae can also reproduce at 5–10 8C, which fact can be used in their selective enrichment (“cold enrichment”).

• Pathogenesis. Studies of the molecular processes involved have used mainly systemically infected mice.

• Adherence. To phagocytic cells (e.g., macrophages) and nonphagocytic cells (e.g., enterocytes).

• Invasion. Endocytosis, induced by the protein internalin on the surface of the listeriae. Formation of the endosome.

• Destruction of the endosome. The virulence factor listeriosis forms pores in the endosomal membrane, releasing the listeriae into the cytoplasm.

• Replication of the listeriae in the cytoplasm of infected cells.

• Local intercellular dissemination. Polymerization of the actin of infected cells at one pole of the listeriae to form so-called actin tails that move the listeriae toward the membrane. Formation of long membrane protuberances (known as litiopids) containing listeriae. Neighboring cells engulf the sisterhoods, whereupon the process of listeria release by means of endosome destruction is repeated.

• Dissemination is generally by means of hematogenous spread.

• Clinical characteristics. Listeriae are classic opportunists. The course of most infections is clinically silent. Symptoms resembling a mild flu do not occur in immunocompetent persons until large numbers of pathogens (106–109) enter the gastrointestinal tract with food. Massive infections frequently cause symptoms of gastroenteritis. Listeriosis can take on the form of a sepsis and/or meningoencephalitis in persons with T cell defects or malignancies, in alcoholics, during cortisone therapy, during pregnancy, in elderly persons and in infants.

• Connatal listeriosis is characterized by sepsis with multiple abscesses and granulomas in many different organs of the infant (granulomatosis unfantastic). The lethality rate in severe cases of listeriosis varies between 10% and 40%. The incubation period can vary from one to three days to weeks.

• Diagnosis requires pathogen identification by means of microscopy and culturing.

• Therapy. Amoxicillin, penicillin G, or cotrimoxazole.

• Epidemiology and prevention. Listeriae occur ubiquitously in soil, surface water, plants, and animals and are also found with some frequency (10%) in the intestines of healthy humans. Despite the fact that contact with listeiae is, therefore, quite normal and even frequent, listeriosis is not at all common. The incidence of severe infections is estimated at six cases per 106 inhabitants per year. Occurrence is generally sporadic. Small-scale epidemics caused by food products—such as milk, milk products (cheese), meat products, and other foods (e.g., coleslaw)—contaminated with very high numbers of listeriae have been described. Preventive measures include proper processing and storage of food products in keeping with relevant hygienic principles.

Erysipelothrix rhizopathies'

• This bacterium is a slender, nonmotile, Gram-positive rod. E. rhizopathies' causes a septic disease in pigs, swine erysipelas. The correlate in humans is now quite rare and is a recognized occupational disease. Following contact with infectious animal material, the pathogens enter body tissues through dermal injuries. After an incubation period of one to three days, the so-called respooled—a hivelike, bluish-red swelling—develops at the site of entry. The lymph nodes are also affected. These benign infections often heal spontaneously and disappear rapidly under treatment with penicillin G. Laboratory diagnostic procedures involve identification of the pathogen in wound secretion using the methods of microscopy and culturing.

Gardnerella vaginalis

• G. vaginalis is a Gram-variable, nonmotile, nonencapsulated rod bacterium. Its taxonomy has changed repeatedly in recent decades. It has thus also been designated as Corynebacterium vaginalis and Hemophilus vaginalis. Based on DNA hybridization, the pathogen is now classified with the regularly shaped, Gram-positive, nonsporting rod bacteria. The natural habitat of this organism is the vagina of sexually mature women. It can also cause vulvovaginitis (vaginosis). G. vaginalis is found in over 90% of women showing the symptoms of this infection, usually together with other bacteria including in particular obligate anaerobes (Mobil uncus, Bacteroides, Pepto streptococcus). The organism can be detected in vaginal discharge by means of microscopy and culturing. In the microscopic analysis, so-called clue cells (vaginal epithelia densely covered with Gram-labile rods) provide evidence of the role played by G. vaginalis. This bacterium can be cultured on blood-enriched agar incubated in an atmosphere containing 5% CO2. The therapeutic agent of choice is metronidazole. 

Corynebacterium, Actinomyces, Other Gram-Positive Rod Bacteria

• Diphtheria bacteria are pleomorphic, club-shaped rod bacteria that often have polar bodies and group in V, Y, or palisade forms. They can be grown on enriched nutrient media. Their pathogenicity derives from diphtheria toxin, which binds to receptors of sensitive cells with the B fragment. Once the binding process is completed, the active A fragment invades the cell. This substance irreversibly blocks translation in the protein biosynthesis chain. The toxin gene is a component of the b prophage. Local and systemic intoxications are differentiated when evaluating the clinical picture. Local infection usually affects the tonsils, on which the diphtherial pseudomembrane develops. Systemic intoxications affect mainly the liver, kidneys, adrenal glands, cardiac muscle, and cranial nerves. Laboratory diagnosis is based on pathogen identification. The most important treatment is antitoxin therapy. Diphtheria occurs only in humans. Thanks to extensive diphtheria toxoid vaccination programs, it is now rare.

• Actinomycetes are part of the normal mucosal flora. These are Gram-positive rods that often occur in the form of branched filaments in young cultures. Conglomerates of microcolonies in pus form so-called sulfur granules. Actinomycetes are obligate anaerobes. The pathogens enter body tissues through mucosa defects. Monoinfections are rare, the most frequent case being actinomycetes-dominated endogenous polyinfections. Cervicofacial actinomycosis, caused by oral cavity colonizer A. israelii, is the most frequent form of actinomycosis. Treatment includes surgical procedures and antibiosis with aminopenicillins.

Corynebacterium diphtheriae (Diphtheria)

• Morphology and culturing. Diphtheria bacteria are Gram-positive, pleomorphic, often club-shaped rods. The individual cells tend to group in V, Y, or palisade arrangements (Fig. 4.9). Neisser staining reveals the polar bodies (polyphosphates stored at one end of the rod). Loffler nutrient medium, which consists of coagulated serum and nutrient broth, is still used for the primary cultures. Selective indicator mediums conTaining tellurite are used in selective culturing. K tellurite is used to inhibit the accompanying flora. The K tellurite is also reduced to tellurium, coloring the colonies a brownish black.

• Extracellular toxin. Diphtheria toxin consists of two functionally distinct fragments, A and B, whereby B stands for binding to receptors of target cells and A stands for toxic activity. Fragment A irreversibly blocks protein synthesis translation in the target cells, which then die. The toxin gene is always a prophage genome component (see lysogenic conversion, p. 186).

Pathogenesis and Clinical Picture

• Local infection. Infection of the mucosa of tonsils, pharynx, nose, and conjunctiva (Fig. 4.10). Wounds and skin lesions can also be infected. The pathogens invade the host through these portals, reproduce, and produce toxin, resulting in local cell damage. The inflammatory reaction leads to collection of a grayish-white exudate, the matrix of the “diphtherial pseudo membrane” consisting of fibrin, dead granulocytes, and necrotic epithelial cells. This coating adheres quite strongly to the mucosa. It may extend into the larynx, thus eventually hindering respiration. Regional lymph nodes are highly swollen.

• Systemic intoxication. Parenchymal degeneration in the cardiac muscle, liver, kidneys, and adrenal glands. Motor cranial nerve paralysis. Late sequel damage due to the intoxication is frequently seen after the acute infection has subsided. Toxin-negative strains of C. diphtheriae are occasionally observed as pathogens in endocarditis or dermal infections. The pathogenicity of such strains corresponds to that of commensal corynebacterial (see Table 4.3, p. 261).

• Diagnosis. The method of choice is detection and identification of the pathogen in cultures from local infection foci. The culture smear, which arrives at the laboratory in transport medium, is plated out on Loffler medium and a selective indicator medium. Identification is based on both morphological and physiological characteristics. The toxin is detected by the Elek-Auchterlonies

• Therapy. Antitoxic serum therapy is the primary treatment, and it must commence as possible if diphtheria is suspected. This treatment is us plummeted by administration of penicillin or erythromycin. immunodiffusion test. A molecular method is now also being used to identify the toxin gene. Toxin detection is necessary for a laboratory diagnosis of diphtheria because of the occurrence of toxin-negative strains.

• Epidemiology and prevention. Humans are the sole pathogen reservoir for diphtheria. Infection sources include infected persons and carriers (rare). The disease is usually transmitted by droplet infection, or less frequently indirectly via contaminated objects. The incubation period is two to five days. Incidence levels in central Europe are low. From 1975 to 1984, only 113 cases were reported in Germany. Incidence levels are higher in other countries (Russia). Protective immunization with diphtheria toxoid is the most important preventive measure (see Table 1.13, p. 33). Exposure prophylaxis involves isolation of infected persons until two cultures from specimens taken at least 24 hours apart are negative.

Actinomyces

• Actinomycetes are Gram-positive bacteria that tend to grow in the form of branched filaments. The resulting mycelial masses are, however, not observed in older cultures, which strongly resemble those of corynebacterial in their morphology.

• Occurrence. Actinomycetes are part of the normal mucosal flora in humans and animals. They colonize mainly the oral cavity, and an actinomycosis infection is therefore always endogenous. Ninety percent of actinomycetes infections in humans are caused by A. Israeli, with far fewer cases caused by A. espundia and other species.

• Morphology and culture. Actinomycetes are Gram-positive, pleomorphic rod bacteria that sometimes also show genuine branching (Fig. 4.11). The yellowish sulfur granules, measuring 1–2 mm, can be observed macroscopically in actinomycetes pus. These particles are conglomerates of small Actinomyces colonies surrounded by a wall of leukocytes. Mycelial filaments extend radially from the colonies (actinium = Greek for raylike). Culturing the organism requires enriched mediums and an anaerobic milieu containing 5–10% CO2. Mycelial microcolonies form only during the first days. Whitish microcolonies, often with a rough surface, begin to appear after two weeks.

• Pathogenesis and clinical picture. The pathogens breach mucosa (perhaps normal dermis as well) and are able to establish themselves in tissue in the presence of a low redox potential. The factors responsible for these conditions include poor blood perfusion and, above all, contributing bate pathogens. Genuine actinomycoses are actually always polymicrobial. The mixed flora found includes mainly the anaerobes of the oral cavity. Actinobacillus actinomycetemcomitans is frequently isolated along with various species of Bacteroides. Facultative anaerobes such as staphylococci, streptococci, and Enterobacteriaceae are, however, also found among the contributing florae.

• Cervicofacial actinomycosis. This is the most frequent form of acrimony Cetes infection (>90%). The abscesses are hard and tumorlike at first, then they necrotize. They may also break through to the dermal surface to create fistulae.

• Thoracic actinomycosis. This rare form results from aspiration of saliva; sometimes this type also develops from an actinomycosis in the throat or hematogenous spread.

• Abdominal actinomycosis. This type results from injuries to the intestine or female genitals.

• Genital actinomycosis. May result from use of intrauterine contraceptive devices.

• Canaliculate. An inflammation of the lacrimal canaliculi caused by any of several Actinomyces species.

• Caries. The Actinomyces species involved in caries development are A. viscos's, A. espundia, and A. odontolites' (p. 243f.). A possible contribution to periodontitis is also under discussion.

• Diagnosis involves identification of the pathogen by microscopy and culturing in pus, fistula secretion, granulation tissue, or bronchial secretion. The samples must not be contaminated with other patient flora, in particular from the oral cavity and must be transported to the laboratory in special anaerobe containers. Microscopic detection of branched rods suffices for a tentative diagnosis. Detection of mycelial microcolonies on enriched nutrient mediums after one to two weeks consolidates this diagnosis. Final identification by means of direct immunofluorescence, cell wall analysis, and metabolic analysis requires several weeks.

• Therapy. Treatment includes both surgical and antibiotic measures. The antibiotic of choice is an aminopenicillin. Antibiosis that also covers the contributing bacterial pathogens is important.

• Epidemiology and prevention. Actinomycoses occur sporadically worldwide. Average morbidity (incidence) levels are between 2.5 and five cases per 100 000 inhabitants per year. Men are infected twice as often as women. Prophylactic considerations are irrelevant due to the endogenous nature of actinomycetes infections.

Other Gram-Positive Rod Bacteria

• Table 4.3 lists bacteria that are rarely involved in infections and normally infect only persons with defective immune defenses. Recent years have seen considerable changes in their classification and nomenclature—still an ongoing process. Many of these bacteria are part of the normal dermal and mucosal flora. They are frequently found in sampled materials as contaminants, but also occasionally cause infections. Some of these bacteria are designated by collective terms such as “diphtheroid rods” or “coryneform bacteria.”

