Chapter 19: Yersinia and Pasteurella

The organisms discussed in this chapter are short, pleomorphic Gram-negative rods that can exhibit bipolar staining. They are catalase positive, oxidase negative, and microaerophilic or facultatively anaerobic. Most have animals as their natural hosts, but they can produce serious disease in humans.

The genus Yersinia includes Yersinia pestis, the cause of plague; Yersinia enterocolitica, an important cause of human diarrheal disease; and several others considered nonpathogenic for humans. Pasteurella are primarily animal pathogens but Pasteurella multocida can also produce human disease.

Plague is an infection of wild rodents transmitted from one rodent to another and occasionally from rodents to humans by the bites of fleas. Serious infection often results, which in previous centuries produced pandemics of “black death” with millions of fatalities. The ability of this organism to be transmitted by aerosol and the severity and high mortality associated with pneumonic plague make Y pestis a potential biological weapon.

Morphology and Identification

Y pestis is a Gram-negative rod that exhibits striking bipolar staining with special stains such as Wright, Giemsa, Wayson, or methylene blue (Figure 19-1). It is nonmotile. It grows as a facultative anaerobe on many bacteriologic media. Growth is more rapid in media containing blood or tissue fluids and fastest at 30°C. In cultures on blood agar at 37°C, colonies may be very small at 24 hours. A virulent inoculum, derived from infected tissue, produces gray and viscous colonies, but after passage in the laboratory, the colonies become irregular and rough. The organism has little biochemical activity, and this is somewhat variable.

Antigenic Structure

All yersiniae possess lipopolysaccharides that have endotoxic activity when released. Y pestis and Y enterocolitica also produce antigens and toxins that act as virulence factors. They have type III secretion systems that consist of a membrane-spanning complex that allows the bacteria to inject proteins directly into cytoplasm of the host cells. The virulent yersiniae produce V and W antigens, which are encoded by genes on a plasmid of approximately 70 kb. This is essential for virulence; the V and W antigens yield the requirement for calcium for growth at 37°C. Compared with the other pathogenic yersiniae, Y pestis has gained additional plasmids. pPCP1 is a 9.5-kb plasmid that contains genes that yield plasminogen-activating protease that has temperature-dependent coagulase activity (20°–28°C, the temperature of the flea) and fibrinolytic activity (35°–37°C, the temperature of the host). This factor is involved in dissemination of the organism from the flea bite injection site. The pFra/pMT plasmid (80–101 kb) encodes the capsular protein (fraction F1) that is produced mainly at 37°C and confers antiphagocytic properties. In addition, this plasmid contains genes that encode phospholipase D, which is required for organism survival in the flea midgut.

Y pestis and Y enterocolitica have a pathogenicity island (PAI) that encodes for an iron-scavenging siderophore (see Chapter 9), yersiniabactin.

Pathogenesis and Pathology

When a flea feeds on a rodent infected with Y pestis, the ingested organisms multiply in the gut of the flea and, helped by the coagulase, block its proventriculus so that no food can pass through. Subsequently, the “blocked” and hungry flea bites ferociously, and the aspirated blood, contaminated with Y pestis from the flea, is regurgitated into the bite wound. The inoculated organisms may be phagocytosed by polymorphonuclear cells and macrophages. The Y pestis organisms are killed by the polymorphonuclear cells but multiply in the macrophages; because the bacteria are multiplying at 37°C, they produce the antiphagocytic protein and subsequently are able to resist phagocytosis. The pathogens rapidly reach the lymphatics, and an intense hemorrhagic inflammation develops in the enlarged lymph nodes, which may undergo necrosis and become fluctuant. Although the invasion may stop there, Y pestis organisms often reach the bloodstream and become widely disseminated. Hemorrhagic and necrotic lesions may develop in all organs; meningitis, pneumonia, and serosanguineous pleuropericarditis are prominent features.

Primary pneumonic plague results from inhalation of infective droplets (usually from a coughing patient), and it is characterized by hemorrhagic consolidation, sepsis, and death.

Clinical Findings

The clinical manifestations of plague depend on the route of exposure. After an incubation period of 2–7 days, there is high fever and painful lymphadenopathy, commonly with greatly enlarged, tender nodes (buboes) in the neck, groin, or axillae. This is the bubonic form of the disease. Vomiting and diarrhea may develop with the early septicemic form of disease. Later, disseminated intravascular coagulation leads to hypotension, altered mental status, and renal and cardiac failure. Terminally, signs of pneumonia and meningitis can appear, and Y pestis multiplies intravascularly and can be seen in blood smears. Primary pneumonic plague results from direct inhalation of organism into the lung. Patients often have a fulminant course with chest pain, cough, hemoptysis, and severe respiratory distress.

Diagnostic Laboratory Tests

Plague should be suspected in febrile patients who have been exposed to rodents in known endemic areas. Rapid recognition and laboratory confirmation of the disease are essential to institute lifesaving therapy.

