Miscellaneous Gram-Negative Bacteria

This chapter covers several Gram-negative but unrelated taxa. We did not mean to make this chapter a dumping ground, but they are all included in this chapter simply for the sake of brevity. The important taxa include the following:

• Brucella melitensis

• Bordetella pertussis

• Francisella spp.

• Pasteurella spp.

• Vibrio cholerae

• Campylobacter spp.

• Helicobacter spp.

• Legionella spp.

• Gardnerella vaginalis

• Chlamydia spp.

• Rickettsia rickettsii

BRUCELLA MELITENSIS

Brucella spp. are Gram-negative, aerobic coccobacilli. Earlier, the genus Brucella was divided into six species but subsequent genetic studies have established that all the six species essentially represent different variants of Brucella melitensis. Brucella melitensis is an important intracellular pathogen commonly associated with cattle, swine, dogs, and rats, which can also serve as the carriers. Goats are believed to be the natural reservoir for B. melitensis. Goats themselves seldom suffer from Brucella infections, but pass on the bacterium in their milk and subsequently in cheese. Human cases of brucellosis are not uncommon, particularly in poor Asian and African countries. The infections are often acquired by consumption of B. melitensis contaminated goat milk and cheese.

Disease

Brucella melitensis causes brucellosis, which is characterized by fever, enlarged spleen, weight loss, and arthritis. The fever shows a high degree of fluctuation with phases when the body temperature is high often for weeks followed by days with a low grade or no fever. Another species, B. abortus (more correctly B. melitensis biotype abortus), causes abortion in the cattle.

Laboratory Diagnosis

Samples of blood or any other clinical specimen should be inoculated into a brain heart infusion agar or trypticase-soy broth. Cultures are incubated in a CO2 incubator for 2–7 days. Subcultures on blood or chocolate agar plates are frequently needed. Selective media and numerous serological tests are also available. Visible translucent colonies develop after 72 hours and turn gray with time. Microscopically, B. melitensis are Gram-negative coccobacilli (Fig. 10.1). In histopathological prepa- rations, they are mostly intracellular. A serum agglutination test is also available and useful.

Figure 10.1.    A Gram-stained smear showing coccobacilli of Brucella melitensis

Antibiotic Sensitivity 

A combination therapy involving rifampin and doxycycline is preferred. Other effective agents include streptomycin and trimethoprim sulfa.

BORDETELLA PERTUSSIS 

Bordetella pertussis is an important pathogen of worldwide occurrence. Annually, more than 50 million persons are known to suffer from B. pertussis infections. This bacterium is found only in humans. Its isolation from other animals or environmental sources has not been reported. This bacterium was first isolated by Bordet and Gengou from a case by whooping cough and later named after Bordet in his honor. The genus has only two species. A second species, B. avium, a causal agent of coryza (rhinotracheitis) in turkeys was added in 1984. Bordetella spp. are aerobic, chemo- organotrophic, nonmotile, flagellated coccobacilli.

Disease 

Bordetella pertussis causes pertussis (whooping cough), which is a highly contagious bioaerosol-borne disease that frequently affects children. Initially, the bacterium attaches to the cells of the respiratory epithelium, causing progressive irritation of the upper trachea. The resulting cough can be so severe that the patient may find it hard to breathe. Death may occur due to airway obstruction. Usually a childhood infection gives lifelong immunity but infections in elderly patients who have had childhood whooping cough is not uncommon. However, such cases only have a persistent cough devoid of whooping. 

The disease is well known to cause high morbidity and mortality, especially in developing countries. Prior to the widespread use of vaccine, pertussis was quite common in United Stated and other industrialized countries. Currently, however, it is being increasingly recognized as a pathogen in adults with waning immunity. 

Virulence Factors 

Bordetella pertussis produces an exotoxin called pertussis toxin. Pili help with the attachment. In addition, two other toxins, dermonecrotic toxin and tracheal cytotoxin, have been identified. The tracheal cytotoxin is believed to kill ciliated cells in the upper respiratory tract area and stimulates release of interleukin-1, which probably plays a role in the fever. The role of dermonecrotic toxin is uncertain at present. 