Mycobacterium

• Mycobacteria are slender rod bacteria that are stained with special differential stains (Ziehl-Nelsen). Once the staining has taken, they cannot be distained with dilute acids, hence the designation acid-fast. In terms of human disease, the most important mycobacteria are the tuberculosis bacteria (TB) M. tuberculosis and M. Bovis and the leprosy pathogen (LB) M. leprae. TB can be grown on lipid-rich culture mediums. Their generation time is 12–18 hours. Initial droplet infection results in primary tuberculosis, localized mainly in the apices of the lungs. The primary disease develops with the Gohn focus (Gohn’s complex), whereby the hilar lymph nodes are involved as well. Ninety percent of primary infection foci remain clinically silent. In 10% of persons infected, primary tuberculosis progresses to the secondary stage (reactivation or organ tuberculosis) after a few months or even years, which is characterized by extensive tissue necrosis, for example pulmonary caverns. The specific immunity and allergy that develop in the course of an infection reflect T lymphocyte functions. The allergy is measured in terms of the tuberculin reaction to check for clinically inapparent infections with TB. Diagnosis of tuberculosis requires identification of the pathogen by means of microscopy and culturing. Modern molecular methods are now coming to the fore in TB detection. Manifest tuberculosis is treated with two to four antitubercular chemotherapeutics in either a short regimen lasting six months or a standard regimen lasting nine months. In contrast to TB, the LB pathogens do not lend themselves to culturing on artificial nutrient mediums. Leprosy is manifested mainly in skin, mucosa, and nerves. In clinical terms, there is a (malignant) lepromatous type leprosy and a (benign) tuberculoid type. Nondifferential forms are also frequent. Humans are the sole infection reservoir. Transmission of the disease is by close contact with skin or mucosa.

Tuberculosis Bacteria (TB)

• History. The tuberculosis bacteria complex includes the species Mycobacterium tuberculosis, M. Bovis, and the rare species M. africanus. The clinical etiology of tuberculosis, a disease long known to man, was worked out in 1982 by R. Koch based on regular isolation of pathogens from lesions. Tuberculosis is unquestionably among the most intensively studied of all human diseases. In view of the fact that tuberculosis can infect practically any organ in the body, it is understandable why a number of other clinical disciplines profit from these studies in addition to microbiology and pathology.

• Morphology and culturing. TB are slender, acid-fast rods, 0.4 lm wide, and 3–4 lm long, nonsporting and nonmotile. They can be stained with special agents (Ziehl-Nielsen, Kanyon, fluorescence, p. 212f.) (Fig. 4.12a).

• TB are obligate anaerobes. Their reproduction is enhanced by the presence of 5–10% CO2 in the atmosphere. They are grown on culture mediums with a high lipid content, e.g., egg-enriched glycerol mediums according to Lo¨wenstein-Jensen (Fig. 4.12b). The generation time of TB is approximately 12–18 hours, so that cultures must be incubated for three to six or eight weeks at 37 8C until proliferation becomes macroscopically visible.

• Cell wall. Many of the special characteristics of TB are ascribed to the chemistry of their cell wall, which features a murein layer as well as numerous lipids, the most important being the glycolipids (e.g., lip arabinogalactan), the mycolic acids, mycosis, and wax D.

• Glycolipids and wax D. — Responsible for resistance to chemical and physical nixie. — Adjuvant effect (wax D), i.e., enhancement of antigen immunogenicity. — Intracellular persistence in nonactivated macrophages by means of inhibition of phagosome-lysosome fusion. — Complement resistance. — Virulence. Cord factor (trehalose 6,6-dimycolate).

• Tuberculoproteins. — Immunogens. The most important of these is the 65 kDa protein. — Tuberculin. Partially purified tuberculin contains a mixture of small proteins (10 kDa). Tuberculin is used to test for TB exposure. Delayed allergic reaction.

• Polysaccharides. Of unknown biological significance.

• Pathogenesis and clinical picture. It is necessary to differentiate between primary and secondary tuberculosis (reactivation or post primary tuberculosis) (Fig. 4.13). The clinical symptoms are based on reactions of the cellular immune system with TB antigens.

• Primary tuberculosis. In the majority of cases, the pathogens enter the lung in droplets, where they are phagocytosed by alveolar macrophages. TB bacteria are able to reproduce in these macrophages due to their ability to inhibit formation of the phagolysosome. Within 10–14 days a reactive inflammatory focus develops, the so-called primary focus from which the TB bacteria move into the regional hilar lymph nodes, where they reproduce and stimulate a cellular immune response, which in turn results in clonal expansion of specific T lymphocytes and attendant lymph node swelling. The Gho’s complex (primary complex, PC) develops between six and 14 weeks after infection. At the same time, granulomas form at the primary infection site and in the affected lymph nodes, and macrophages are activated by the cytokine MAF (macrophage activating factor). A tuberculin allergy also develops in the microorganism.

• Secondary tuberculosis. In about 10% of infected persons the primary tuberculosis reactivates to become an organ tuberculosis, either within months (5%) or after a number of years (5%). Exogenous reinfection is rare in the populations of developed countries. Reactivation begins with a caseation necrosis in the center of the granulomas (also called tubercles) that may progress to cavitation (formation of caverns). Tissue destruction is caused by cytokines, among which tumor necrosis factor a (TNFa) appears.

• Immunity. Humans show a considerable degree of genetically determined resistance to TB. Besides this inherited faculty, an organism acquires an (incomplete) specific immunity during initial exposure (first infection). This acquired immunity is characterized by localization of the TB at an old or new infection focus with limited dissemination (Koch’s phenomenon). This immunity is solely a function of the T lymphocytes. The level of immunity is high while the body is fending off the disease but falls off rapidly afterwards. It is therefore speculated that resistance lasts only as long as TB or the immunogens remain in the organism (= infection immunity).

• Tuberculin reaction. Parallel to this specific immunity, an organism infected with TB shows an altered reaction mechanism, the tuberculin allergy, which also develops in the cellular immune system only. The tuberculin reaction, positive six to 14 weeks after infection, confirms the allergy. The tuberculin proteins are isolated as purified tuberculin (PPD = purified protein derivative). Five tuberculin units (TU) are applied intracutaneously in the tuberculin test (Mantoux tuberculin skin test, the “gold standard”). If the reaction is negative, the dose is sequentially increased to 250 TU. A positive reaction appears within 48 to 72 hours as an inflammatory reaction (induration) at least 10 mm in diameter at the site of antigen application. A positive reaction means that the person has either been infected with TB or vaccinated with BCG. It is important to understand that a positive test is not an indicator for an active infection or immune status. While a positive test person can be assumed to have a certain level of specific immunity, it will by no means be complete. One-half of the clinically manifest cases of tuberculosis in the population are secondary reactivation tuberculosed that develop in tuberculin positive persons.

• Diagnosis requires microscopic and cultural identification of the pathogen or pathogen-specific DNA.

Traditional method

• Workup of test material, for example with N-acetyl-L-cysteine-NaOH (NALC-NaOH method) to liquefy viscous mucus and eliminate rapidly proliferous of test material, for example with N-acetyl-L-cysteine-NaOH (NALC-NaOH method) to liquefy viscous mucus and eliminate rapidly prolife.

• Microscopy. Ziehl-Nielsen and/or auramine fluorescent staining (p. 212). This method produces rapid results but has a low level of sensitivity (>104–105/ml) and specificity (acid-fast rods only).

• Culture on special solid and in special liquid mediums. Time requirement: four to eight weeks.

• Identification. Biochemical tests with pure culture if necessary. Time requirement: one to three weeks.

• Resistance test with pure culture. Time requirement: three weeks.

• Rapid methods. A number of different rapid TB diagnostic methods have been introduced in recent years that require less time than the traditional methods.

• Culture. Early-stage growth detection in liquid mediums involving identification of TB metabolic products with highly sensitive, semi-automated equipment. Time requirement: one to three weeks. Tentative diagnosis.

• Identification. Analysis of cellular fatty acids by means of gas chromatography and of mycolic acids by means of HPLC. Time requirement: 12 days with a pure culture.

• DNA probes. Used to identify M. tuberculosis complex and other mycobacteria. Time requirement: several hours with a pure culture.

• Resistance test. Use of semi-automated equipment (see Proliferation/nonproliferation determination in liquid mediums containing standard antituberculotic agents (Table 4.4). Time requirement: 7–10 days.

• Direct identification in patient material. Molecular methods used for direct detection of the M. tuberculosis complex in (uncultured) test material. These methods involve amplification of the search sequence.

• Therapy. The previous method of long-term therapy in sanatoriums has been replaced by a standardized chemotherapy (see Table 4.4 for examples), often on an outpatient basis.

• Epidemiology and prevention. Tuberculosis is endemic worldwide. The disease has become much less frequent in developed countries in recent decades, where its incidence is now about five to 15 new infections per 100 000 inhabitants per year and mortality rates are usually below one per 100 000 inhabitants per year. Seen from a worldwide perspective, however, tuberculosis is still a major medical problem. It is estimated that every year approximately 15 million persons contract tuberculosis and that three million die of the disease. The main source of infection is the human carrier. There are no healthy carriers. Diseased cattle are not a significant source of infection in the developed world. Transmission of the disease is generally direct, in most cases by droplet infection. Indirect transmission via dust or milk (udder tuberculosis in cattle) is the exception rather than the rule. The incubation period is four to 12 weeks.

• Exposure prophylaxis. Patients with open tuberculosis must be isolated during the secretory phase. Secretions containing TB must be disinfected. Tuberculous cattle must be eliminated.

• Disposition prophylaxis. An active vaccine is available that reduces the risk of contracting the disease by about one-half. It contains the live vaccine BCG (lyophilized bovine TB of the Calmette-Gue´ ran type). Vaccination of tuberculin-negative persons induces allergy and (incomplete) immunity that persist for about five to 10 years. In countries with low levels of tuberculosis prevalence, the advisory committees on immunization practices no longer recommend vaccination with BCG, either in tuberculin-negative children at high risk or in adults who have been exposed to TB. Preventive chemotherapy of clinically inapparent infections (latent tuberculosis bacterial infection, LTBI) with INH (300 mg/d) over a period of six months has proved effective in high-risk persons, e.g., contact persons who therefore became tuberculin-positive, in tuberculin-positive persons with increased susceptibility (immunosuppressive therapy, therapy with corticosteroids, diabetes, alcoholism) and in persons with radiologically confirmed residual tuberculosis. Compliance with the therapeutic regimen is a problem in preventive chemotherapy.

Leprosy Bacteria (LB)

• Morphology and culture. Mycobacterium leprae (Hansen, 1873) is the causa tie pathogen of leprosy. In morphological terms, these acid-fast rods are identical to tuberculosis bacteria. They differ, however, in that they cannot be grown on nutrient mediums or in cell cultures.

• Pathogenesis. The path mechanisms of LB are identical to those of TB. The host organism attempts to localize and isolate infection foci by forming granulomas. Leprous granulomas are histopathologic Ally identical to tuberculous granulomas. High counts of leprosy bacteria are often found in the macrophages of the granulomas.

• Immunity. The immune defenses mobilized against a leprosy infection are strictly of the cellular type. The lepromin skin test can detect a post infection allergy. This test is not, however, very specific (i.e., positive reactions which no leprosy infection is present). The clinically differentiated infection course forms observed are probably due to individual immune response variants. 

• Clinical picture. Leprosy is manifested mainly on the skin, mucosa, and peripheral nerves. A clinical differentiation is made between tuberculoid leprosy (TL, Fig. 4.14) and lepromatous leprosy (LL, Fig. 4.15). There are many intermediate forms. TL is the benign, nonprogressive form characterized by spotty dermal lesions. The LL form, on the other hand, is characterized by a malignant, progressive course with nodular skin lesions and cordlike nerve thickenings that finally lead to neuroparalytic. The inflammatory foci contain large numbers of leprosy bacteria.

• Diagnosis. Detection of the pathogens in skin or nasal mucosa scrapings under the microscope using Ziehl-Neilsen staining (p. 212). Molecular confirmation of DNA sequences specific to leprosy bacteria in a polymerase chain reaction is possible.

• Therapy. Paucibacillary forms are treated with dapsone plus rifampicin for six months. Multibacillary forms require treatment with Dapson, rifampicin, and clofazimine over a period of at least two years.

• Epidemiology and prevention. Leprosy is now rare in socially developed countries, although still frequent in developing countries. There are an estimated 11 million victims worldwide. Infected humans are the only source of infection. The details of the transmission pathways are unknown. Discussion of the topic is considering transmission by direct contact with skin or mucosa injuries and aerogenic transmission. The incubation period is 2–5–20 years. Isolation of patients under treatment is no longer required. An effective epidemiological reaction requires early recognition of the disease in contact persons by means of periodical examinations every six to 12 months up to five years following contact.

Nontuberculous Mycobacteria (NTM)

• Mycobacteria that are neither tuberculosis nor leprosy bacteria are categorized as atypical mycobacteria (old designation), nontuberculous mycobacteria (NTM) or MOTT (mycobacteria other than tubercle bacilli).

• Morphology and culture. In their morphology and staining behavior, NTM are generally indistinguishable from tuberculosis bacteria. With the exception of the rapidly growing NTM, their culturing characteristics are also similar to TB. Some species proliferate only at 30 8C. NTM are frequent inhabitants of the natural environment (water, soil) and also contribute to human and animal mucosal flora. Most of these species show resistance to the antituberculotic agents in common use.

• Clinical pictures and diagnosis. Some NTM species are apathogenic, others can cause dysbacteriosis in humans that usually follow a chronic course (Table 4.5). NTM infections are generally rare. Their occurrence is encouraged by compromised cellular immunity. Frequent occurrence is observed together with certain malignancies, in immunosuppressed patients and in AIDS patients, whereby the NTM isolated in 80% of cases are M. avium or M. intercellular. As a rule, NTM infections are indistinguishable from tuberculous lesions in clinical, radiological, and histological terms. Diagnosis therefore requires culturing and positive identification. The clinical significance of a positive result is difficult to determine due to the ubiquitous occurrence of these pathogens. They are frequent culture contaminants. Only about 10% of all persons in whom NTM are detected actually turn out to have a mycobacterium

• Therapy. Surgical removal of the infection focus is often indicated. Chemotherapy depends on the pathogen species, for instance a triple combination (e.g., INH, ethambutol, rifampicin) or, for resistant strains, a combination of four or five antituberculosis agents...

