Enteropathogenic Yersiniae: Classification, Structure, Virulence factor, Pathogenesis and Laboratory Diagnosis


Classification


Yersinia genus belongs to the family Enterobacteriaceae and contains more than 10 species. Yersinia pestis, a causative agent of plague is the most virulent among them. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause self-limiting diarrheal diseases and food poisonings in humans generally termed as yersinioses. However, enteropathogenic yersiniae, especially Yersinia pseudotuberculosis, sometimes produce persistent chronic infections that can disable patients for a long time.

Causative agents of yersinioses have multiple biotypes and serovars.


Structure and Properties of Y. pseudotuberculosis and Y. enterocolitica


Morphology

All yersiniae are similar to some extent with other enterobacteria: small polymorphic gram-negative rods that exhibit bipolar stain. They look like short chains or diplobacteria under microscopy. Enteric yersiniae carry peritrichous flagella with numerous pili.

Cultivation

Yersiniae readily grow on ordinary nutrient media. The best growth temperature for yersiniae is about 25°C. At these conditions bacteria render motility; temperature rise to 37°C leads to motility loss.

Microbial growth exhibits small convex semi-transparent polymorphic colonies with slight brownish pigmentation.

Various lactose-containing media (McConkey, or EMB agar) and several special media are used for yersiniae cultivation. During cultivation the bacteria produce lactose-negative translucent colonies. Colonies of yersiniae can dissociate into S- or R forms.


Biochemical properties


Yersiniae are facultatively anaerobic bacteria. They display marked biochemical diversity. These bacteria are not lactose-fermenting, but they can utilize many other carbohydrates (glucose, maltose, mannitol, dextrin, glycerin, etc.) with acid formation. Y. enterocolitica ferments sucrose, while Y. pseudotuberculosis not. According to carbohydrate fermentation, Y. enterocolitica is divided into 5 biotypes.

Yersiniae have weak proteolytic activity. As other enterobacteria, they are oxidase-negative, but catalase positive. Yersiniae produce urease and can reduce nitrates into nitrites.


Antigenic structure


Yersiniae possess flagellar H-antigen and several somatic cell wall antigens. Y. pseudotuberculosis contains S- and R-somatic antigens, Y. enterocolitica has somatic lipopolysaccharide O-antigen.

More than 50 serovars are described within Y. enterocolitica species; serovars 03, 08, and 09 most frequently cause the disease in humans. Y. pseudotuberculosis has at least 6 distinct serovars; but serovar 01 is a predominant human pathogen.


Virulence factors


Yersiniae express different virulence factors. After microbial cell lysis lipopolysaccharide endotoxin is released, which exhibits pyrogenic, hemolytic and proinflammatory activity. Certain serovars of Y. pseudotuberculosis can produce enterotoxin.

Bacterial adhesin invasin is encoded by genes of nucleoid. Large number of virulence factors is encoded by mobile genetic elements.

A 72-kb virulence plasmid (pYV – plasmid of yersinia virulence) is responsible for microbial adherence, invasiveness, intercellular spread, and ability to survive and propagate within host lymphoid tissues. This plasmid contains yersinia pathogenicity island or Yop virulon.

Yop virulon encodes yersinia invasive effector proteins (Yop proteins or yersinia outer proteins). Also it codes for structures of type III bacterial secretion system (injectisome, or needle complex), composed of translocator proteins (so-called Ysc proteins). Needle complex promotes microbial adherence to host epithelial or immune cells and delivers invasive Yop proteins into them. When injected, Yop molecules impair the dynamics of the cytoskeleton, allow microbial penetration and intracellular spread, thereby promoting further bacterial invasion. Yop proteins sharply diminish the production of proinflammatory cytokines by macrophages and other immune cells that maintain bacterial survival within lymphoid tissues.


Resistance


Yersiniae are rather stable in the environment. They can readily withstand cooling up to -250С. Bacteria survive for several months in water and some foodstuffs, contaminated by bacteria (e.g., in butter for 150 days and in water for 200 days). They stay viable and even propagate in milk, fruits and vegetables.

Yersiniae are sensitive to heating, desiccation and also susceptible to disinfectant actions.


Pathogenesis and Clinical Findings in Yersinioses


Yersiniae inhabit the intestinal tract of many animals (rodents, hares, foxes, cattles, etc.), in which they occasionally cause diseases. Rodents as common sources of infection discharge the bacteria into environment with feces and urine.

The disease is transmitted from animals by fecal-oral route predominantly through contaminated foodstuffs, especially raw vegetables, salads, etc.

Infectious dose of bacteria is rather high (up to 107-109 microbial cells). More often the disease affects infants and young children.

Incubation period lasts about 5-10 days.

Yersiniae enter the gastrointestinal tract and bind to enterocytes and intestinal M cells by means of needle complex and number of microbial adhesins (e.g., by invasin). Yersinia virulon complex ensures microbial invasion and intracellular persistence. The bacteria propagate within intestinal mucosa, affecting ileum and other parts of gut.

Then yersiniae spread into intestinal lymphoid tissues (lymph nodes and lymphatic follicles) and suppress cytokine synthesis by immune cells, thereby inhibiting phagocytosis and maintaining microbial survival. Bacterial propagation results in inflammation and ulceration of the intestine.

Inflammatory tissue damage and enterotoxin production elicit watery or bloody diarrhea.

Yersiniosis, caused by Y. enterocolitica is characterized mainly by modest diarrheal syndrome and usually self-limited.

Y. pseudotuberculosis can produce more severe disease with a tendency to microbial spread and persistency. Further invasion of bacteria results in their appearance in mesenteric lymph nodes and next in bloodstream with microbial dissemination and endotoxemia.

Slow elimination of bacteria may cause chronic disease, which is manifested by infectious allergy and autoimmune reactions with arthritis, skin lesions (erythema nodosum), and inner organ dysfunction. The acquired immunity is low specific and weak, thereby recurrent diseases occur.


Laboratory Diagnosis of Yersinioses


For laboratory diagnosis of yersinioses stool specimens, vomit, blood, food remnants can be examined.

As the number of yersiniae in stool is often small, the sensitivity of cultural method can be enhanced by “cold enrichment”. In that case the stool specimen is placed into phosphate buffered saline, pH 7.6, and incubated at 4°C for 2-4 weeks. The majority of intestinal flora dies but yersiniae can grow. Similar result may be obtained by primary yersinia infection on mice.

Sub-culture is produced on Endo or MacConkey agar. Microbial isolate is further identified by its biochemical and antigenic properties.

For serological diagnosis the specific antibodies against microbial antigens are determined at the second week of disease by indirect hemagglutination test or ELISA. The assays are considered to be positive in titers 1:100-1:200.


Treatment and Prophylaxis of Yersinioses


Yersinioses are usually treated with antibiotics that affect gram-negative bacteria (third generation cephalosporins, fluoroquinolones, tetracyclines, or trimethoprim-sulfamethoxazole). Bacteria are resistant to ampicillin and first-generation cephalosporins.

Vaccines are currently not available for disease prophylaxis, thus all measures of sanitary control with prevention of food and water resources from microbial contamination should be maintained to limit disease spread.