Yersinia enterocolitica O9 as a Possible Barrier Against Y. pestis in Natural Plague Foci in Ningxia, China

A survey of Yersinia spp, as related to plague control, was made in Haiyuan of Ganning loess plateau plague focus, Yanchi of Inner Mongolia plateau plague focus, and Yinchuan city, as a control area, in Ningxia, China. In Haiyuan, where the main plague reservoir was Mongolian ground squirrel (Citellus alaschanicus) living in the prairie, Y. enterocolitica O9 was frequently isolated from pigs, dogs, rodents living in and around houses, but only rarely from hare and Mongolian ground squirrel. In Yanchi, where the main plague reservoir was Mongolian gerbil (Meriones unguiculatus) living in the prairie and Y. pestis, which was isolated from rodents up to 1991, Y. enterocolitica O9 was sometimes isolated from pigs and rodents. In all areas, some strains of Y. enterocolitica O3 and Y. pseudotuberculosis serotypes 3 and 4b were also isolated from pigs, dogs, and from rodents. We propose that an epidemiological link exists between the prevalence of Y. pestis and Y. enterocolitica O9 in domestic and rodents living in these areas in China. The residential area in Haiyuan may be protected against Y. pestis by the domestic animals and rodents which acquired cross-protection against Y. pestis by infection with Y. enterocolitica O9, but this is not the case in the Yanchi district. Received: 14 February 2000 / Accepted: 17 July 2000     

virF-Positive Yersinia pseudotuberculosis and Yersinia enterocolitica Found in Migratory Birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Somatic and flagellar antigens ofSerratia ficaria from the United States and the Mediterranean region

Somatic (O) and flagellar (H) antigens of 37Serratia ficaria strains were studied. All strains shared a common H antigen (H1). Four O antigens were identified that defined four serovars (O1:H1, O2:H1, O3:H1, and O4:H1). All American strains studied (isolated from the fig-fig wasp biological cycle or from a human patient) belonged to serotype O1:H1. Strains from the Mediterranean region (Sicily, Tunisia, France) were not so antigenically uniform and all four serotypes were found in figs from Sicily.     

Enterotoxigenic porcine and human isolates ofYersinia enterocolitica in Sweden

The production of heat-stable and heat-labile enterotoxins byYersinia enterocolitica was studied in 69 strains from healthy swine and in 24 strains from humans with acute diarrhea. All of the human strains were of serotype O3, and 20 (83%) of them produced heat-stable enterotoxin detectable in the infant mouse assay. All were negative in the Chinese Hamster Ovary (CHO) cell test for detection of heat-labile enterotoxin. Of the 69 porcine strains, which were of twelve serotypes plus 9 nontypable strains, 26 (38%) gave a positive infant mouse test. Of the porcine isolates of serotype O3, 42% were enterotoxigenic. A high incidence of enterotoxigenicity was also apparent among six other serotypes (53%). All porcine strains were negative in the CHO cell test. However, of seven culture supernatants from these porcine strains, three gave positive reactions in rabbit skin permeability tests, two of which were also positive in rabbit loop tests. Heat treatment of the supernatants abolished the reactivity in both tests. It is concluded that production of a heatstable enterotoxin is fairly common in porcine and human strains ofY. enterocolitica of serotype O3 in Sweden.     

Kinetics of infection induced byYersinia

Yersinia enterocolitica of different serotypes andY. intermedia, Y. frederiksenii, andY. kristensenii, in a total of nine strains, were inoculated intragastrically and intravenously into Swiss mice. The animals were observed daily to check for clinical alterations. Groups of five were killed intermittently at 6-h, and 3-, 6-, 10-, 15-,and 21-day periods or more after the inoculation; possible macroscopic alterations of the organs and tissues were checked. Development of infection at these periods was followed by performing viable bacterial counts on homogenates of selected tissues and the kinetics of infection was established. Clinical and pathologic alterations occurred only in the animals inoculated with the human strains ofY. enterocolitica 0:3 and 0:8, independent of the route of infection. After intragastric inoculation, theY. enterocolitica strains considered to be adapted to man were isolated from all organs and tissues, with the exception of the blood, from which only serotype 0:8 was isolated; otherYersinia strains were found only in the cecal content. After intravenous challenge, all the strains infected the organs and tissues at different times and in varied intensity, with exception of Peyer's patches and mesenteric lymph nodes, which were not infected by all theYersinia strains.     