A. Specimens

Blood is taken for culture and aspirates of enlarged lymph nodes for smear and culture. Acute and convalescent sera may be examined for antibody levels. In pneumonia, sputum is cultured; in possible meningitis, cerebrospinal fluid is taken for smear and culture.

B. Smears

Y pestis are small gram-negative bacilli that appear as single cells or as pairs or short chains in clinical material. Wright, Giemsa, or Wayson stains may be more useful when staining material from a suspected buboe or a positive blood culture result because of the striking bipolar appearance (safety pin shape) of the organism using these stains that is not evident on a direct Gram stain. More specific direct staining methods (possibly available through reference laboratories) include the use of fluorescent antibody stains targeting the capsular F1 antigen.

C. Culture

All materials are cultured on blood agar, chocolate, and MacConkey agar plates and in brain–heart infusion broth. Growth on solid media may be slow, requiring more than 48 hours, but blood culture results are often positive in 24 hours. Cultures can be tentatively identified by biochemical reactions. Y pestis produces nonlactose-fermenting colonies on MacConkey agar, and it grows better at 25°C than at 37°C. The organism is catalase positive; indole, oxidase, and urease negative; and nonmotile. The last two reactions are useful in differentiating Y pestis from other pathogenic yersiniae. An organism with these characteristics should be referred to a public health laboratory for more confirmatory testing. Definite identification of cultures is best done by immunofluorescence or by lysis by a specific Y pestis bacteriophage (confirmation available through state health department laboratories and by consultation with the Centers for Disease Control and Prevention [CDC], Plague Branch, Fort Collins, CO).

All cultures are highly infectious and must be handled with extreme caution inside a biological safety cabinet.

D. Serology

In patients who have not been previously vaccinated, a convalescent serum antibody titer of 1:16 or greater is presumptive evidence of Y pestis infection. A titer rise in two sequential specimens confirms the serologic diagnosis.

Treatment

Unless promptly treated, plague may have a mortality rate of nearly 50%; pneumonic plague has a mortality rate of nearly 100%. The drug of choice is streptomycin, but the more readily available aminoglycoside gentamicin has been shown to be as effective. Doxycycline is an alternative drug as is the fluoroquinolone antibiotic ciprofloxacin. These agents are sometimes given in combination with streptomycin or gentamicin. Drug resistance has rarely been noted in Y pestis.

Epidemiology and Control

Plague is an infection of wild rodents (field mice, gerbils, moles, skunks, and other animals) that occurs in many parts of the world. The chief enzootic areas are India, Southeast Asia (especially Vietnam), Africa, and North and South America. The western states of the United States and Mexico also contain reservoirs of infection. Epizootics with high mortality rates occur intermittently; at such times, the infection can spread to domestic rodents (eg, rats) and other animals (eg, cats), and humans can be infected by flea bites or by contact. The commonest vector of plague is the rat flea (Xenopsylla cheopis), but other fleas may also transmit the infection.

The control of plague requires surveys of infected animals, vectors, and human contacts; in the United States, this is done by county and state agencies with support from the Plague Branch of the CDC and by destruction of plague-infected animals. If a human case is diagnosed, health authorities must be notified promptly. All patients with suspected plague should be isolated, particularly if pulmonary involvement has not been ruled out. All specimens must be treated with extreme caution. Contacts of patients with suspected plague pneumonia should receive doxycycline as chemoprophylaxis.

Killed whole-cell vaccines are no longer available. Because of concern for bioterrorism, numerous vaccines are currently under development.

These are nonlactose-fermenting, Gram-negative rods that are urease positive and oxidase negative. They grow best at 25°C and are motile at 25°C but nonmotile at 37°C. They are found in the intestinal tracts of a variety of animals, in which they may cause disease, and are transmissible to humans, in whom they can produce a variety of clinical syndromes.

Y enterocolitica exists in more than 70 serotypes; most isolates from human disease belong to serotypes O:3, O:8, and O:9. There are striking geographic differences in the distribution of Y enterocolitica serotypes. Y enterocolitica can produce a heat-stable enterotoxin, but the role of this toxin in diarrhea associated with infection is not well defined.

Y enterocolitica has been isolated from rodents and domestic animals (eg, sheep, cattle, swine, dogs, and cats) and waters contaminated by them. Transmission to humans probably occurs by contamination of food, drink, or fomites. Person-to-person transmission with this organism is probably rare.

Pathogenesis and Clinical Findings

An inoculum of 10⁸–10⁹ yersiniae must enter the alimentary tract to produce infection. During the incubation period of 4–7 days, yersiniae multiply in the gut mucosa, particularly the ileum. This leads to inflammation and ulceration, and leukocytes appear in feces. The process may extend to mesenteric lymph nodes and, rarely, to bacteremia.