Laboratory Diagnosis 

Nasal and throat swabs should be streaked on special media, such as Bordet-Gengou medium. Incubation is done at 35°C, aerobically, or in the presence of 3%–8% CO2. 

Several serological tests and molecular genetics-based techniques are also available for rapid diagnosis and species identification. Bordetella pertussis strains are oxidase positive and urease negative, aerobic, Gram-negative coccobacilli.

Antibiotic Sensitivity

Bordetella pertussis is sensitive to erythromycin. Excellent preventive vaccines are available and widely used in the industrialized countries.

FRANCISELLA TULARENSIS

Like Bordetella and Brucella spp., Francisella are aerobic, nonfermentative coccobacilli that are almost always associated with an infection. The genus Francisella is believed to have two species, F. tularensis and F. philomiragia. Of the two, F. tularensis is of a greater clinical significance.

Disease

Francisella tularensis causes tularemia, also known as rabbit fever, tick fever, and glandular fever. The disease is considered zoonotic and mostly results from exposure to rodents, rabbits, or ticks. The infection results from the handling of infected animals, or following a tick bite. The incubation period is generally 3–5 days. The symptoms develop rather abruptly and may include fever, chill, and malaise. In some cases, painful lesions develop on the skin near the enlarged lymph nodes, leading to lymphadenopathy and bacteremia. Another clinical manifestation of tularemia, called oculoglandular tularemia, develops following a direct infection of the eyes by exposure to the bioaerosols from infected patients or through contaminated fingers. Infected individuals develop painful conjunctivitis and regional lymphadenopathy. Pneumonic tularemia, yet another type of clinical manifestation, may result from the inhalation of infectious bioaerosol and can be fatal if not treated in a timely manner.

Virulence Factors

Not much is known about the virulence factors associated with F. tularensis except for the presence of antiphagocytic capsules. By virtue of being an intracellular pathogen, the bacterium resists complement-mediated killing and phagocytosis.

Laboratory Diagnosis

Clinical materials, such as exudates from the lesions, biopsied lymph nodes, and respiratory secretion, should be collected with utmost care. Because F. tularensis is a highly contagious pathogen, proper safety precautions must be observed when collecting or processing clinical specimens. The bacterium can be isolated on glucose-cysteine blood (GCB) agar supplemented with thiamine. Francisella tularensis can also be isolated, albeit less successfully, on charcoal-yeast extract agar and Thayer-Martin medium. The incubation is done aerobically at 35°C for 3–5 days. On GCB agar, small colonies measuring about 3 mm develop after 3–4 days.

Antibiotic Sensitivity 

Cases of tularemia can be treated with streptomycin, chloramphenicol, or tetracycline. However, streptomycin is generally considered the drug of choice. Most strains of F. tularensis produce beta lactamases; therefore, penicillins are not indicated.

PASTEURELLA SPECIES 

Similar to Brucella, Bordetella, and Francisella spp., members of the genus Pasteurella are also Gram-negative aerobic coccobacilli. However, unlike the other three, members of the genus Pasteurella are fermentative. Most strains of Pasteurella spp. grow slightly faster than those belonging to the other three genera. Clinically, important species include P. multocida, P. pneumotropica, P haemolytica, and P. ureae (now Actinobacillus ureae). Based on the results of genetic studies, currently a process to revisit genus Pasteurella is under way, and it is quite possible that some of the species will be merged or split to create newer species. 

Species belonging to genus Pasteurella have been isolated from wild and domesticated animals, but most isolates of P. ureae have originated from human sources only. A majority of strains of P. ureae have been isolated from the respiratory tract of apparently healthy persons, but it has been occasionally incriminated in the cases of septicemia and meningitis. Pasteurella multocida, which is normally present in the respiratory tract of numerous wild and domestic animals, has been occasionally implicated in cellulitis-like lesions or abscesses developing at the site of an animal bite. The infection is known to progress into osteomyelitis and arthritis. In rare instances, P. multocida has been noted in association with pneumonia, emphysema, and lung abscess. Pasteurella ureae, though frequently isolated from human sources, has been only rarely found in association with septicemia and meningitis in humans. 

Virulence Factors 

Members of the genus Pasteurella are encapsulate and that certainly interferes with phagocytosis. Also, several strains produce soluble toxins which could play a role in its pathogenesis. 