Identification of distinct lipopolysaccharide patterns among <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> and <Emphasis Type="Italic">Y. enterocolitica</Emphasis>-like bacteria

The lipopolysaccharide (LPS) of strains representing various serotypes of Yersinia enterocolitica and Y. enterocolitica-like bacteria was studied by deoxycholate-PAGE and silver staining analysis. Four main types of LPS were detected based on the O-polysaccharide (O-PS): (i) LPS with homopolymeric O-PS, (ii) LPS with ladder-forming heteropolymeric O-PS, (iii) LPS with single-length O-PS, and (iv) semi-rough LPS without O-PS. Within the first three types, several subvariants were detected. Selected serotypes representing all above LPS types are sensitive to bacteriophage ϕR1-37 indicating that they share the phage receptor, a hexasaccharide called outer core in Y. enterocolitica O:3. Whereas phage ϕR1-37-resistant mutants of homopolymeric O-PS have lost only the outer core, those of ladder-forming or single-length O-PS have lost also the O-PS suggesting that in the latter ones the outer core is bridging between O-PS and lipid A-core. This work forms a basis of further structural, biochemical and genetic studies of these LPSs.     

Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have the eae gene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among the eae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. The ehxA gene, which encodes enterohemolysin, was found in ∼60% of the isolates, and the saa and subAB genes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. The stx_1a and stx_2a subtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. The stx_2d subtype was the second most common subtype (28% overall), followed by stx_2c (7.5%), and only 2 to 3% of the produce STEC strains had the stx_2e and stx_2g subtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.     

Biochemical heterogeneity of serotype 03 strains of 700Yersinia strains isolated from humans,other mammals,flies, animal feed,and river water

A total of 700 serotype 03 strains of yersiniae were recovered from patients, households withYersinia enterocolitica-infected persons, healthy humans, pigs, dogs, flies, feed, and river water, from 1977 to 1983. Of these isolates, 695 belonged toYersinia enterocolitica, three toYersinia intermedia, and two toYersinia frederiksenii. The 695Y. enterocolitica strains were classified into 484 biotype 4 serotype 03 phage type VIII, 17 biotype 4A (ornithine decarboxylase-negative) serotype 03 phage type VIII, 15 biotype 4B (maltosenegative) serotype 03 phage type VIII, and 179 biotype 3B (VP-, sorbose-, and inositolnegative) serotype 03 phage type II. These four biochemical heterogeneous types, including three new types, ofY. enterocolitica probably have long existed in Japan. There was a close relation between human infection withY. enterocolitica and the harboring ofY. enterocolitica in pigs and dogs.     

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods

Aim: To evaluate whole cell protein profiling vis‐à‐vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. Methods and Results: Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS–PAGE. Twenty‐one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30–6,31 strains were clustered into two discrete subgroups. Conclusions: Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. Significance and impact of the study: Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.     

Assessment of virulence determinants in Yersinia enterocolitica 1A,O:5 strains isolated from chicken carcasses

The virulence potential associated with 42–45 MDa plasmid pYV, of three Yersinia enterocolitica, 1A, O:5, strains isolated previously from chicken carcasses was tested by plasmid extraction, and by four in vitro tests: (1) Auto-agglutination; (2) Calcium dependence for growth at 37 °C; (3) Congo Red and Crystal Violet binding; and (4) Resistance to the bactericidal effect of serum. The in vitro tests were negative in all strains tested, but from strain PP131 it was possible to extract a plasmid which was quite similar in size to the wild type pYV, but heavier and not observed in any other isolated strains. These contradictions between the presence of a plasmid of approximately 42–45 MDa and the in vitro tests negative results may suggest that there is no homology between the PP131 plasmid and pYV. From these results we conclude that the plasmid found in PP131 is not pYV and that the three Y. enterocolitica 1A, O:5, strains are non-pathogenic on pYV classical mechanism basis, which do not exclude the possibility of them becoming infectious by a different mechanism, especially in children or immune-depressed individuals.     

Halophilic Black Sea vibrios and their role in human pathology

During the period of 4 years 558 halophil vibrio strains (258 V. parahaemolyticus strains and 300 V. angiolyticus strains) were isolated from the water and from the representatives of the hydrobios of the Black Sea. The vibrios were isolated the whole year round, but during the winter and spring months their lowest concentration in water was observed, while in September-October it reached its maximum. During these 4 years 102 cases of gastroenteric diseases were registered, and in some of the patients halophil vibrios were isolated. In most cases the diseases developed after the consumption of sea fish and other sea products, as well as salted vegetables. The diseases had mainly a sporadic character. 90% of the strains isolated from humans belonged to serotypes O4 : K12 and O4 : K8; besides, the presence of serotypes O3 : K33, O3 : K57, O5 : K47, O6 : K46 and O10 : K52 was revealed.     

Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica

An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4°C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone—soya broth yielded better results than in—frequently used—phosphate buffer, pH 7.6.While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29°C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results.It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.     

