Role of the fly in the transport of Yersinia enterocolitica.

Yersinia enterocolitica were isolated from flies collected from a piggery and a kitchen of farm and from ham hung in a piggery. The cultures were identified as Y. enterocolitica biovar 4 and serovar 3 by biochemical and serological characteristics. From these results it is suggested that flies may play an important role in food contamination by Y. enterocolitica. In this study, the probable donors of Y. enterocolitica to the flies were swine.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA

The Yersinia adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus. For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four beta-strands. To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants. Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis. We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain. Thus, YadA meets all the criteria of an autotransporter. The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters. Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk. However, the YadA-binding module did not confer serum resistance. Mutants lacking the head and neck domain were resistant to complement-mediated lysis. In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model.     

Yersinia enterocolitica in Danish pigs

Yersinia enterocolitica serotype 0:3, the predominating pathogenic serotype in Danish pigs, was isolated consistently from the tonsils of pigs in six farms but not from those in another four farms during a one-year survey, indicating a herd-wise distribution. Only one positive culture was obtained from four specific-pathogen-free herds. The organisms were not recovered from samples of fodder, water and faeces from any of the infected farms. Strains of Y. enterocolitica were tested for sensitivity to antimicrobial agents.     

Survival of Yersinia enterocolitica in the environment

When Yersinia enterocolitica was introduced into soils (or physiological saline), very little decrease in the population was observed throughout the test period. If the soil was allowed to air dry slowly, only 0.1% (2.8 x 10(3) colony forming units/g of soil) of the original population added still remained viable by day 10. On the other hand, the introduced organisms disappeared rapidly in river water but their longevities could be extended significantly if a eucaryote inhibitor was added to the river water or the river water was passed through a 0.8-micron membrane filter to remove eucaryotic predators. Furthermore, the rapid decrease of the Yersinia population coincided with an increase in numbers of protozoans. However, when Yersinia was added to filter-sterilized river water or when small numbers of the organism, below the threshold level believed necessary for active predation to occur, were added to the river water, no response in predators was observed; nevertheless, the population of Yersinia still showed a continued decline. When the organism was introduced into sephadex-treated river water or groundwater, its survival improved significantly compared with its survival in nontreated water samples. Low ambient temperature dramatically increased its ability to survive in the aquatic environment. It is concluded that, in addition to the temperature factor, the longevity of Y. enterocolitica in river water is chiefly regulated by predators and toxin producers.     

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

RAPD analysis of Yersinia enterocolitica

A total of 87 isolates of Yersinia enterocolitica were examined with randomly amplified polymorphic DNA (RAPD) by use of three different primers. Based on the RAPD profiles, the strains could be divided into three major groups: (1) the pathogenic American serotypes, O: 8, O: 13ab, O: 20 and O: 21; (2) the pathogenic European serotypes, O: 3, O: 5,27 and O: 9; and (3) the nonpathogenic serotypes. Five tested strains of the American serotype O: 4 gave unique profiles with YCPEL, but did not give reproducible profiles with the other primers. The European serotypes could be further subdivided into a group consisting of strains of O: 3 and O: 5,27 and a group of strains of O: 9. RAPD profiling provides an easy approachable method to divide isolates of Y. enterocolitica into pathogenic and nonpathogenic strains and further to differentiate between the pathogenic isolates.     

Septicemia due to Yersinia enterocolitica

Despite the increasing number of reports of Yersinia enterocolitica infection in humans, septicemia with this organism has remained a rare complication. A 73-year-old woman presented with fever, jaundice, hepatomegaly and cellulitis. Microorganisms isolated from both skin lesion and blood were biochemically and serologically identified as Yersinia enterocolitica, biotype 4, serotype 3 and lysotype 9b. High agglutinating titres against this organism were demonstrated in the patient''s serum. Complete recovery followed a course of gentamicin sulfate. A household pet was considered, but not proved, to be the source of this infection.     

Beta-lactamases from Yersinia enterocolitica.

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.     

Role of bone marrow in inflammatory response to experimental Yersinia enterocolitica infection in mice

Abstract Mice infected intraperitoneally (i.p.) with Yersinia enterocolitica developed an inflammatory response, as revealed by a large influx of leukocytes in the peritoneal cavity. When the infection was preceded by the administration of Y. enterocolitica by the same route 4 days before, this resulted in a poor inflammatory reaction. On the other hand, the response of previously immunized animals to infection resembled to those of primoinfected mice. Bone marrow cellularity was decreased after the infection with Y. enterocolitica . Since bone marrow depletion by pre-treatment with cyclophosphamide decreased the inflammatory response to Y. enterocolitica , we concluded that marrow cell reserve was necessary for the inflammatory reaction, whereas specific immunity did not affect this response.     

Studies on the Pathogenicity of Yersinia enterocolitica

Analysis of the pathogenicity of Yersinia enterocolitica was performed with an experimental model successfully produced in rabbits by intraduodenal inoculation with strains isolated from various sources. Pathogenic strains easily penetrated the epithelial linings of the intestinal mucous membrane into the target reticuloendothelial tissues of the intestine, such as the lamina propria and lymph follicles, where they multiplied within mononuclear cells and produced granuloma. Granuloma, in severe infections, underwent necrobiosis and sometimes progressed to ulceration accompanied by colony formation of the organisms. In mild infections, granulomatous lesions were localized in lymph follicles and never progressed to ulceration. Nonpathogenic strains were rapidly excreted without penetration of epithelial linings. Y. enterocolitica should be within the category of invasion type enteropathogenic bacteria such as Shigella and Salmonella. Pathogenic behavior of Y. enterocolitica is discussed in comparison with that of Shigella and Salmonella.     

Detection of modification-restriction systems in Yersinia enterocolitica

Four Y. enterocolitica strains (10166, 10373, 2119, 5513) have been studied for the presence of the enzymatic systems of modification-restriction (M-R). As revealed with the use of cross titration, strains 10166 and 10373 contain M-R systems, supposedly of type II.     

Phenomenon of pili formation in Yersinia enterocolitica

In Y. enterocolitica strain, serovar 0:10, the capacity for the formation of pili inducing the mannose-resistant hemagglutination (MRHA) of formolated sheep red blood cells was due to the presence of plasmid pYE10. MRHA-inducing pili differed serologically from Y. pestis and Y. tuberculosis adhesion pili. Plasmid pYE10 was immobilized for transfer to cells of Escherichia coli strain HB101 (rec A) by means of pRP 4. The expression of MRHA-inducing pili in the new host the rec A-independent character of the synthesis. Y. enterocolitica cells containing pYE10 agglutinated in tissue-culture media with 10% of serum added at 37 degrees C.