Laboratory Diagnosis 

Pasteurella spp. grow well on blood agar and chocolate agar. Cultures are generally incubated aerobically at 35°C. Visible colonies develop after 24 hours. The colonies are generally smooth with a grayish appearance. As stated above, Pasteurella spp. are nonmotile, Gram-negative bacilli that are fermentative, and mostly oxidase and catalase positive. 

Antibiotic Sensitivity 

Pasteurella spp. are generally sensitive to penicillin and possibly to tetracycline and cephalosporins. They are mostly resistant to aminoglycosides.

VIBRIO CHOLERAE 

Members of the genus Vibrio are abundantly present in the fresh and marine waters. It is a large genus that is generally divided into 26 species, 12 of which are believed to be clinically significant. Of these, V. cholerae, being the causal agent of cholera, is perhaps the most important and best known. Other species, such as V. mimicus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and V. furnissii are mostly implicated in gastroenteritis, and occasionally in the cases of ear and wound infections, conjunctivitis, and other complications. 

Like other members of the genus Vibrio, V. cholerae is primarily an aquatic bacterium and is frequently found in the sewage contaminated water. It is a facultative anaerobe, Gram-negative curved rod with a polar flagellum (Fig. 10.2). Tests

Figure 10.2.    Photomicrograph of Vibrio cholerae showing a single polar flagellum in a digitally colorized Leifson flagella stain (source: CDC). See color insert

for indole are positive, urease negative, and oxidase positive, and most strains reduce nitrate to nitrite.  

Disease 

Vibrio cholera is the principal causal agent of the deadly disease cholera, responsible for millions of deaths and debilitating morbidity each year in most of the developing countries. The affected areas include much of Asia, Africa, and Latin America. During the period 1991–1995, more than a million people suffered from cholera in Latin America alone, resulting in the deaths of nearly 10,000 persons. No clear data are available from the endemic areas of Asia and Africa, but the extent of mortality and morbidity is believed to be quite high. Infection is usually acquired by ingestion of food or beverages contaminated with the fecal matter. The incubation period is often less than 3 days. Symptoms include diarrhea and vomiting that quickly lead to severe loss of fluid and electrolytes. More than 70% of untreated patients die. Among the most important biotypes are V. cholerae O1 and V. cholerae O139. Other species of genus Vibrio that are often indicated in human diseases, such as V. parahaemolyticus, V. alginolyticus, V. mimicus and several others, will not be discussed here. 

Virulence Factor 

Cholera toxin is commonly noted in the strains of V. cholera biotype O1. Other virulence factors include pili, adhesion factors, siderophores, and neuraminidase. The strains of V. cholerae other than biotype O1 produce heat stable enterotoxins, heat labile hemolysin, and proteinases. Other species are also known to produce enterotoxins and hemolysin. 

Laboratory Diagnosis 

Vibrio cholera is not a fastidious bacterium. Thiosulfate–citrate–bile salt sucrose (TCBS) agar is an excellent isolation medium. However, not all species of Vibrio grow on this medium. Incubation in enrichment broth (alkaline peptone water) may be required. The cultures can be incubated aerobically or anaerobically at 35°C for 24 hours. On TCBS medium, colonies of V. cholerae are yellow and measure about 2 mm in diameter. Colonies of V. mimicus and V. parahaemolyticus that do not ferment sucrose develop a green color. Vibrio spp. generally grow well on sheep blood agar and develop a greenish color. Serologically, V. cholerae O1 are believed to have three antigenic factors (A, B, and C). Gene probes are also available for specific identification. 

Antibiotic Sensitivity 

Fluid and electrolyte balance should be restored promptly. Antibiotics help to decrease the duration of diarrhea by 50% and decrease the excretion of the affected person by 1 day. Tetracylcines have been used in most cases to treat V. cholera infections. However, some areas have strains that are resistant to tetracyclines. In such cases, macrolides and fluoroquinolones are acceptable alternatives. Vaccines that provide protection for a limited period are also available.