Biochemical,serological, and phage typing characteristics of 459Yersinia strains isolated from a terrestrial ecosystem

The origins of human contamination withYersinia enterocolitica are still unknown. We have investigated the major components of a terrestrial ecosystem (soil, earthworms, field voles, shrews, crops, hares, rabbits, and birds) for the presence ofYersinia. Four hundred fifty-nine strains ofYersinia were isolated. We report the first isolations of typicalY. enterocolitica belonging to classical or new biotypes and ofY. enterocolitica-like organisms (sucrose negative; rhamnose positive; melibiose and rhamnose positive) from soil samples, earthworms, crops, and birds. Sucrose-negativeY. enterocolitica strains and biotypes 1, 2, and 3, usually associated with human nonmesenteric syndromes, are predominant in soil, which can be considered as a reservoir for these biotypes.Y. enterocolitica serogroups O∶3 and O∶9, strains of which are responsible in Europe for human mesenteric syndromes, were not found in this study. The epidemiology ofY. enterocolitica infections is discussed.     

Prevalence of Very Low Numbers of Potential Pathogenic Isolates of <Emphasis Type="Italic">Yersinia</Emphasis><Emphasis Type="Italic">enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia intermedia</Emphasis> in Traditional Fast Foods of India

In this study, an incidence pattern of 1.7% for Yersinia enterocolitica and 2.5% for Y. intermedia were observed in an analysis of 120 diversified food samples collected from the local market of Mysore, Southern India. Two native isolates characterized as Y. enterocolitica belonged to biotype 1B and revealed the presence of major virulence related traits such as regulator of virulence, mucoid Yersinia factor regulator, attachment invasion locus, heat stable enterotoxin, Yersinia type II secretory system and phospholipase A in PCR. Force type neighbor-joining phylograms generated for Y. enterocolitica based on PCR amplicons of rovA and ypl showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis.     

Yersinia enterocolitica Isolates from Wild Boars Hunted in Lower Saxony,Germany

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified.     

Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli

Aims: The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes. Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml^?1 reductions at 37°C. Multiplex‐PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes. Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes. Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain.     

An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

Seropathotypes, Phylogroups, Stx subtypes, and intimin types of wildlife-carried, shiga toxin-producing escherichia coli strains with the same characteristics as human-pathogenic isolates

The objectives of this study were to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) strains in wildlife that have spread in Europe, living near human settlements; to analyze their epidemiological role in maintenance and transmission to domestic livestock; and to assess the potential health risk of wildlife-carried strains. STEC strains were recovered from 53% of roe deer, 8.4% of wild boars, and 1.9% of foxes sampled in the northwest of Spain (Galicia). Of the 40 serotypes identified, 21 were classified as seropathotypes associated with human disease, accounting for 81.5% of the wildlife-carried STEC strains, including the enterohemorrhagic serotypes O157:H7-D-eae-γ1, O26:[H11]-B1-eae-β1, O121:H19-B1-eae-ε1, and O145:[H28]-D-eae-γ1. None of the wildlife-carried strains belonged to the highly pathogenic serotype O104:H4-B1 from the recent Germany outbreak. Forty percent of wildlife-carried STEC strains shared serotypes, phylogroups, intimin types, and Stx profiles with isolates from human patients from the same geographic area. Furthermore, wildlife-carried strains belonging to serotypes O5:HNM-A, O26:[H11]-B1, O76:H19-B1, O145:[H28]-D, O146:H21-B1, and O157:H7-D showed pulsed-field gel electrophoresis (PFGE) profiles with >85% similarity to human-pathogenic STEC strains. We also found a high level of similarity among STEC strains of serotypes O5:HNM-A, O26:[H11]-B1, and O145:HNM-D of bovine (feces and beef) and wildlife origins. Interestingly, O146:H21-B1, the second most frequently detected serotype in this study, is commonly associated with human diarrhea and isolated from beef and vegetables sold in Galicia. Importantly, at least 3 STEC isolates from foxes (O5:HNM-A-eae-β1, O98:[H21]-B1-eae-ζ1, and O146:[H21]-B1) showed characteristics similar to those of human STEC strains. In conclusion, roe deer, wild boar, and fox in Galicia are confirmed to be carriers of STEC strains potentially pathogenic for humans and seem to play an important role in the maintenance of STEC.     

Incidence of non-cholera vibrios in Hungary.

In the period 1971 to 1976, 200 non-cholera vibrio (NCV) strains were isolated in Hungary; 18 of the cultures were derived from 34 729 faecal and 182 from 237 surface water samples. Ninety-two strains belonged to the Heiberg-Smith group I and 108 to group II. Two strains failed to give the string test and 3 were pteridine resistant. The strains were classified into 48 serotypes according to Smith's system. Faecal NCV strains belonged to serotypes 46 and 328; these serotypes did not occur in water. Of the 18 faecal strains 13 were isolated from 18 048 persons who had travelled in cholera-infected areas, and 5 strains from persons who had never left Hungary (2 from 4559 patients with diarrhoea and 3 from 6061 healthy individuals). These data indicate that although NCV are present in the environment, they play an insignificant role in enteric infections in Hungary.     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。