Nocardia

• Occurrence. The genus Nocardia includes species with morphology similar to that of the actinomycetes, differing from them in that the natural habitat of these obligate aerobes is the soil and damp biotopes. The pathogens known for involvement in nocardiosis, a generally very rare type of infection, include N. asteroids, N. Brasiliense's, N. farina, N. nova, and N. otitidiscaviarum.

• Morphology and culture. Nocardia are Gram-positive, fine, pleomorphic rods that sometimes show branching. They can be cultured on standard nutrient mediums and proliferate particularly well at 30 8C. Nocardia are obligate aerobes.
• Pathogenesis and clinical picture. Nocardia penetrate from the environment into the microorganism via the respiratory tract or dermal wounds. An infection develops only in patients with predisposing primary diseases directly affecting the immune defenses. Mono infections are the rule. There are no typical clinical symptoms. Most cases of infection involve pyogenic inflammations with central necroses. The following types have been described: pulmonary nocardiosis (bronchial pneumonia, pulmonary abscess), systemic nocardiosis (sepsis, cerebral abscess, abscesses in the kidneys and musculature), and surface nocardiosis (cutaneous and subcutaneous abscesses, lymphocutaneous syndrome)

• Actinomycetes' are tumorlike processes affecting the extremities, including bone. An example of such an infection is Madura foot, caused by Nocardia species, the related species Actinometers maduro, and Streptomyces soapiness. Fungi (p. 355) can also be a causal factor in this clinical picture.

• Diagnosis. Detection of the pathogen by means of microscopy and culturing techniques is required in materials varying with the specific disease. Due to the long generation time of these species, cultures have to be incubated for at least one week. Precise identification to differentiate pathogenic and apathogenic species is desirable, but difficult.

• Therapy. The anti-infective agents of choice are sulfonamides and cotrimoxazole. Surgery may be required.

• Epidemiology and prevention. Nocardiosis are rare infections. Annual incidence levels range from about 0.5 to 1 case per 1 000 000 inhabitants. The pathogens, which are present in the natural environment, are carried by dust to susceptible patients. There are no practicable prophylactic measures.

Neisseria, Moraxella, and Acinetobacter

• Neisseria are Gram-negative, aerobic cocci that are often arranged in pairs. They are typical mucosal parasites that die rapidly outside the human organism. Culturing on enriched nutrient mediums is readily feasible. Neisseria gonorrhea is the pathogen responsible for gonorrhea (“clap”). Infection results from sexual intercourse. The organisms adhere to cells of the urogenital tract by means of attachment pili and the protein Opa, penetrate into the organism using parasite-directed endocytosis and cause a pyogenic infection, mainly of the urogenital epithelium. An infection is diagnosed mainly by means of microscopy and culturing of purulent secretions. The therapeutic of choice is penicillin G. Alternatives for use against penicillinase-positive gonococci include third generation cephalosporins and 4-quinolones. N. meningitidis is a parasite of the nasopharyngeal mucosa. These meningococci cause meningitis and sepsis. Diagnosis involves detection of the pathogens in cerebrospinal fluid and blood. The disease occurs sporadically or in the form of minor epidemics in children, youths, and young adults. The antibiotics of choice are penicillin G and third generation cephalosporins.

Neisseria gonorrheal (Gonorrhea)

• Morphology and culture. Gonococci are Gram-negative, coffee-bean-shaped cocci that are usually paired and have a diameter of approximately 1 lm (Fig. 4.16). Attachment pili on the bacterial cell surface are responsible for their adhesion to mucosal cells. Gonococci can be grown on moist culture mediums enriched with protein (blood). The atmosphere for primary culturing must contain 5–10% CO2.

• Pathogenesis and clinical picture. Gonorrhea is a sexually transmitted disease. The pathogens penetrate into the urogenital mucosa, causing a local purulent infection. In men, the prostate and epididymis can also become infected. In women, the gonococci can also cause salpingitis, oophoritis, or even peritonitis. Gonococci reaching the conjunctival membrane may cause a purulent conjunctivitis, seen mainly in newborn children. Gonococci can also infect the rectal or pharyngeal mucosa. Hematogenous Lý disseminated gonococci may also cause arthritis or even endocarditis.

• Diagnosis. The method of choice is detection of the pathogens by means of methylene blue and gram staining and culturing. Gonococci are sensitive in cultures and the material must be used immediately after they are obtained to inoculate Thayer-Martin blood agar with antibiotics added to eliminate accompanying flora, on which medium the cultures are then transported to the laboratory. The identification procedure involves both morphology and biochemical characteristics. Techniques developed recently utilize immunofluorescence or conglutinations methods (p. 217) utilizing monoclonal antibodies to the main protein of the outer membrane, Por. Direct detection in pus and secretion samples is possible using an enzymatic immunosorbence test or detection of gonococcus-specific DNA sequences coding for rRNA using a gene probe.

• Therapy. The agent of choice used to be penicillin G. In recent years, however, the percentage of penicillinase-producing strains has increased considerably all over the world. For this reason, third generation cephalosporins are now used to treat uncomplicated cases of gonorrhea. They are applied in a single dose (e.g., ceftriaxone, 250–500 mg i.m.). Good results have also been reported with single-dose oral application of fluorinated 4-quinolones (e.g., 0.5 g ciprofloxacin or 0.4 g ofloxacin).

• Epidemiology and prevention. Gonorrhea is a worldwide sexually transmitted disease that occurs only in humans. Its level of annual incidence in developed countries is estimated at 12 cases per 1000 inhabitants. The actual figures are likely to be much higher due to large numbers of unreported cases. A reduction in incidence seen in recent years may be due to AIDS prophylaxis. Protective immunization for high-risk persons is not feasible due to the antigen variability of the organism as described above. Stopping the spread of gonorrhea involves mainly rapid recognition of infections and treatment accordingly. One hundred percent prevention of ophthalmia neonatorum is possible with a single parenteral dose of 125 mg ceftriaxone. Local prophylaxis is also practiced using a 1% solution of silver nitrate or eye ointments containing 1% tetracycline or 0.5% erythromycin.

Neisseria meningitidis (Meningitis, Sepsis)

• Morphology and culture. Meningococci are Gram-negative, coffee-bean shaped cocci that are frequently pleomorphic and have a diameter of 1 lm (Fig. 4.16b). They are nonmotile and feature a polysaccharide capsule. Growing meningococci in cultures requires mediums containing blood. A concentration of 5–10% CO2 encourages proliferation.

• Antigen structure. Serogroups A, B, C, D, etc. (a total of 12) are differentiated based on the capsule chemistry. Epidemics are caused mainly by strains of serogroup A, sometimes by B strains as well and, more rarely, by group C strains. Serogroups are divided into serovars based on differences in the outer membrane protein antigens.

• Pathogenesis and clinical picture. Meningococci are parasites of the nasopharynx. These microorganisms are carried by 5–10% of the population. If virulent meningococci colonize the nasopharyngeal mucosa of a host lacking the antibodies, pathogen invasion of the mucosa by means of “parasite directed endocytosis” becomes possible (see p. 12). The CNS is doubtless the preferred compartment for secondary infections, although hematogenous Lý disseminated pathogens can also infect the lungs, the endocardium, or major joints. Onset of the meningitis is usually sudden, after an incubation period of two to three days, with severe headache, fever, neck stiffness, and severe malaise. Severe hemorrhagic sepsis sometimes develops (Waterhouse-Friedrichsen syndrome).

• Diagnosis requires detection of the pathogen in cerebrospinal fluid or blood by means of microscopy and culturing techniques. For success in culturing, the material must be used to inoculate blood agar without delay. Identification of the pathogen is based on identification of metabolic properties. The slide agglutination test is used to determine the serogroup. Latex agglutination or conglutinations (p. 217) can be used for direct antigen detection in cerebrospinal fluid.

• Therapy. The antibiotic of choice is penicillin G. Very good results have also been obtained with third generation cephalosporins, e.g., cefotaxime or ceftriaxone. It is important to start treatment as quickly as possible to prevent delayed damage. The advantage of cephalosporins is that they are also effective against other meningitis pathogens due to their broad spectrum of action (with the exception of Listeria monocytogenes).

• Epidemiology and prevention. Meningococcal infections are more frequent in the winter and spring months. Transmission of meningococci is by droplet infection. Humans are the only pathogen reservoir. Sources of infection include both carriers and infected persons with manifest disease. In developed countries, meningitis occurs sporadically or in the form of minor epidemics in more or less isolated collectives (work camps, recruiting camps, school camping facilities). The incidence level is approximately 12 cases per 100 000 inhabitants per year. In parts of the developing world (African meningitis belt) the level is higher. Lethality runs to 85% if the disease is left untreated but is reduced to less than 1% if treatment is begun early enough. Prophylactic antibiosis is indicated for those in close contact with diseased persons (e.g., in the same family). Prophylactic measures also include treatment of carriers to eliminate this reservoir, whereby myocilin or rifampicin must be used instead of penicillin G. Prophylactic immunization can be achieved with a vaccine made from the purified capsule polysaccharides A, C, Y, and W-135. There is no serogroup B vaccine, since the capsule in serogroup B consists of Poly neuraminic acid, which the immune system does not recognize as a foreign substance.

Moraxella and Acinetobacter

• The taxonomic definitions of these genera are still inconclusive. Bergey’s Manual of Systematic Bacteriology groups under the family Moraxella. These bacteria are short, rounded rods, often coccoid, sometimes also diplococcic. Their natural habitat is either human mucosa (Moraxella) or the natural environment (Acinetobacter).

• Moraxella. The genus comprises two medically important species: — Moraxella catarrhalis. Component of the normal flora of the upper respiratory tract. May be responsible for: pneumonia, acute exacerbation of chronic bronchitis, otitis media (up to 20% in children), and sinusitis. About 90% of all strains produce one of the so-called BRO penicillinases, so that therapy with a penicillinase-stable beta lactam antibiotic is indicated.

• Moraxella lacuna. Formerly Diprotactinium Morax-Axenfeld. Can cause conjunctivitis and keratitis. The reason why this organism is now rarely found as a pathogen in these eye infections is unknown.

• Acinetobacter. In immunodeficient persons, A. Baumann Ii, A. calcoaceticus, and other species can cause nosocomial infections (urinary tract infections, pneumonias, wound infections, sepsis). Clinical strains of these species often show ultradistance to antibiotics, so that treatment of these infections may prove difficult.

Enterobacteriaceae, Overview

• The most important bacterial family in human medicine is the Enterobacteriaceae. This family includes genera and species that cause well-defined diseases with typical clinical symptoms (typhoid fever, dysentery, plague) as well as many opportunists that cause mainly nosocomial infections (urinary tract infections, pneumonias, wound infections, sepsis). Enterobacteriaceae are Gram-negative, usually motile, facultatively anaerobic rod bacteria. The high levels of metabolic activity observed in them are made use of in identification procedures. The species are subdivided into epidemiologically.

• Definition and significance. Together with the families Vibrionaceae and others (p. 224), the Enterobacteriaceae form the group of Gram-negative, facultatively anaerobic rod bacteria. Their natural habitat is the intestinal tract of humans and animals. Some species cause characteristic diseases. While others are facultatively pathogenic, they are still among the bacteria most frequently isolated as pathogens (e.g., E. coli). They are often responsible for nosocomial diseases (see p. 343ff.).

• Taxonomy. The taxonomy of the Enterobacteriaceae has seen repeated changes in recent decades and has doubtless not yet assumed its final form. The family includes 41 genera with hundreds of species. Table 4.6 provides an overview of the most important Enterobacteriaceae in the field of human medicine. The taxonomic system applied to Enterobacteriaceae is based on varying patterns of metabolic processes (Fig. 3.36, p. 214). One of the important characteristics of this bacterial family is lactose breakdown (presence of the lac operon). The lac operon includes the genes lacZ (codes for b-galactosidase), lacy (codes for b-galactose permease), and Laca (codes for transacetylase). Lactose-positive Enterobacteriaceae are grouped together as coliform Enterobacteriaceae. Salmonellae and most of the shigella are lactose negative.

• Morphology and culture. Enterobacteriaceae are short Gram-negative rods with rounded ends, 0.5–1.5 lm thick, and 24 lm long (Fig. 4.17a). Many have peritrichous flagellation. Species with many flagella (e.g., Proteus species) show motility on the agar surface, which phenomenon is known as “swarming.” Some Enterobacteriaceae possess a capsule. All bacteria in this family can readily be cultured on simple nutrient mediums. They are rapidly growing facultative anaerobes. Their mean generation time in vitro is 20–30 minutes. They show resistance to various chemicals (bile salts, crystal violet), which fact is made use of in selective culturing. Endo agar is an important selective indicator medium; it allows only Gram-negative rod bacteria to grow and indicates lactose breakdown (Fig. 4.17b).

• Antigen structure. The most important antigens of the Enterobacteriaceae are: & O antigens. Specific polysaccharide chains in the lipopolysaccharide complex of the outer membrane (p. 156). & H antigens. Flagellar antigens consisting of protein. & K antigens. Linear polymers of the outer membrane built up of a repeated series of carbohydrate units (sometimes proteins as well). They can cover the cell densely and render them O in agglutinable (p. 155). & F antigens. Antigens of protein attachment fimbriae.

• Pathogenicity determinants. A number of factors are known to play a role in the pathogenicity of various Enterobacteriaceae infections. The most important are.