AEROMONAS SPECIES 

Members of the genus Aeromonas are a group of dynamic bacteria in the sense that they can grow at pH 4.5 to 9.0 and at temperatures ranging from 10 to 45°C. Six species, including A. hydrophila, A. caviae, A. veronii, A. jandaei, A. schubertii, and A. trota, are considered medically significant. Aeromonas spp. cause gastroenteritis characterized by watery diarrhea, often accompanied by fever, nausea, and abdominal pain. The infections are usually food and waterborne. These bacteria have also been indicated in cases of septicemia, respiratory and urinary tract infections, peritonitis, and conjunctivitis. Aeromonas infections appear to be more common in Southeast Asia. No specific virulence factor has been identified except that many strains produce cytotoxins and enterotoxins. 

Laboratory Diagnosis 

Aeromonas spp. grow well on blood agar, MacConkey agar, and Hektoen agar. Selective media are also available for their isolation from feces and other heavily contaminated specimens. Certain specimens may require enrichment, which can be achieved by incubating the sample in alkaline peptone water or Gram-negative broth. Aeromonas spp. are facultative anaerobes. Visible colonies appear within 24 hours. Except for A. caviae strains, most species cause a strong beta hemolysis on blood agar. 

Antibiotic Sensitivity 

Aeromonas spp. produce a variety of beta lactamases and are generally resistant to penicillins and first-generation cephalosporins. Antibiotic sensitivity tests are strongly recommended.

CAMPYLOBACTER SPECIES 

Campylobacter are Gram-negative, microaerophilic, aerobic, nonspore forming curved rods that are motile and have one polar flagellum (Fig. 10.3). They are oxidase positive. The main pathogenic species are C. jejuni and C. pylori. Campylobacter pylori is also referred to as Helicobacter pylori by some microbiologists. Campylobacter is similar to Vibrio in appearance, but they are nonfermentative and their DNA base ratio is lower (29% compared with 45% in Vibrio). The natural reservoirs of C. jejuni include poultry, pigs, and cattle.

Figure 10.3.    Electron micrograph of C. jejuni (note the curved rod with a polar flagellum).

Table 10.1    Major Similarities and Differences between Campylobacter jejuni and C. pylori

Strains

Properties (tests)	C. jejuni	C. pylori (H. pylori)
Oxidase Catalase Nitrate reduction Sensitivity to cephalosporins	Positive Positive Positive Resistant	Positive Positive Negative Sensitive

Disease

Campylobacter jejuni is the leading cause of enteritis all over the world. Its incubation period is about 2–10 days. Symptoms include diarrhea with bloody stool, abdominal pain, and fever. Campylobacter pylori is generally associated with type B gastritis (gastric ulcer or peptic ulcer). There appears to be a strong relationship between chronic gastritis and gastric carcinoma.

Laboratory Diagnosis

Suitable clinical specimens are streaked on blood agar and campy agar; the latter is a selective medium. Incubation is done at 35°C in the presence of 5%–10% CO2. Currently, dependable campy kits are available for the rapid diagnosis. Some of the physiological differences among C. jejuni and C. (Helicobacter) pylori are summarized in Table 10.1.

Figure 10.4.    Gram-negative bacilli with some unidentified filamentous structures seen in a clinical specimen obtained from a patient suffering from Legionnaire’s disease (source: CDC).

Antibiotic Sensitivity

Campylobacter are sensitive to erythromycin, but antibiotic therapy is usually not indicated in C. jejuni infections. Clinical management of C. pylori infection requires a combination of antibiotics and a proton pump inhibitor. Tetracyclines and macrolides are often used for therapy, but worldwide resistance is increasing.

LEGIONELLA SPECIES

Legionella spp. are aerobic, nonsporulating, motile, Gram-negative rods (Fig. 10.4). Unlike to other Gram-negative bacteria, but similar to Corynebacterium and Mycobacterium, Legionella strains contain branched fatty acid in the cytoplasmic membrane. They are widely distributed in environment and commonly present in cooling towers. There are 32 recognized species, but L. pneumophila is the most important pathogenic species.

Disease

Legionella pneumophila is an intracellular pathogen. The incubation period may range from 2 to 10 days. The disease variously known as Legionnaires’ disease, legionellosis, or Pontiac fever may present two distinct clinical conditions:

• Pneumonia often accompanied by fever, chills, and malaise. This form of the infection can be fatal if not treated.