Early symptoms include fever, abdominal pain, and diarrhea. Diarrhea ranges from watery to bloody and may be caused by an enterotoxin or due to invasion of the mucosa. At times, the abdominal pain is severe and located in the right lower quadrant, suggesting appendicitis. One to 2 weeks after onset, some patients with histocompatibility antigen HLA-B 27 develop arthralgia, arthritis, and erythema nodosum, suggesting an immunologic reaction to the infection. Very rarely, Yersinia infection produces pneumonia, meningitis, or sepsis; in most cases, it is self-limited.

Y enterocolitica has also been associated with transfusion-related infections caused by contaminated red blood cells. This is a consequence of the ability of the organism, transmitted by an asymptomatic donor, to multiply at refrigeration temperatures.

Diagnostic Laboratory Tests

A. Specimens

Specimens may be stool, blood, or material obtained at surgical exploration. Stained smears are not contributory.

B. Culture

The number of yersiniae in stool may be small and can be increased by “cold enrichment”: a small amount of feces or a rectal swab is placed in buffered saline with a pH of 7.6 and kept at 4°C for 2–4 weeks; many fecal organisms do not survive, but Y enterocolitica multiplies. Subcultures made at intervals on MacConkey agar may yield yersiniae. Alternatively, most clinical laboratories use a Yersinia-selective agar such as cefsulodin-irgasan-novobiocin (CIN) agar incubated at room temperature for several days. Y enterocolitica colonies have a bull’s eye appearance with a red center on CIN agar.

C. Serology

In paired serum specimens taken 2 or more weeks apart, a rise in agglutinating antibodies can be shown; however, cross-reactions between yersiniae and other organisms (vibrios, salmonellae, and brucellae) may confuse the results.

Treatment

Most Yersinia infections with diarrhea are self-limited, and the possible benefits of antimicrobial therapy are unknown. Y enterocolitica is generally susceptible to aminoglycosides, chloramphenicol, tetracycline, trimethoprim–sulfamethoxazole, piperacillin, third-generation cephalosporins, and fluoroquinolones; it is typically resistant to ampicillin and to first-generation cephalosporins. Proved Yersinia sepsis or meningitis has a high mortality rate, but deaths occur mainly in immunocompromised patients. Yersinia sepsis can be successfully treated with third-generation cephalosporins (possibly in combination with an aminoglycoside) or a fluoroquinolone (possibly in combination with another anti­microbial). In cases in which clinical manifestations strongly point to either appendicitis or mesenteric adenitis, surgical exploration has been the rule unless several simultaneous cases indicate that Yersinia infection is likely.

Prevention and Control

Contact with farm and domestic animals, their feces, or materials contaminated by them probably accounts for most human infections. Meat and dairy products have occasionally been indicated as sources of infection, and group outbreaks have been traced to contaminated food or drink. Conventional sanitary precautions are probably helpful. There are no specific preventive measures.

Concept Checks

• Yersinia species are zoonotic pathogens that cause disease in humans ranging from mild gastrointestinal infections to serious diseases with high mortality such as plague.

• Y pestis is transmitted to humans usually through the bite of an infected flea, although inhalation is another potential route. Y pestis possesses virulence factors transmitted by plasmids that allow it to survive in the gut of the flea and that contribute to severe clinical manifestations in humans.

• A bubo (an enlarged suppurative lymph node) forms close to the bite wound accompanied by fever and is the most common form of plague. From the localized lesion, the infection may disseminate causing the septicemic form of the disease.

• Treatment consists of supportive care and antibiotic treatment with streptomycin, gentamicin, doxycycline, or a fluoroquinolone antibiotic.

• Y enterocolitica causes gastroenteritis or mesenteric lymphadenitis after ingestion of contaminated food or water.

• Y enterocolitica can be recovered from the stool of infected patients using selective media called CIN agar incubated at room temperature.

• Treatment for gastroenteritis caused by Y enterocolitica consists of trimethoprim–sulfamethoxazole, doxycycline, or a fluoroquinolone antibiotic.

Pasteurellae are nonmotile, Gram-negative coccobacilli with a bipolar appearance on stained smears. They are aerobes or facultative anaerobes that grow readily on ordinary bacteriologic media at 37°C. They are all oxidase positive and catalase positive but diverge in other biochemical reactions.

P multocida occurs worldwide in the respiratory and gastrointestinal tracts of many domestic and wild animals. It is perhaps the most common organism in human wounds inflicted by bites from cats and dogs. It is one of the common causes of hemorrhagic septicemia in a variety of animals, including rabbits, rats, horses, sheep, fowl, cats, and swine. It can also produce human infections in many systems and may at times be part of normal human microbiota.

Clinical Findings

The most common presentation is a history of animal bite followed within hours by an acute onset of redness, swelling, and pain. Regional lymphadenopathy is variable, and fever is often low grade. Pasteurella infections sometimes present as bacteremia or chronic respiratory infection without an evident connection with animals.

P multocida is susceptible to most antibiotics. Penicillin G is considered the drug of choice for P multocida infections resulting from animal bites. Tetracyclines and fluoroquinolones are alternative drugs.