• Adhesion factors. Attachment fimbriae, attachment pili, colonizing factor antigens (CFAs). & Invasive factors. Proteins localized in the outer membrane (invasions') that facilitate the invasion of target cells. & Exotoxins. — Enterotoxins disturb the normal functioning of enterocytes. Stimulation of adenylate or guanylate cyclase; increased production of cAMP (see p. 298). This results in the loss of large amounts of electrolytes and water. — Cytotoxins exert a direct toxic effect on cells (enterocytes, endothelial cells). & Endotoxin. Toxic effect of lipoid A as a component of LPS (p. 156). & Serum resistance. Resistance to the membrane attack complex C5b6789 of the complement system (p. 86ff.). & Phagocyte resistance. Makes survival in phagocytes possible. Resistance against defensins and/or oxygen radicals (p. 23). & Cumulation of Fe2+. Active transport of Fe2+ by siderophores in the bacterial cell (p. 13).

Salmonella (Gastroenteritis, Typhoid Fever, Paratyphoid Fever

• All salmonellae are classified in the species Salmonella enterica with seven subspecies. Nearly all human pathogen salmonellae are grouped under S. enterica, subsp. enterica. Salmonellae are further subclassified in over 2000 serovars based on their O and H antigens, which used to be (incorrectly) designated as species

• Typhoid salmonellosis is caused by the serovars typhi and paratyphoid A, B, and C. The salmonellae are taken up orally and the invasion pathway is through the intestinal tract, from where they enter lymphatic tissue, first spreading lymphopenias, then hematogenous. A generalized septic clinical picture result. Human carriers are the only source of infection. Transmission is either direct by smear infection or indirect via food and drinking water. Anti-infective agents are required for therapy (ampicillin, cotrimoxazole, 4-quinolones). An active vaccine is available to protect against typhoid fever.

• Enteric salmonellosis develops when pathogens are taken up with food. The primary infection source is usually livestock. These relatively frequent infections remain restricted to the gastrointestinal tract. Treatment with anti-infective agents is necessary in exceptional cases only.

• Taxonomy. The salmonellae that cause significant human disease are classified in most countries under the taxon Salmonella enterica, subsp. enterica (synonymous with S. cholerae's, subsp. cholerae's). However, this nomenclature has still not been officially adopted by the Enterobacteriaceae Subcommittee. Salmonella enterica, spp. enterica includes over 2000 serovars, which were formerly (incorrectly) designated with species names. The serovars are capitalized to differentiate them from species.

• Pathogenesis and clinical pictures. Salmonellae are classified as either typhoid or enteric regarding the relevant clinical pictures and epidemiology. It is not known why typhoid salmonellae only cause systemic disease in humans, whereas enteric salmonella infections occur in animals as well and are usually restricted to the intestinal tract.

• Typhoid salmonellosis. Attachment of typhoid salmonellae to cells of the jejunum (M cells). Invasion by means of endocytosis, transfer, and exocytosis. Phagocytosis in the subserosa by macrophages and translocation into the mesenteric lymph nodes. Proliferation occurs. Lymphopenias and hematogenous dissemination. Secondary foci in the spleen, liver, bone marrow, bile ducts, skin (roseola), Peyer’s patches. Manifest illness begins with fever, rising in stages throughout the first week to 39/40/41 8C. Further symptoms: stupor (typhons [Greek] = fog), leukopenia, bradycardia, splenic swelling, abdominal roseola, beginning in the third week diarrhea, sometimes with intestinal bleeding due to ulceration of the Peyer’s patches.

• Enteric salmonellosis. Attachment to enterocytes of the ileum and colon. Invasion of mucosa induced by invasion proteins on the surface of the salmonella cells. Persistence in epithelial cells, possibly in macrophages as well. Production of Salmonella enterotoxin. Local inflammation. Manifest illness usually begins suddenly with diarrhea and vomiting, accompanied in some cases by high fever. The symptoms abate after several days without specific therapy. In cases of massive diarrhea, symptoms may be observed that result from the loss of water and electrolytes (Table 4.8).

• Diagnosis. The method of choice is detection of the pathogens in cultures. Selective indicator mediums are used to isolate salmonellae in stool. Identification is done using metabolic patterns (see Fig. 3.36, p. 214). Serovar classification is determined with specific antisera in the slide agglutination test. Culturing requires at least two days. Typhoid salmonellosis can be diagnosed indirectly by measuring the titer of agglutinating antibodies to O and H antigens (according to Gruber-Widal). To provide conclusive proof the titer must rise by at least fourfold from blood sampled at disease onset to a sample taken at least one week later.

• Therapy. Typhoid salmonellosis must be treated with anti-infective agents, whereas symptomatic treatment will suffice for enteric infections. Symptomatic treatment encompasses slowing down intestinal activity (e.g., with loperamide) and replacing fluid and electrolyte losses orally as required (WHO formula: 3.5 g NaCl, 2.5 g NaHCO3, 1.5 g Kc, 20 g glucose per liter of water).

Shigella (Bacterial Dysentery)

• Shigella is the causative pathogen in bacterial dysentery. The genus comprises the species S. dysenteries', S. flexier, S. boydii, and S. sonnet. Shigellas are nonmotile. The three primary species can be classified in serovars based on the fine structure of their O antigens. Shigellas are characterized by invasive properties. They can penetrate the colonic mucosa to cause local necrotic infections. Humans are the sole source of infection since shigellas are pathologically active in humans only. The pathogens are transmitted directly, more frequently indirectly, via food and drinking water. Antibiotics can be used therapeutically.

• Classification. The genus Shigella includes four species: S. dysenteries', S. flexier, S. boydii, and S. sonnet. The first three are subdivided into 10, six, an 15 serovars, respectively, based on their antigen structures. Shigella are nonmotile and therefore have no flagellar (H) antigens.

• Pathogenesis. Shigella are only pathogenic in humans. The pathogens are ingested orally. Only a few hundred bacteria suffice for an infective dose. Shigella enter the terminal ileum and colon, where they are taken up by the M cells in the intestinal mucosa, which in turn are in close vicinity to the macrophages. Following phagocytosis by the macrophages, the shigella lyse the phagosome and actively induce macrophage apoptosis. The shigellas released from the dead macrophages are then taken up by enterocytes via the basolateral side of the mucosa (i.e., retrograde transport). The invasion is facilitated by outer membrane polypeptides, the invasion's, which are coded by inv genes localized on 180–240 kb plasmids. Adjacent enterocytes are invaded by means of lateral transfer from infected cells. In the enterocytes, the shigellas reproduce, finally destroying the cells. Shigella dysenteries' produces Shiga toxin, the prototype for the family of Shiga like toxins (or verocytotoxins), which also occur in several other Enterobacteriaceae. The toxin inhibits protein synthesis in eukaryotic cells by splitting the 23S rRNA at a certain locus. Shigatoxin contributes to the colonic epithelial damage, the small intestine diarrhea with watery stools at the onset of shigellosis and (less frequent) the hemolytic-uremic syndrome (HUS).

• Clinical picture. Following an incubation period of two to five days, the disease manifests with profuse watery diarrhea (= small intestine diarrhea). Later, stools may contain mucus, pus, and blood. Intestinal cramps, painful stool elimination (tenesmus), and fever are observed in the further course of the infection. Complications include massive intestinal bleeding and perforation peritonitis. These severe effects are caused mainly by S. dysenteries', whereas S. sonnet infections usually involve only diarrhea.

• Diagnosis requires identification of the pathogen in a culture. Combined selective/indicator mediums must be used for the primary culture. Suspected colonies are identified by using indicator media to detect certain metabolic characteristics (p. 214). The serovar is determined with specific antisera in the slide agglutination test.

• Therapy. Anti-infective agents are the first line of treatment (aminopenicillins, 4-quinolones, cephalosporins). Losses of water and electrolytes may have to be replaced.

• Epidemiology and prevention. Bacterial dysentery occurs worldwide, although it is usually seen only sporadically in developed countries. In developing countries, its occurrence is more likely to be endemic and even epidemic. The source of infection is always humans, in most cases infected persons whose stools contain pathogens for up to six weeks after the disease has abated. Transmission is by direct contact (smear infection) or indirect uptake via food, surface water, or flies. Control of dysentery includes exposure prophylaxis measures geared to prevent susceptible persons from coming into contact with the pathogen.

Yersinia (Plague, Enteritis)

• Y. pestis is the causative pathogen of plague (black death, bubonic plague). Plague is a classic rodent zoonosis. It occurred in epidemic proportions in the Middle Ages but is seen today only sporadically in persons who have had direct contact with diseased wild rodents. The pathogens penetrate into the skin through micro traumata, from where they reach regional lymph nodes in which they proliferate, resulting in the characteristic buboes. In the next stage, the pathogens may enter the bloodstream, or the infection may generalize to affect other organs. Laboratory diagnosis involves isolation and identification of the organism in pus, blood, or other material. Therapy requires use of antibiotics. Y. enterocolitica and Y. pseudotuberculosis cause generalized zoonoses in wild animals and livestock. Diseased animals contaminate their surroundings. Humans then take up the pathogens orally in water or food. The organisms penetrate the mucosa of the lower intestinal tract, causing enteritis accompanied by mesenteric lymphadenitis. Extra mesenteric forms are observed in 20% of infected persons (sepsis, lymphadenopathies, various focal infections). Secondary immunopathological complications include arthritis and erythema nodosum. Diagnosis involves identification of the pathogen by means of selective culturing.

Yersinia pestis

• Morphology and culture. Y. pestis is a monoflagellated, short, encapsulated, Gram-negative rod bacteria that often shows bipolar staining. This bacterium is readily cultured on standard nutrient mediums at 30 8C. Pathogenesis and clinical picture. The plague is primarily a disease of rodents (rats). It spreads among them by direct contact or via the rat flea. plague epidemics in humans resulted from these same transmission pathways. The rare human infections seen today result from contact with rodents that are infected with or have died of plague. The pathogen breaches the skin through dermal injuries. From such a location, the bacteria reach regional lymph nodes in which they proliferate. Two to five days after infection, hemorrhagically altered, blue, and swollen lymph nodes (buboes) are observed. Over 90% of pestis infections show the “bubonic plague” course. In 50–90% of untreated cases, the organisms break out into the bloodstream to cause a clinical sepsis, in the course of which they may invade many different organs. Dissemination into the pulmonary circulation results in secondary pulmonary plague with bloody, bacteria-rich, highly infectious sputum. Contact with such patients can result in primary pulmonary plague infections due to direct, aerogenic transmission. Left untreated, this form of plague is lethal in nearly 100% of cases.

• Diagnosis. The pathogen must be identified in bubo punctate, sputum, or blood by means of microscopy and culturing. Therapy. In addition to symptomatic treatment, antibiotics are the primary method (streptomycin, tetracyclines, in the case of meningitis, chloramphenicol). Incision of the buboes is contraindicated. Epidemiology and prevention. Plague still occurs endemically in wild rodents over large areas of Asia, Africa, South America, and North America. Human plague infections have been reduced to sporadic instances. The sources of infection are mainly diseased rodents. Transmission of the disease is mainly via direct contact with such animals. Prevention involves exposure prophylactic measures. Persons with manifest disease, in particular the pulmonary form, must be isolated. Contact persons must be quarantined for six days (= incubation period). Cases of plague infection must be reported to health authorities.

Yersinia enterocolitica and Yersinia pseudotuberculosis

• Occurrence and significance. Y. enterocolitica and Y. pseudotuberculosis cause generalized infections in domestic and wild animals, especially rodents. The pathogens can be transmitted from animals to humans. Y. enterocolitica is responsible for about 1% of acute enteritis cases in Europe. Y. pseudotuberculosis is insignificant in terms of human pathology. Morphology, culture, and antigen structure. These are pleomorphic, short rods with peritrichous flagellation. They can be cultured on all standard mediums. These Yersinia bacteria grow better at 20–30 8C than at 37 8C. Pathogenesis and clinical pictures. All of the strains isolated as human pathogens bear a 70 kb virulence plasmid with several Vir determinants. They code for polypeptides that direct the functions cell adhesion, phagocytosis resistance, serum resistance, and cytotoxicity. Yersinia also have chromosomal virulence genes, for example markers for invasions', enterotoxins, and an iron capturing system. Exactly how these virulence factors interact to produce the disease is too complex to be described in detail here. Yersinia are usually ingested indirectly with food. Although much less frequent, infections can also occur by way of direct contact with diseased animals or animal carriers. The bacteria enter the lower intestinal tract, penetrate the mucosa and are transported with the macrophages into the mesenteric lymph nodes. A simplified overview of the resulting clinical pictures follows:

• Intestinal yersinioses. The clinically dominant symptom is enteritis together with mesenteric lymphadenitis. This form is frequently observed in youths and children. Other enteric forms include pseudo appendicitis in youths and children, ileitis (pseudo-Crohn disease), and colitis in adults. & Extraintestinal yersinioses. These infections account for about 20% of cases, usually adults. Notable features of the clinical picture include sepsis, lymphadenopathy, rarely hepatitis, and various local infections (pleuritis, endocarditis, osteomyelitis, cholecystitis, localized abscesses). & Other sequelae. The immunopathological complications observed in about 20% of acutely infected patients one to six weeks after onset of the intestinal symptoms include reactive arthritis and erythema nodosum. Diagnosis. A confirmed diagnosis is only possible with identification of the pathogen in a culture based on physiological characteristics. Special mediums are used to isolate the pathogen from stool. The agglutination reaction, an ELISA or immunoblot assay can be used to detect the antibodies. Therapy. Generally, favorable courses require no chemotherapy. Clinically difficult cases can be treated with cotrimoxazole, second- or third generation cephalosporins, or fluorinated 4-quinolones. Epidemiology and prevention. Prevalence of Y. enterocolitica and Y. pseudotuberculosis in animals is widespread. The most important reservoirs in epidemiological terms are mammals that are diseased or carry latent infections. From these sources, vegetation, soil, and surface water are contaminated. Transmission is by the oral pathway in food. Contact zoonosis is possible, but rare. There are no specific prophylactic measures.