• Febrile illness, which can be self-limiting.

Virulence Factors 

No particular virulence factor is known except that L. pneumophila can grow in alveolar macrophages and is capable of resisting phagolysosome fusion. 

Laboratory Diagnosis 

Legionella pneumophila does not grow on routine laboratory media. Throat swabs should be streaked on buffered charcoal-yeast extract agar containing alpha–ketoglutarate (BCYE-alpha). The bacterium can survive at temperatures from 4°C to 55°C. Incubation is done at 35°C for 5–7 days under aerobic condition. The colonies grow to about 3–4 mm in 7 days. They are convex and circular, grayish in color with a glistening texture and a ground glass appearance. Some of the significant differences among the commonly isolated species of Legionella are depicted in Table 10.2. 

Antibiotic Sensitivity 

Most strains of L. pneumophila are sensitive to fluoroquinolones and macrolides.

GARDNERELLA VAGINALIS 

Gardnerella vaginalis is an endogenous opportunistic “pathogen.” The bacterium is an anaerobic, fastidious, and Gram-variable bacillus. 

Disease 

Gardnerella vaginalis causes vaginosis, a disease characterized by thick vaginal discharge with an offensive odor. The condition must be differentiated from vaginitis, which is an inflammatory disease caused by yeast Candida spp. or protozoa Trichomonas vaginalis. Vaginosis is devoid of inflammatory symptoms, such as itching and burning sensations. The condition may be caused by a decline in the vaginal population of normal resident microbiota, especially Lactobacillus acidophilus.

Laboratory Diagnosis

A vaginal swab, preferably collected in the morning, immediately after the subject gets up, is the most suitable clinical specimen. It should be streaked on human blood bilayers agar with Tween 80 or agar containing 2% human blood, and incubated at 35°C in a CO2 incubator for 48 hours. The colonies are β-hemolytic, opaque, convex, and grayish in color and measuring about 0.5 mm in diameter. Confirmatory tests include a positive hippurate and starch hydrolysis. Tests for α- glucosidase are positive and for the β-glucosidase are negative.

Antibiotic Sensitivity

The drug of choice is metronidazole, either orally or intravaginally, which leads to a high rate of cure. Gardnerella vaginalis infection can recur. Tinidazole is a second-generation nitroimidazole which can be used in these cases. Lactobacillus spp., used to restore normal vaginal microbiota, may also be employed. Men may serve as carriers. However, simultaneous treatment of the male partner has not been proven to be beneficial in preventing recurrence of bacterial vaginosis in female partners, though additional trials are needed.

CHLAMYDIA SPECIES

Members of the genus Chlamydia are very small (<1 µm in size), Gram-negative, irregular in shape, intracellular pathogens. Important pathogenic species include C. trachomatis and C. pneumoniae, which are known to infect humans only.

Disease

Chlamydia trachomatis is the well-known cause of nongonococcal urethritis, and perhaps the most common sexually transmitted disease in the industrialized world, affecting nearly 1.5 million women in the United States alone. The infection may eventually involve the bladder, kidneys, and cervix, leading to infertility. Trachoma, a chronic form of eye infection, is another common clinical condition that is noted worldwide.

Chlamydia pneumoniae causes a milder form of pneumonia, and also infections of the pharynx or throat. Additional modes of transmission may include contact with infectious droplets, but it has not been well defined. The infection is usually mild and self-limited. Sero-prevalence rates are high by adulthood. 

Lab Diagnosis 

Fluorescence antibody technique, ELISA, and tissue culture using HeLa (Henrietta Latousche) cells can be useful. Chlamydia strains cannot be cultured on standard laboratory media. 

Antibiotic Sensitivity 

Most strains of Chlamydia spp. are sensitive to tetracyclines, quinolones, and macrolides. 

RICKETTSIA RICKETTSII 

Rickettsia rickettsii are Gram-negative, irregularly shaped, ultra-small (less than 0.1 µm in diameter), and obligate intracellular parasites. Their natural habitats are wood ticks (Dermacentor andersoni), dog ticks (Dermacentor variabilis), and certain other tick species in Latin America. Rickettsia rickettsii causes Rocky Mountain spotted fever. The term can be misleading because the disease is most common in the Eastern and Southeastern United States. A large number of cases are reported from Texas and Oklahoma. 