Escherichia coli

• The natural habitat of E. coli is the intestinal tract of humans and animals. It is therefore considered an indicator organism for fecal contamination of water and foods. E. coli is the most frequent causative pathogen in human bacterial infections. Extraintestinal infections include urinary tract infections, which occur when the tract is obstructed or spontaneously caused by the pathovar UPEC. The most important other coli infections are cholecystitis, appendicitis, peritonitis, postoperative wound infections, and sepsis. Intestinal infections are caused by the pathovars EPEC, ETEC, EIEC, EHEC, and Egged. EPEC and egged frequently cause diarrhea in infants. ETEC produce enterotoxins that cause a choler Alike clinical picture. EIEC cause a dysentery like infection of the large intestine. EHEC produce verocytotoxins and cause a hemorrhagic colitis as well as the rare hemolytic-uremic syndrome. E. coli bacterial infections are diagnosed by means of pathogen identification.

• General characteristics. The natural habitat of E. coli is the intestines of animals and humans. This bacterium is therefore used as an indicator for fecal contamination of drinking water, bathing water, and foods. Guideline regulations: 100 ml of drinking water must not contain any E. coli. Surface water approved for bathing should not contain more than 100 (guideline value) to 2000 (absolute cutoff value) E. coli bacteria per 100 ml. E. coli is also an important human pathogen. It is the bacterial species most frequently isolated from pathological materials.

• Morphology, culture, and antigen structure. The Gram-negative, straight rods are peritrichously flagellated. Lactose is broken down rapidly. The complex antigen structure of these bacteria is based on O, K, and H antigens. Fimbrial antigens have also been described. Specific numbers have been assigned to the antigens, e.g., serovar O18:K1:H7.

• Pathogenesis and clinical picture of extraintestinal infections. Extraintestinal infections result from relocation of E. coli bacteria from one’s own flora to places on or in the microorganism where they are not supposed to be but where conditions for their proliferation are favorable.

• Urinary tract infection. Such an infection manifests either solely in the lower urinary tract (urethritis, cystitis, erythrocytosis) or affects the renal pelvis and kidneys (cyst pyelitis, pyelonephritis). In acute urinary tract infections, E. coli is the causative organism in 70–80% of cases and in chronic, persistent infections in 40–50% of cases.

• Sepsis. E. coli causes about 15% of all cases of nosocomial sepsis (S. aureus 20%). An E. coli sepsis is frequently caused by the pathovar SEPEC, which shows serum resistance (p. 13).

• Other E. coli infections. Wound infections, infections of the gallbladder and bile ducts, appendicitis, peritonitis, meningitis in premature infants, neonates, and very elderly patients.

• Pathogenesis and clinical pictures of intestinal infections. E. coli that causes intestinal infections are now classified in five pathovars with differing pathogenicity and clinical picture.

• Enteropathogenic E. coli (EPEC). These bacteria cause epidemic or sporadic infant diarrheas, now rare in industrialized countries but still a main contributor to infant mortality in developing countries. EPEC attach themselves to the epithelial cells of the small intestine by means of the EPEC adhesion factor (EAF), then inject toxic molecules into the enterocytes by means of a type III secretion system (see p. 17).

• Enterotoxin E. coli (ETEC). The pathogenicity of these bacteria is due to the heat-labile enterotoxin LT (inactivation at 60 8C for 30 minutes) and the metastable toxins Stab and Stab (can tolerate temperatures up to 100 8C). Some strains produce all of these toxins, some only one. LT is very similar to cholera toxin. It stimulates the activity of adenylate cyclase (see p. 298). Stab stimulates the activity of guanylate cyclase. (cGMP mediates the inhibition of Na+ absorption and stimulates Cl– secretion by enterocytes.) ETEC pathogenicity also derives from specific fimbriae, so-called colonizing factors (CFA)that allow these bacteria to attach themselves to small intestine epithelial cells, thus preventing their rapid removal by intestinal peristalsis. The enterotoxins and CFA are determined by plasmid genes. The clinical picture of an ETEC infection is characterized by massive watery diarrhea. The disease can occur at any age. Once the illness has abated, a local immunity is conferred lasting several months.

• Enter invasive E. coli (EIEC). These bacteria can penetrate into the colonic mucosa, where they cause ulcerous, inflammatory lesions. The pathogenesis and clinical picture of EIEC infections are the same as in bacterial dysentery (p. 288). EIEC strains are often lac negative.

Opportunistic Enterobacteriaceae

• Many Enterobacteriaceae with minimum pathogenicity are classic opportunists. The most frequent opportunistic infections caused by them are: urinary tract infections, respiratory tract infections, wound infections, dermal and subcutaneous infections, and sepsis. Such infections only occur in predisposed hosts, they are frequently seen in patients with severe primary diseases. Another reason why opportunistic Enterobacteriaceae have become so important in hospital medicine is the frequent development of resistance to anti-infective agents, which ability enables them to persist at locations where use of such agents is particularly intensive, i.e., in hospitals. Occurrence of multiple resistance in Enterobacteriaceae is due to the impressive genetic variability of these organisms (p. 170). Table 4.9 provides an overview of the most important opportunistic Enterobacteriaceae.

Vibrio, Aeromonas, and Plaisimonds

• Vibrio cholerae is the most important species in this group from a medical point of view. Cholera vibrios are Gram-negative, comma-shaped, monoicous flagellated rods. They show alkali tolerance (pH 9), which is useful for selective culturing of V. cholerae in alkaline peptone water. The primary cholera pathogen is serovar O:1. NonO:1 strain (e.g., O:139) cause the typical clinical picture in rare cases. O:1 vibrions are further subdivided into the biovars cholerae and lector. The disease develops when the pathogens enter the intestinal tract with food or drinking water in large numbers (> –108). The vibrio's multiply in the proximal small intestine and produce an enterotoxin. This toxin stimulates a series of reactions in enterocytes, the end result of which is increased transport of electrolytes out of the enterocytes, whereby water is also lost passively. Massive watery diarrhea (up to 20 l/day) results in exsiccosis. The initial therapeutic focus is thus on replacement of lost electrolytes and water. Cholera occurs only in humans. Preventive measures concentrate on protection from exposure to the organism. A killed whole cell vaccine and an attenuated live vaccine are available. They provide only a moderate degree of protection over a period of only six months. International healthcare sources report an incubation period of five days.

• Morphology and culture. Cholera vibrions are Gram-negative rod bacteria, usually slightly bent (comma-shaped), 1.5–2 lm in length, and 0.3–0.5 lm wide, with monoicous flagellation (Fig. 4.19). Culturing of V. cholerae is possible on simple nutrient mediums at 37 8C in a normal atmosphere. Owing to its pronounced alkali stability, V. cholerae can be selectively cultured out of bacterial mixtures at pH 9.

• Antigens and classification. V. cholerae bacteria are subdivided into serovars based on their O antigens (lipopolysaccharide antigens). The serovar pathogen is usually serovar O:1. Strains that do not react to an O:1 antiserum are grouped together as nonO:1 vibrions. NonO:1 strain were recently described in India (O:139) as also causing the classic clinical picture of cholera. O:1 vibrios are further subclassified in the biovars cholerae and elder based on physiological characteristics. The var elder has a very low level of virulence

• Cholera toxin. Cholera toxin is the sole cause of the clinical disease. This substance induces the enterocytes to increase secretion of electrolytes, above all Cl– ions, whereby passive water loss also occurs. The toxin belongs to the group of AB toxins (see p. 16). Subunit B of the toxin binds to enterocyte receptors, the active toxin subunit A causes the adenylate cyclase in the enterocytes to produce cAMP continuously and in large amounts (Fig. 4.20). cAMP in turn acts as a second messenger to activate protein kinase A, which then activates the specific cell proteins that control secretion of electrolytes. The toxin genes ctxA and ctxB are components of the so-called CTX element, which is integrated in the nucleoid of toxic cholera vibrios (see lysogenic conversion, p. 186) as part of the genome of the filamentous prophage CT. The CTX element also includes several regulator genes that regulate both produces.

• Pathogenesis and clinical picture. Infection results from oral ingestion of the pathogen. The infective dose must be large (> –108), since many vibrios are killed by the hydrochloric acid in gastric juice. Based on their pronounced stability in alkaline environments, vibrios are able to colonize the mucosa of the proximal small intestine with the help of TCP (see above) and secrete cholera toxin (see Fig. 4.20). The pathogen does not invade the mucosa. The incubation period of cholera is two to five days. The clinical picture is characterized by voluminous, watery diarrhea and vomiting. The number of fluids lost per day can be as high as 20 l. Further symptoms derive from the resulting exsiccosis: hypotension, tachycardia, anuria, and hypothermia. Lethality can be as high as 50% in untreated cases.

• Diagnosis requires identification of the pathogen in stool or vomit. Some a rapid microscopical diagnosis succeeds in finding numerous Gram-negative, bent rods in swarm patterns. Culturing is done on liquid or solid selective mediums, e.g., alkaline peptone water or taurocholate gelatin agar. Suspected colonies are identified by biochemical means or by detection of the O:1 antigen in an agglutination reaction.

• Therapy. The most important measure is restoration of the disturbed water and electrolyte balance in the body. Secondly, tetracyclines and cotrimoxazole can be used, above all to reduce fecal elimination levels and shorten the period of pathogen secretion.

• Epidemiology and prevention. Nineteenth-century Europe experienced several cholera pandemics, all of which were caused by the classic cholerae biovar. An increasing number of cases caused by the biovar lector, which is characterized by a lower level of virulence, have been observed since 1961. With the exception of minor epidemics in Italy and Spain, Europe, and the USA have been spared major outbreaks of cholera in more recent times. South America has for a number of years been the venue of epidemics of the disease. Humans are the only source of infection. Infected persons in particular eliminate large numbers of pathogens. Convalescents may also shed V. cholerae for weeks or even months after the infection has abated. Chronic carriers as with typhoid fever are very rare. Transmission of the disease is usually via foods, and in particular drinking water. This explains why cholera can readily spread to epidemic proportions in countries with poor hygiene standards.

Other Vibrio Bacteria

• Vibrio parahaemolyticus is a halophilic (salt-friendly) species found in warm ocean shallows and brackish water. These bacteria can cause gastroenteritis epidemics. The pathogen is transmitted to humans with food (seafood, raw fish). The illness is transient in most cases and symptomatic therapy is sufficient. Vibrio vulnificus is another aquatic organism that produces a very small number of septic infections, mainly in immunosuppressed patients.

Aeromonas and Pressimone's

• The bacteria of these two genera live in freshwater biotopes. Some are capable of causing infection in fish (A. salmonoids). They are occasionally observed as contaminants of moist parts of medical apparatus such as dialysis equipment, vaporizers, and respirators. They can cause nosocomial infections in hospitalized patients with weakened immune systems. Cases of gastroenteritis may result from eating foods contaminated with large numbers of these bacteria.

Haemophiles and Pasteurella

• The most important species of Pasteurella from the medical point of view is Hemophilus influenzae. This is a nonmotile, Gram-negative rod that is often encapsulated. Capsule serovar b is the main pathogenic form. H. influenzae is a facultative anaerobe that requires growth factors X (hemin) and V (NAD, NADP) in its culture medium. H. influenzae is a typical parasite of the respiratory tract mucosa. It occurs only in humans. It causes infections of the upper and lower respiratory tract in individuals with weakened immune defenses and in children under the age of four or five. Invasive infections—meningitis and sepsis—are also observed in small children. A betalactamasestable beta lactam antibiotic is required for treatment since the number of beta lactamase-producing strains observed is increasing. Conjugate vaccines in which the capsule polysaccharide is coupled with proteins are available for prophylactic immunization. These vaccines can be administered beginning in the third month of life.

Hemophilus influenzae

• Hemophilic bacteria are so designated because they require growth factors contained in blood. The most important human pathogen in this genus is H. influenzae. Other Haemophile's species either infect only animals or are found in the normal human mucosal flora. These latter include H. parainfluenzas, H. hemolytic us, H. sengis', H. amphophiles, and H. preprophases. These species can cause infections on occasion. Morphology and culture. Hemophilus are small (length: 1.0–1.5 lm, width: 0.3 lm), often encapsulated, nonmotile, Gram-negative rods (Fig. 4.21a). The encapsulated strains are subclassified in serovars a-f based on the fine structure of their capsule polysaccharides. Serovar b (Hib) causes most Haemophile's infections in humans.

• Pathogenesis and clinical pictures. H. influenzae is a mucosal parasite of the upper respiratory tract present in 30–50% of healthy persons. The strains usually found are nonencapsulated and therefore hardly virulent. The capsule protects the cells from phagocytosis and is thus the primary determinant of pathogenicity. Others include the affinity of H. influenzae to respiratory tract mucosa and meninges and production of an IgA1 protease (see p. 15).

• Diagnosis. The method of choice is identification of the pathogen in cerebrospinal fluid, blood, pus, or purulent sputum using microscopy and culture assays. Satelliting on blood agar is an indication of a V factor requirement. An X factor requirement is confirmed most readily by the porphyrin test, with a negative result in the presence of H. influenzae.

• Therapy. In view of the increasing number of beta-lactamase-producing H. influenzae strains observed in recent years, penicillinase-stable Beta lactam antibiotics should be used to treat these infections. The likelihood that a strain produces beta lactamase is 5–30% in most countries. 4-quinolones are an alternative to Beta lactams that should not, however, be used in children. The agent of choice in meningitis is ceftriaxone.

• Epidemiology and prevention. H. influenzae is found only in humans. The incidence of severe invasive infections (meningitis, sepsis, epiglottitis) in children has been reduced drastically—to about one in 10 of the numbers seen previously—since a vaccination program was started and will continue to fall assuming the vaccinations are continued (see vaccination schedule, p. 33).

Hemophilus dacrya and Hemophilus Aegyptus

• H. decry are short, Gram-negative, nonmotile rods that are difficult to culture and require special mediums. This bacterium causes sulcus Molle (soft chancre) a tropical venereal disease seen rarely in central Europe. The infection locus presents as a painful, readily bleeding ulcer occurring mainly in the genital area. Regional lymph nodes are quite swollen. Identification of the pathogen by means of microscopy and culturing are needed to confirm the diagnosis. Therapeutic alternatives include sulfonamides, streptomycin, and tetracyclines. H. Aegyptus (possibly identical with biovar III of Hemophilus influenzae) causes a purulent conjunctivitis occurring mainly in northern Africa, in particular Egypt. A raised incidence of Brazilian purpuric fever, a systemic infection with this organism, has been observed in Brazil in recent year.

Pasteurella

• Various different species belonging to the genus Pasteurella occur in the normal mucosal flora of animals and humans; some are pathogenic in animals. Their significance as human pathogens is minor. Infections by Pasteurella multicides are described here as examples of human pictureless. The bacteria invade the organism through bite or scratch injuries or in droplets during contact with infected animals. Weakened immune defenses may then result in either local wound infections with lymphadenitis, subacute to chronic infections of the lower respiratory tract, or CNS infections (after cerebral trauma or brain surgery). Diagnosis is based on pathogen identification.

Gram-Negative Rod Bacteria with Low Pathogenic Potential

• The bacterial species listed in Table 4.10 are typical opportunists that occasionally cause infections in persons with defective specific or nonspecific immune defenses. When they are isolated from infective material, their pathological significance is in most cases difficult to interpret.

Campylobacter, Helicobacter, Spirillum

• & Campylobacter, Helicobacter, and Spirillum belong to the group of spirals, motile, Gram-negative, microaerophilic bacteria. C. jejunal causes a form of enteritis. The sources of infection are diseased animals. The pathogens are transmitted to humans in food. The diseases are sometimes also communicable among humans. The pathogens are identified for diagnostic purposes in stool cultures using special selective mediums. Helicobacter pylori contribute to the pathogenesis of type B gastritis and peptic ulcers. Spirillum minus causes rat bite fever, known as sudokus in Japan where it is frequent. & The genera Campylobacter, Helicobacter, and Spirillum belong to the group of aerobic, microaerophilic, motile, Gram-negative rod bacteria with a helical/ vibrioid form (p. 220). Human pathogens are found in all three genera.

Campylobacter

• Classification. For several years now, Campylobacter bacteria have been classified together with Arcobacter (medically insignificant) in the new family Campylobacteria (fam. Nov.). The genus Campylobacter comprises numerous species, among which C. jejune (more rarely C. coli, C. lair) as well as C. fetus have been observed as causative pathogens in human infections. Morphology and culture. Campylobacters are slender, spirally shaped rods 0.2–0.5 lm thick and 0.5–5 elm long. Individual cells may have one spiral winding or several. A single flagellum is attached to either one or both poles. Campylobacter can, under microaerophilic conditions, and in an atmosphere containing 5% O2 and 10% CO2, be cultured on blood agar plates. The optimum proliferation temperature for C. fetus is 25 8C and for C. jejuna 42 8C. Pathogenesis and clinical pictures. The details of the pathogenic mechanisms of these pathogens are largely unknown. C. jejunal produces an enterotoxin similar to the Stab produced by E. coli as well as a number of cytotoxins. C. jejunal causes a form of enterocolitis with watery, sometimes bloody diarrhea and fever. The incubation period is two to five days. The manifest illness lasts less than one week. C. fetus has been identified in isolated cases as a pathogen in endocarditis, meningitis, peritonitis, arthritis, cholecystitis, salpingitis, and sepsis in immunocompromised patients.

• Diagnosis. To isolate C. jejunal in stool cultures, mediums are used containing selective supplements (e.g., various anti-infective agents). The cultures are incubated for 48 hours at 42 8C in a microaerophilic atmosphere. Identification is based on growth requirements as well as detection of catalase and oxidase. C. fetus is readily isolated in most cases, since it is usually the only organism found in the material (e.g., blood, cerebrospinal fluid, joint punctate, pus, etc.). Therapy. Severe Campylobacter infections are treated with macrolides or 4- quinolones. Resistance is known to occur. Epidemiology and prevention. Campylobacter jejunal is among the most frequent enteritis pathogens worldwide. The bacteria are transmitted from animals to humans via food and drinking water. Direct smear infection transmission among humans is possible, especially in kindergarten or family groups. There are no specific preventive measures.

Helicobacter pylori

• Morphology and culture. H. pylori are spirally shaped, Gram-negative rods with lipoteichoic flagellation. Cultures from stomach biopsies are grown on enriched mediums and selective mediums under microaerobic conditions (90% N2, 5% CO2, and 5% O2) for three to four days. Identification is based on detection of oxidase, catalase, and urease. Pathogenesis and clinical pictures. H. pylori occurs only in humans and is transmitted by the fecal-oral pathway. The pathogen colonizes and infects the stomach mucosa. The pathogenicity factors include pronounced motility for efficient target cell searching, adhesion to the surface epithelial cells of the stomach, urease that releases ammonia from urea to facilitate survival of the cells in a highly acidic environment and a vacuolizing cytotoxin (Vacca) that destroys epithelial cells. Once the pathogen has infected the stomach tissues an acute gastritis results, the course of which may or may not involve overt symptoms. Potential sequelae include: 1. Mild chronic gastritis type B that may persist for years or even decades and is often asymptomatic. 2. Duodenal ulceration, sometimes gastric ulceration as well. 3. Chronic atrophic gastritis from which a gastric adenocarcinoma sometimes develops. 4. Rarely B cell lymphomas of the gastric mucosa (MALToma).

• Diagnosis. Histopathological, cultural and, mole gestion of 13C-labeled urea and measurement of 13CO2 in the expelled air. Antigen detection in stool. Antibodies can be identified with an ELISA or Western blotting. Therapy. In patients with ulcers and/or gastritis symptoms, a triple combination therapy with omeprazole (proton pump blocker), metronidazole, and clarithromycin lasting seven days is successful in 90% of cases. Epidemiology. Based on Sero epidemiological studies we know that H. pylori occur worldwide. Generalized contamination of the population begins in childhood and may reach 100% in adults in areas with poor hygiene. The contamination level is about 50% among older adults in industrialized countries. Transmission is by the fecal-oral route.

Spirillum minus

• This species is a motile bacterium only 0.2 lm thick and 3–5 lm long with two to three spiral windings. It cannot be grown on culture mediums. S. minus causes spirally rat bite fever, also known as sudokus. This disease occurs worldwide, with a high level of incidence in Japan. The organism is transmitted to humans by the bites of rats, mice, squirrels, and domestic animals that eat rodents. Following an incubation period of seven to 21 days a febrile condition develops with lymphangitis and lymphadenitis. Ulcerous lesions develop at the portal of entry. Diagnosis can be done by using dark field or phase contrast microscopy to detect the spirilla in blood or ulcerous material. Penicillin G is used to treat the infection.

Pseudomonas, Stenotrophomonas, Burkholder a

• Pseudomonads are Gram-negative, aerobic, rod-shaped bacteria with widespread occurrence in nature, especially in damp biotopes. The most important species from a medical point of view is Pseudomonas aeruginosa. Free O2 is required as a terminal electron acceptor to grow the organism in cultures. The pathogenesis of Pseudomonas infections is complex. The organism can use its attachment pili to adhere to host cells. The relevant virulence factors are: exotoxin A, exoenzyme S, cytotoxin, various metal proteases, and two types of phospholipase C. Of course, the lipopolysaccharide of the outer membrane also plays an important role in the pathogenesis. Pseudomonas infections occur only in patients with weakened immune defense systems, notably pneumonias in cystic fibrosis, colonization of burn wounds, endocarditis in drug addicts, postoperative wound infection, urinary tract infection, sepsis. P. aeruginosa frequently contributes to nosocomial infections. Diagnosis requires identification of the pathogen in cultures. Multiple resistance to anti-infective agents presents a therapeutic problem. Numerous other Pseudomonas species and the species of the genera Burkholder Ia and Stenotrophomonas are occasionally found in pathogenic roles in immunosuppressed patients. B. mallei causes malleus (glanders) and B. pseudo mallei causes melioidosis.

Pseudomonas aeruginosa

• Occurrence, significance. All pseudomonads are widespread in nature. They are regularly found in soils, surface water, including the ocean, on plants and, in small numbers, in human and animal intestines. They can proliferate in a moist milieu containing only traces of nutrient substances. The most important species in this group from a medical point of view is P. aeruginosa, which causes infections in person with immune defects. Morphology and culture. P. aeruginosa are plump, 2–4 lm long rods with one to several polar flagella. Some strains can produce a viscous extracellular slime layer. These mucoid strains are frequently isolated in material from cystic fibrosis patients. P. aeruginosa possesses an outer membrane as part of its cell wall. The architecture of this membrane is responsible for the natural resistance of this bacterium to many antibiotics. P. aeruginosa can only be grown in culture mediums containing free O2 as a terminal electron acceptor. In nutrient broth, the organism therefore grows at the surface to form a so-called pellicle. Colonies on nutrient agar often have a metallic sheen (P. aeruginosa; Latin: aces = metal ore). Given suitable conditions, P. aeruginosa can produce two pigments, i.e., both yellow green fluorescein and blue green pyocyanin.

• Pathogenesis and clinical pictures. The path mechanisms involved are highly complex. P. aeruginosa usually enters body tissues through injuries. It attaches to tissue cells using specific attachment fimbriae. The most important virulence factor is exotoxin A (ADP ribosyl transferase), which blocks translation in protein synthesis by inactivating the elongation factor eEF2. The exoenzyme S (also an ADP ribosyl transferase) inactivates cytoskeletal proteins and GTP-binding proteins in eukaryotic cells. The so-called cytotoxin damages cells by creating transmembrane pores. Various different metalloproteases hydrolyze elastin, collagen, or laminin. Two type C phospholipases show membrane activity. Despite these pathogenic determinants, infections.

• Diagnosis. Laboratory diagnosis includes isolation of the pathogen from relevant materials and its identification based on a specific pattern of metabolic properties. Therapy. The antibiotics that can be used to treat P. aeruginosa infections are aminoglycosides, acyl ureidopenicillins, carboxypenicillin's, group 3b cephalosporins (see p. 190), and carbapenems. Combination of an aminoglycoside with a beta lactam is indicated in severe infections. Susceptibility tests are necessary due to frequent resistance. Epidemiology and prevention. Except in cystic fibrosis, P. aeruginosa is mainly a hospital problem. Since this ubiquitous organism can proliferate under the sparest of conditions in a moist milieu, a number of sources of infection are possible: sinks, toilets, cosmetics, vaporizers, inhalers, respirators, anesthesiology equipment, dialysis equipment, etc. Infected patients and staff carrying the organism are also potential primary sources of infection. Neutropenic patients are particularly susceptible. Preventive measures i.e., above all disinfection and clinical hygiene, concentrate on avoiding exposure.

Other Pseudomonas species, Stenotrophomonas and Burkholder a

• Opportunistic pseudomonads. Other Pseudomonas species besides P. aeruginosa are capable of causing infections in immunosuppressed patients. These nosocomial infections are, however, infrequent. It would therefore not be particularly useful here to list all of the species that occasionally come to the attention of physicians. Classic opportunists also include Stenotrophomonas melophilia (formerly Xanthomonas algophilia) and Burkholder cepacian (formerly Pseudomonas cepacian). These species all occur in hospitals and frequently show resistance to anti-infective agents. Antibiotic therapy must therefore always be based on a resistance test. Burkholder a malleus. This species is the causative organism in malleus or glanders, a disease of sloped. The bacteria invade the human organism through microtraumas, e.g., in the skin or mucosa, and form local ulcers. Starting from these primary infection foci they can move to other organs.

• Burkholder a pseudo malleus. This species is the causative organism in melioidosis, a disease of animals and humans resembling malleus. The natural reservoirs of B. pseudo mallei are soil and surface water. The pathogen invades the body through injuries of the skin or mucosa and causes multiple subcutaneous and subserous abscesses and granulomas. Starting from primary foci, the infection can disseminate and cause abscesses in a number of different organs. This disease is observed mainly in Asia.