Disease 

Rocky Mountain spotted fever often begins with a rash that is present between the third and fifth day following the tick bite, and is generally accompanied by fever, nausea, and headache. Death may occur due to multiorgan failure, with the highest mortality being in the very young and the elderly. It is an important zoonotic disease. 

Laboratory Diagnosis 

Rocky Mountain spotted fever is usually diagnosed on the basis of clinical symptoms and case history. Serological tests are of a limited value. Since the causal agent is an obligate parasite, it cannot be cultured on laboratory media. 

Antibiotic Sensitivity 

Infections by R. rickettsii response well to tetracycline and macrolides.

BACTEROIDES SPECIES

Bacteroides spp. are anaerobic, Gram-negative bacilli that account for a large number of anaerobic bacteria isolated from human sources. They are generally present in the mouth and gastrointestinal tract, and are occasionally associated with periodontal lesions. A majority of Bacteroides isolates are clustered together under one umbrella called Bacteroides fragilis group that consists of 10 species, namely B. distasonis, B. caccae, B. eggerthii, B. fragilis, B. merdae, B. vulgatus, B. ovatus, B. stercoris, B. thetaiotaomicron, and B. uniformis. They all hydrolyze esculin and grow in the presence of 20% bile salt. Members of the B. fragilis group are resistant to colistin, kanamycin, and vancomycin and grow on media specially formulated for anaerobic bacteria.

CALYMMATOBACTERIUM GRANULOMATIS

Calymmatobacterium granulomatis, formerly known as Donovania granulomatis, are facultative anaerobic, Gram-negative bacilli that are believed to be closer to Klebsiella spp. because of their genomic similarities. They are known to cause granuloma inguinale, which is a chronic disease involving subcutaneous tissues in the general vicinity of the genital, anal, and inguinal areas. The infection is some what rare in the United States, but not uncommon in tropical countries. The laboratory diagnosis can be made by demonstration of the bacterium in histiocytes in Giemsa or Wright stained smears. Cultures are usually not needed but can be made on coagulated egg yolk medium. Granuloma inguinale can be treated with tetracycline or macrolides.

CARDIOBACTERIUM HOMINIS

Cardiobacterium hominis strains are slow growing, Gram-negative bacilli that are facultative anaerobes. They are normally present in the upper respiratory tract from where they can enter the blood and attach to heart tissue. Though they usually appear in a rather low number, C. hominis are known to cause endocarditis, mostly in patients with preexisting conditions. Diagnosis is often based on its isolation in blood cultures at 35°C in the presence of 3%–5% carbon dioxide. Visible growth can be seen in 48–72 hours. Strains of C. hominis are generally sensitive to penicillin or ampicillin.

STREPTOBACILLUS MONILIFORMIS

Streptobacillus moniliformis are Gram-negative bacilli that are nonmotile, asporogenous facultative anaerobes. They are normally present in the nasopharynx of rats and known to cause rat-bite fever. The clinical condition may be accompanied by granules, bulbous swelling, and recurrent fever. The bacterium can be isolated from blood, joint fluid, and pus when cultured on media containing 15% defibrinated rabbit blood and incubated at 35°C in a carbon dioxide incubator. Most strains of S. moniliformis are sensitive to penicillins and tetracycline. 

SPIRILLUM MINUS 

Like S. moniliformis, S. minus is also a causal agent of rat-bite fever or spirillar fever, which follows bites by rats, mice, or some other animals including dogs and cats. The incubation period is usually 4 weeks, as opposed to 2 weeks for S. moniliformis infection. The clinical condition is characterized by inflammation, and indura- tion, accompanied by lymphangitis. Spirillum minus cannot be cultured on most laboratory media. The diagnosis is generally based on the microscopic demonstration of the bacterium in the relevant clinical specimens, including the blood, exudates from the bite wound, or cutaneous eruptions near the site of the initial bite. These are Gram-negative small spirals that can also be stained with Giemsa stain. Direct examination of the clinical specimens using dark field microscopy is also useful. Also, like S. moniliformis, S. minus infections are treated with penicillin and tetracycline.