Legionella (Legionnaire’s Disease)

• Legionella is the only genus in the family Legionellae. The species Legionella pneumophila is responsible for most legionellosis in humans. Legionellae are difficult to stain. They are Gram-negative, aerobic rod bacteria. Special mediums must be used to grow them in cultures. Infections with Legionella occur when droplets containing the pathogens are inhaled. Two clinically distinct forms are on record: legionnaire’s disease leading to a multifocal pneumonia and nonpneumatic legionellosis or Pontiac fever. The person's most likely to contract legionnaire’s disease are those with a primary cardiopulmonary disease and generally weakened immune defenses. Laboratory diagnostic methods include microscopy with direct immunofluorescence, culturing on special mediums and antibody assays. The antibiotics of choice are the macrolides. The natural habitat of legionellae is damp biotopes. The sources of infection listed in the literature include hot and cold-water supply systems, cooling towers, moisturizing units in air conditioners, and whirlpool baths. Legionellosis' can occur both sporadically and in epidemics.

Brucella, Bordetella, Francise'll

• The genera Brucella, Bordetella, and Francise'll are small, coccoid, Gram-negative rods. They can be cultured under strict aerobic conditions on enriched nutrient mediums. Brucella abortus, B. meltiness, and B. suis cause brucellosis, a classic zoonosis that affects cattle, goats, and pigs. The pathogens can be transmitted to humans directly from diseased animals or indirectly in food. They cause characteristic granulomas in the organs of the RES. The primary clinical symptom is the undulant fever. Diagnosis is by means of pathogen identification or antibody assay using a standardized agglutination reaction. Bordetella pertussis is the causative organism of whooping cough, which affects only humans. The pathogens are transmitted by aerosol droplets. The organism is not characterized by specific invasive properties, although it is able to cause epithelial and subepithelial necroses in the mucosa of the lower respiratory tract. The catarrhal phase, paroxysmal phase, and convalescent phase characterize the clinical picture of whooping cough (pertussis), which is usually diagnosed clinically. During the catarrhal and early paroxysmal phases, the pathogens can be cultured from nasopharyngeal secretions. The most important prophylactic measure is the vaccination in the first year of life. Franc Isella tularemia causes tularemia. This disease, rare in Europe, affects wild rodents and can be transmitted to humans by direct contact, by arthropod vectors, and by dust particles.

Brucella (Brucellosis, Bang’s Disease)

• Occurrence and classification. The genus Brucella includes three medically relevant species—B. abortus, B. militances, and B. suis—besides a number of others. These three species are the causative organisms of classic zoonoses in livestock and wild animals, specifically in cattle (B. abortus), goats (B. militances), and pigs (B. suis). These bacteria can also be transmitted from diseased animals to humans, causing a uniform clinical picture, so-called undulant fever or Bang’s disease. Morphology and culture. Brucellae are slight, coccoid, Gram-negative rods with no flagella. They only reproduce aerobically. In the initial isolation the atmosphere must contain 5–10% CO2. Enriched mediums such as blood agar are required to grow them in cultures.

Bordetella (Whooping Cough, Pertussis)

• The genus Bordetella, among others, includes the species B. pertussis, B. Para pertussis, and B. bronchiectasis. Of the three, the pathogen responsible for whooping cough, B. pertussis, is of greatest concern for humans. The other two species are occasionally observed as human pathogens in lower respiratory tract infections. Morphology and culture. B. pertussis bacteria are small, coccoid, nonmotile, Gram-negative rods that can be grown aerobically on special culture mediums at 37 8C for three to four days. Pathogenesis. Pertussis bacteria are transmitted by aerosol droplets. They are able to attach themselves to the cells of the ciliated epithelium in the bronchi. They rarely invade the epithelium. The infection results in (sub-) epithelial inflammations and necroses.

Franc Isella tularemia (Tularemia)

• F. tularemia bacteria are coccoid, nonmotile, Gram-negative, aerobic rods. They cause a disease similar to plague in numerous animal species, above all in rodents. Humans are infected by contact with diseased animals or ectoparasites or dust. The pathogens invade the host either through microtraumas in the skin or through the mucosa. An ulcerous lesion develops at the portal of entry that also affects the local lymph nodes (ulcer glandular, glandular, or oculoglandular form). Via hypogenous and hematogenous dissemination, the pathogens then spread to parenchymatous organs, in particular RES organs such as the spleen and liver. Small granulomas develop, which develop central caseation or purulent abscesses. In pneumonic tularemia, as few as 50 CFU cause disease. The incubation period is three to four days. Diagnostic procedures aim to isolate and identify the pathogen in cultures and under the microscope. Agglutinating antibodies can be detected beginning with the second week. A seroconversion is the confirming factor. Antibiosis is carried out with streptomycin or gentamicin.

Gram-Negative Anaerobes

• The obligate anaerobic, Gram-negative, pleomorphic rods are components of the normal mucosal flora of the respiratory, intestinal, and genital tracts. Among the many genera, Bacteroides, Prevo Tella, Prohormonal, and Fusobacterium, each of which comprises numerous species, are of medical significance. They cause endogenous necrotic infections with subacute to chronic courses in the CNS, head, lungs, abdomen, and female genitals. A typical characteristic of such infections is that a mixed flora including anaerobes as well as aerobes is almost always found to be causative. Laboratory diagnostic procedures seek to identify the pathogens. Special transport vessels are required to transport specimens to the laboratory. Identification is based on morphological and physiological characteristics. A special technique is GC organic acid assay. Potentially effective antibiotics include certain penicillin's and cephalosporins, clindamycin, and metronidazole.

Treponema (Syphilis, Yaws, Pinta)

• Treponema pallidum, subsp. pallidum is the causative pathogen of syphilis. Treponemes feature 10–20 primary spiral windings and can be viewed using dark field microscopy. They cannot be grown on artificial nutrient culture mediums. Syphilis affects only humans. The pathogens are transmitted by direct contact, in most cases during sexual intercourse. They invade the subcutaneous and subserous connective tissues through micro traumata in skin or mucosa. The disease progresses in stages designated as primary, secondary, and tertiary syphilis or stages I, II, and III. Stage I is characterized by the painless primary affect and local lymphadenitis. Dissemination leads to stage II, characterized by Poly lymphadenopathy as well as generalized exanthem and enanthem. Stage III is subdivided into neurosyphilis, cardiovascular syphilis, and gummites syphilis. In stages I and II the lesion pathogens can be viewed under a dark field microscope. Antibody assays include the VDRL flocculation reaction, TP-PA particle agglutination, and the indirect immunofluorescence test FTA-ABS. The therapeutic of choice is penicillin G. This disuse is known in all parts of the world. Preventive measures concentrate on protection from exposure. Other Treponema-caused diseases that do not occur in Europe include nonvenereal syphilis, caused by T. pallidum, subsp. endemism, yaws, caused by T. pallidum, subsp. per tenue, and Pinta, caused by Treponema carat Eum.

Treponema pallidum, subsp. pallidum (Syphilis)

• Morphology and culture. These organisms are slender bacteria, 0.2 lm wide and 5–15 lm long; they feature 10–20 primary windings and move by rotating around their lengthwise axis. Their small width makes it difficult to render them visible by staining. They can be observed in vivo using dark field microscopy. In-vitro culturing has not yet been achieved. Pathogenesis and clinical picture. Syphilis affects only humans. The disease is normally transmitted by sexual intercourse. Infection comes about because of direct contact with lesions containing the pathogens, which then invade the host through microtraumas in the skin or mucosa. The incubation period is two to four weeks. Left untreated, the disease manifests in several stages.

Treponema pallidum, subsp. endemic Um (Nonvenereal Syphilis)

• This subspecies is responsible for nonvenereal syphilis, which occurs endemically in certain circumscribed areas in the Balkans, the eastern Mediterranean, Asia, and Africa. The disease manifests with maculae to populous, often hypertrophic lesions of the skin and mucosa. These lesions resemble the venereal efflorescence's. The pathogens are transmitted by direct contact or indirectly on everyday objects such as clothes, tableware, etc. The incubation period is three weeks to three months. Penicillin is the therapy of choice. Serological syphilis tests are positive.

Treponema pallidum, subsp. pretense (Yaws)

• This species causes yaws (German “Frambo¨ sei,” French “pian”), a chronic disease endemic in moist, warm climates characterized by epidermal proliferation and ulceration. Transmission is by direct contact. The incubation period is three to four weeks. Treponemes must be found in the early lesions to confirm diagnosis. Serological syphilis reactions are positive. Penicillin G is the antibiotic of choice.

Treponema carenum (Pinta)

• This species causes Pinta, an endemic treponematosis that occurs in parts of Central and South America, characterized by marked dermal depigmentation. The pathogens are transmitted by direct contact. The incubation period is one to three weeks. The disease often has a chronic course and can persist for years. Diagnosis is confirmed by identification of treponemes from the skin lesions. Penicillin G is used in therapy.

Borrelia (Relapsing Fever, Lyme Disease)

• Borrelia recurrent is is the pathogen of an epidemic relapsing fever transmitted by body lice that no longer occurs in the population of developed countries. B. dittoing, B. hermit, and other borrelia are the causative pathogens of the endemic, tickborne relapsing fever, so called for the periodic relapses of fever characterizing the infection. The relapses are caused by borelian that have changed the structure of the variable major protein in their outer membranes so that the antibodies produced by the host in the previous episode are no longer effective against them. Laboratory diagnostic confirmation requires identification of the borrelial in the blood. Penicillin G is the antibiotic of choice. B. burgdorferi is the causative pathogen in Lyme disease, a tickborne infection. Left untreated, the disease has three stages. The primary clinical symptom of stage I is the erythema chronicum migraines. Stage II in the European variety is clinically defined by chronic lymphocytic meningitis Bannwart. Meningitis is frequent in children. The primary symptoms of stage III are acrodermatitis chronic a atrophicans Herxheimer and Lyme arthritis. Laboratory diagnostics comprises detection of specific antibodies by means of immunofluorescent or EIA methods. Beta lactam antibiotics are used to treat the infection. Lyme disease is the most frequent tickborne disease in central Europe.

Borrelia that Cause Relapsing Fevers

• Taxonomy and significance. The genus Borrelia belongs to the family Spirochetosis. The body louse Borne epidemic form of relapsing fever is caused by the species B. recurrencies. The endemic form, transmitted by various tick species, can be caused by any of a number of species (at least 15), the most important being B. dittoing and B. Hermia. Morphology and culture. Borelian are highly motile spirochetes with three to eight windings, 0.3–0.6 lm wide, and 8–18 lm in length. They propel themselves forward by rotating about their lengthwise axis. They can be rendered visible with Giemsa stain (Fig. 4.24). It is possible to observe live borrelia using dark field or phase contrast microscopy.

Borrelia burgdorferi (Lyme Disease)

• Classification. The etiology of an increase in the incidence of acute cases of arthritis among youths in the Lyme area of Connecticut in 1977 was at first unclear. The illness was termed Lyme arthritis. It was not until 1981 that hitherto unknown borrelial were found to be responsible for the disease. They were classified as B. burgdorferi in 1984 after their discoverer. Analysis of the genome of various isolates has recently resulted in a proposal to subclassify B. burgdorferi sense Lato in three species: B. burgdorferi sense strict, B. carinii, B. afzelin. Morphology and culture. These are thin, flexible, helically wound, highly motile spirochetes. They can be rendered visible with Giemsa staining or by means of dark field or phase contrast microscopy methods. These borrelia can be grown in special culture mediums at 35 8C for five to 10 days, although culturing these organisms is difficult and often unsuccessful. Pathogenesis and clinical picture. The pathogens are transmitted by the bite of various tick species (see p. 607). The incubation period varies from three to 30 days. Left untreated, the disease goes through three stages (Table 4.12), though individual courses often deviate from the classic pattern. The presenting symptom in stage I is the erythema chronicum migrants (Fig. 4.25)

Leptospira (Leptospirosis, Weil Disease)

• The pathogenic species Leptospira interrobangs is subclassified in over 100 serovars reflecting different surface antigens. The serovars are divided into 19 serogroups (icterohemorrhagiae, capicola, Pomona, etc.). These organisms are in the form of spiral rods and can be grown in in-vitro cultures. Leptospirosis is a zoonosis that occurs worldwide. The sole sources of infection are diseased rodents and domestic animals (pigs), which excrete the pathogen in their urine. Upon contact, Leptospira penetrate skin or mucosa, are disseminated hematogenous and cause a generalized vasculitis in various organs. The in-curation period is seven to 12 days. The disease at first presents as a sepsis, followed after three to seven days by the so-called immune stage. In the milder form, anicteric leptospirosis, the most frequent manifestation in stage two is an aseptic meningitis. The icteric form of leptospirosis (Weil disease) can cause dysfunction of liver and kidneys, cardiovascular disruptions, and hemorrhages. The method of choice in laboratory diagnostics is antibody identification in a lysis-agglutination reaction. The therapeutic agent of choice is penicillin G.

Rickettsia, Coxiella, Orientin, and Ehrlich Ia (Typhus, Spotted Fever, Q Fever, Ehrlichiosis)

• The genera of the Rickettsiaceae and Oxaloacetate contain short, coccoid, small rods that can only reproduce in host cells. With the exception of Coxiella (aerogenic transmission), they are transmitted to humans via the vector's lice, ticks, fleas, or mites. R. prowazekii and R. typhi cause typhus, a disease characterized by high fever and a spotty exanthem. Several rickettsia species cause spotted fever, a milder typhus like disease. Oriente tsutsugamushi is transmitted by mite larvae to cause tsutsugamushi fever. This disease occurs only in Asia. Coxiella burnetiid is responsible for Q fever, an infection characterized by a pneumonia with an atypical clinical course. Several species of Ericaceae cause ehrlichiosis in animals and humans. The method of choice for laboratory diagnosis of the various rickettsioses and ehrlichiosis is antibody assay by any of several methods, in most cases indirect immunofluorescence. Tetracyclines represent the antibiotic of choice for all of these infections. Typhus and spotted fever no longer occur in Europe. Q fever infections are reported from all over the world. Sources of infection include diseased sheep, goats, and cattle. The prognosis for the rare chronic form of Q fever (syn. Q fever endocarditis) is poor. Ehrlichiosis infects mainly animals, but in rare cases humans as well.

Bartonella and Afilias

Bartonella

• Classification. The genus Bartonella includes, among others, the species B. bacilliformis, B. Quintana, B. Henslie, and B. Clarridge Ia. Morphology and culture. Bartonella bacteria are small (0.6–1 lm), Gram-negative, frequently pleomorphic rods. Bartonellae can be grown on culture mediums enriched with blood or serum.

Afipia Felis

• The bacterial species Afilias (Armed Forces Institute of Pathology) Felis was discovered several years ago. At first, it appeared that most cases of cat scratch disease were caused by this pathogen. Then it turned out that the culprit in those cases was usually either B. Henslee or B. Clarridge and that Afilias felids was responsible for only a small number. Africia Felis and B. Henslie cat scratch infections present with the same clinical symptoms. Most cases of A. Felis infections clear up spontaneously without antibiotic therapy. Should use of an antibiotic be clinically indicated, a tetracycline (or in severe cases a carbapenem or aminoglycoside) would be appropriate.

Chlamydia

• Chlamydial are obligate cell parasites. They go through two stages in their reproductive cycle: the elementary bodies (EB) are optimized to survive outside of host cells. In the form of the initial bodies (IB), the chlamydial reproduce inside the host cells. The three human pathogen species of chlamydial are C. psittacine, C. trachomatis, and C. pneumoniae. Tetracyclines and macrolides are suitable for treatment of all chlamydial infections. C. psittacine is the cause of psittacosis or ornithosis. This zoonosis is a systemic disease of birds. The pathogens enter human lungs when dust containing chlamydia is inhaled. After an incubation period of one to three weeks, pneumonia develops that often shows an atypical clinical course.

Overview and General Characteristics of Chlamydial

• Definition and classification. The bacteria in the taxonomic family Chlamydiosis are small (0.3–1 lm) obligate cell parasites with a Gram-negative cell wall. The reproductive cycle of the chlamydia comprises two developmental stages: The elementary bodies are optimally adapted to survival outside of host cells. The initial bodies, also known as reticulate bodies, are the form in which the chlamydia reproduce inside the host cells by means of transverse fission. Three human pathogen species of chlamydia are known: C. psittacine, C. trachomatis (with the biovars trachoma and lymphogranuloma venereum), and C. pneumoniae. Morphology and developmental cycle. Two morphologically and functionally distinct forms are known: & Elementary bodies. The round to oval, optically dense elementary bodies have a diameter of approximately 300 nm. They represent the infectious form of the pathogen and are specialized for the demands of existence outside the host cells. Once the elementary bodies have attached themselves to specific host cell receptors, they invade the cells by means of endocytosis (Fig. 4.27). Inside the cell, they are enclosed in an endocytose membrane vesicle or inclusion, in which they transform themselves into the other form—initial bodies—within a matter of hours. & Initial bodies. Chlamydial in this spherical to oval form are also known as reticular bodies. They have a diameter of approximately 1000 nm. The initial bodies reproduce by means of transverse fission and are not infectious while in this stage. At the end of the cycle, the initial bodies are transformed back into elementary bodies. The cell breaks open and releases the elementary bodies to continue the cycle by attaching themselves to new host cells.

Chlamydia psittacine (Ornithosis, Psittacosis)

• Pathogenesis and clinical picture. The natural hosts of C. psittacine are birds. This species causes infections of the respiratory organs, the intestinal tract, the genital tract, and the conjunctiva of parrots and other birds. Humans are infected by inhalation of dust (from bird excrements) containing the pathogens, more rarely by inhalation of infectious aerosols. After an incubation period of one to three weeks, ornithosis presents with fever, headache, and a pneumonia that often takes an atypical clinical course. The infection may, however, also show no more than the symptoms of a common cold, or even remain clinically silent. Infected persons are not usually sources of infection. Diagnosis. The pathogen can be grown from sputum in special cell cultures. Direct detection in the culture is difficult and only possible in specially equipped laboratories. The complement binding reaction can be used to identify antibodies to a generic antigen common to all chlamydial, so that this test would also have a positive result in the presence of other chlamydial infections. The antibody test of choice is indirect micro immunofluorescence. Therapy. Tetracyclines (doxycycline) and macrolides. Epidemiology and prevention. Ornithosis affects birds worldwide. It is also observed in poultry. Diagnosis of an ornithosis in a human patient necessitates a search for and elimination of the source, especially if the birds in question are household pets.

Chlamydia trachomatis (Trachoma, Lymphogranuloma venereum)

• C. trachomatis is a pathogen that infects only humans. Table 4.16 lists the relevant diseases, biovars, and serovars. Trachoma is a follicular keratoconjunctivitis. The disease occurs in all climatic zones, although it is more frequent in warmer, less-developed countries. It is estimated that 400 million people carry this chronic infection and that it has caused blindness in six million. The pathogen is transmitted by direct contact and indirectly via objects in daily use. Left untreated, the initially acute in Foamation can develop a chronic course lasting months or years and leading to formation of a corneal scar, which can then cause blindness. The laboratory diagnostics procedure involves detection of C. trachomatis in conjunctival smears using direct immunofluorescence microscopy. The fluorochrome-marked monoclonal antibodies are directed against the MOMP (major outer membrane protein) of C. trachomatis. The pathogen can also

•  1. Detection under the microscope in smear material using direct immunofluorescence (see under trachoma).

• 2. Direct identification by means of amplification of a specific DNA sequence in smear material and urine. 

• 3. Growing in special cell cultures.

• Lymphogranuloma venereum. This venereal disease (syn. lymphogranuloma inguinale, lymphatic venerea (Favre-Durand-Nicolas's disease) not to be confused with granuloma inguinale, see p. 305) is frequently observed in the inhabitants of warm climatic zones. A herpetiform primary lesion develops at the site of invasion in the genital area, which then becomes an sulcus with accompanying lymphadenitis. Laboratory diagnosis is based on isolating the proliferating pathogen in cell cultures from purulent material obtained from the ulus or from matted lymph nodes. The antibodies can be identified using the complement binding reaction or the micro immunofluorescence test. Tetracyclines and macrolides are the potentially useful antibiotic types.

Chlamydia pneumoniae

• This new chlamydial species (formerly TWAR chlamydia) causes infections of the respiratory organs in humans that usually run a mild course: influenzalike infections, sinusitis, pharyngitis, bronchitis, pneumonias (atypical). Clinically silent infections are frequent. C. pneumoniae is pathogenic in humans only. The pathogen is transmitted by aerosol droplets. These infections are probably among the most frequent human chlamydial infections. Serological studies have demonstrated antibodies to C. pneumoniae in 60% of adults. Specific laboratory diagnosis is difficult. Special laboratories can grow and identify the pathogen in cultures and detect it under the microscope using marked antibodies to the LPS (although this test is positive for all chlamydial infections). C. pneumoniae-specific antibodies can be identified with the micro immunofluorescence method. In a primary infection, a measurable titer does not develop for some weeks and is also quite low. The antibiotics of choice are tetracyclines or macrolides. There is a growing body of evidence supporting a causal contribution by C. pneumoniae to atherosclerotic plaque in the coronary arteries, and thus to the pathogenesis of coronary heart disease.

Mycoplasma

• Mycoplasmas are bacteria that do not possess rigid cell walls for lack of a murein layer. These bacteria take on many different forms. They can only be rendered visible in their native state with phase contrast or dark field microscopy. Mycoplasmas can be grown on culture mediums with high osmotic pressure levels. M. pneumoniae frequently causes pneumonias that run ictal courses, especially in youths. Ten to twenty percent of pneumonias contracted outside of hospitals are caused by this pathogen. M. hominis and Urea plasma real-time contribute to nonspecific infections of the urogenital tract. Infections caused by Mycoplasmataceae can be diagnosed by culture growth or antibody assays. The antibiotics of choice are tetracyclines and macrolides (macrolides not for M. hominis). Mycoplasmas show high levels of natural resistance to all beta lactam antibiotics.

Nosocomial Infections

• Nosocomial infections occur in hospitalized patients as complications of their primary disease. Such infections are reported in an average of approximately 3.5% (Germany) to 5% (USA) of all hospitalized patients, in tertiary care hospitals in about 10% and in the intensive care units of those in about 15–20% of cases. The most frequent types of infection are urinary tract infections (42%), pneumonia (21%), surgical wound infections (16%), and sepsis (8%). The pathogen types most frequently involved are opportunistic, Gram-negative rods, staphylococci and enterococci, followed by fungi. The bacteria are often resistant to many different antibiotics. The hands of medical staff play a major role in transmission of the infections. Control of nosocomial infections requires a number of operational measures (disinfection, asepsis, rationalized antibiotic therapies, isolation), organizational measures (hygiene committee, recognition of infections, procedural guidelines, training programs), and structural measures.

Definition

• The term nosocomial infection designates infections contracted by hospitalized patients 48 hours or more from the beginning of hospitalization. These are secondary infections that occur as complications of the primary diseases to be treated in the hospital.

Pathogens, Infections, Frequency

• The significance of the different human pathogens in nosocomial infections varies widely: & Subcellular entities. Isolated cases of Creutzfeldt-Jakob disease due to unsterilized instruments have been described in the literature. Such accidents now no longer occur.

• 1 Data (modified) acc. to “Nosocomial Infection in Deutschland – Erasing und Prevention (NIDEP–Studie).” Vol. 56, Publication Series of the German Federal Health Office. Nomos Verlagsgesellschaft, Baden-Baden, 1995. 2 UTI = urinary tract infections, RTI = lower respiratory tract infections, PWI = postoperative wound infections, SEP = primary sepsis, Other = all other infections.

• There are no reliable figures available on viral nosocomial infections. A rough estimate puts viral nosocomial infections at less than 1% of the total. An example of a viral nosocomial infection is infectious hepatitis transmitted by blood or blood products. & Bacteria are the main pathogens involved in nosocomial infections. Most of the causative organisms are facultatively pathogenic (opportunistic) bacteria, which are frequently resistant to many different antibiotics. These bacteria have found niches in which they persist as so-called hospital flora. The resistance patters seen in these bacteria reflect the often wide variations between anti-infective regimens as practiced in different hospitals. & Fungi. Fungal nosocomial infections have been on the increase in recent years. It can be said in general that they affect immunocompromised patients and that neutropenic patients are particular susceptible. Table 4.17 lists the pathogens that cause the most significant nosocomial infections as determined in a prevalence study done in Germany (East and West) in 1995 (NIDEP Study). Table 4.18 shows the prevalence levels at which nosocomial infections occurred in 72 selected hospitals on a given date in the above study. The prevalence and pathogen data shown here approximate what other studies have found. Prevalence and incidence levels can vary considerably from hospital to.

Sources of Infection, Transmission Pathways

• Nosocomial infections originate either from the patient’s own flora (endogenous infections) or from external sources (exogenous infections). Endogenous infections are the more frequent type. In such cases, the patient may have brought the pathogens into the hospital. It is, however, frequently the case that a patient’s skin and mucosa are colonized within one to two days by bacteria of the hospital flora, which often shows multiple resistance to antibiotics and replaces the patient’s individual flora, and that most endogenous infections are then actually caused by the specific hospital flora. The source of infection for exogenous infections is most likely to lie with the medical staff. In most cases, the pathogens are transmitted from patient to patient during medical and nursing activities. Less frequently, the staff is either also infected or colonized by the hospital flora. Another important cause of nosocomial infections is technical medical measures that facilitate passage of the pathogens into the body. All invasive diagnostic measures present infection risks. The patient’s surroundings, i.e., the air, floor, or walls of the hospital room, are relatively unimportant as sources of infection.

Control

• The measures taken to control and prevent nosocomial infections correspond in the wider sense to the general methods of infection control. The many different individual measures will not be listed here. The infection control program varies depending on the situation in each particular hospital and can be summarized in three general groups: Operational measures. This category includes all measures pertaining to treatment and care of patients and cleaning measures. This includes asepsis, disinfection, sterilization, and cleaning. Further precautionary operational measures include isolation of patients that would be sources of infection and the economical and specific administration of antibiotic therapies. Organizational measures. The organization of hospital infection control must be adapted to the structure of each particular hospital. Realization of the necessary measures, which of course always involve working time and expense, is best realized by establishing an infection control committee charged with the following tasks: determination and analysis of the situation, definition of measures required to improve infection control by issuing binding guidelines, cooperation in the planning and acquisition of operational and structural facilities, cooperation on functional procedures in the various sections of the hospital, contributions to staff training in matters of hospital infection control. In order to carry out these tasks efficiently, the committee should have access to a working group of specialists. In larger hospitals, a hospital epidemiologist, and staff as required, are retained for these functions.

• Structural measures. These measures refer above all to new structures, which must be built in accordance with hygienic criteria. It is therefore the obligation of the planning architect to consult experts when planning the hygienically relevant parts of a construction measure. Hygienic aspects must of course also be considered in reconstruction and restoration of older building substance.