High prevalence of Yersinia enterocolitica 4:O3 on pig offal in southern Germany: a slaughtering technique problem

Prevalence and contamination routes of pathogenic Yersinia enterocolitica were studied in Southern Germany. Tonsil and faeces samples of 50 fattening pigs, 140 offal samples and 120 minced meat samples were examined. Pig and offal samples were collected from a slaughterhouse approved by the European Union, and minced meat samples from two large meat factories. Yersinia enterocolitica was isolated using direct plating, overnight enrichment and selective enrichment in MRB and ITC broth. The isolates were bio- and serotyped, and pathogenicity was studied using two plasmid-encoded virulence markers: calcium dependence and Congo red absorption. The genotypes were studied with pulsed-field gel electrophoresis using NotI enzyme. Prevalence of pathogenic Y. enterocolitica 4:O3 was 60% and 10% in tonsils and faeces of fattening pigs, respectively. Besides tonsils, prevalence of pathogenic Y. enterocolitica 4:O3 was also high in other pluck set samples, including tongues, lungs, hearts, diaphragms and livers. However, the highest isolation rate was obtained from the tonsils. Kidneys, which were not attached to the pluck set and did not hang together with tonsils on the rack, had the lowest isolation rate. Yersinia enterocolitica 4:O3 was isolated from 12% of minced meat samples. A total of 25 NotI profiles were obtained from porcine samples. The most common genotype, NBI, found in tonsils was also the most common type recovered from offal and minced meat samples. The high contamination rate of tonsils, and the indistinguishable NotI profiles obtained from tonsils and offal indicate that the tonsils contaminate offal when they are removed and hung on the rack together. When the head, with the tonsils and tongue, is not removed prior to evisceration and is not handled and inspected separately, it is difficult to control the spread of Y. enterocolitica 4:O3 from tonsils to the carcass, and subsequently, to meat.     

In vitro antibiotic susceptibilities of Yersinia enterocolitica biotype 1A

Antibiotic susceptibilities of 80 strains of Yersinia enterocolitica biotype 1A isolated from human, swine and various aquatic sources to 20 β-lactam and 26 non-β-lactam antibiotics were studied. Most isolates were resistant to penicillins, first-generation cephalosporins, macrolides and lincosamides, while sensitive to aminoglycosides and quinolones. In comparison to earlier studies, the majority of the strains were either intermediately sensitive or resistant to piperacillin. Relatively decreased susceptibility or resistance to third-generation cephalosporins was also observed in several Y. enterocolitica isolates. This revised version was published online in August 2006 with corrections to the Cover Date.     

Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1–2 mg mg^-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al³⁺ chelate, which was found to be stable in solution for several weeks. To purify Al³⁺-yersiniophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al³⁺-yersiniophore was eluted with 0.01 m AlCl₃ and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C₂₁H₂₄N₃O₄S₃Al and a molecular weight of 505.07404 when chelated to Al³⁺. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.     

Destabilization of phospholipid model membranes by YplA, a phospholipase A2 secreted by Yersinia enterocolitica

Yersinia enterocolitica produces a virulence-associated phospholipase A(2) (YplA) that is secreted via its flagellar type-III secretion apparatus. When the N-terminal 59 amino acids of YplA are removed (giving YplA(S)), it retains phospholipase activity; however, it is altered with respect to the apparent kinetics of hydrolysis using fluorescent phospholipid substrates in micellar form. To explore the physical properties of YplA more carefully, Langmuir phospholipid monolayers were used to study the association of YplA with biological membranes. YPlA and YplA(S) both associate with Langmuir monolayers, but YplA(S) appears to interact better at low initial lipid densities while YplA interacts better at higher densities. This may indicate that the N-terminus of YplA has a role in mediating its initial interaction with compact cellular membranes, which is consistent with spectroscopic observations that fluorescein-labeled YplA may interact more readily with the nonpolar region of liposomes than does YplA(S).     

Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

Summary A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.     

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

长春地区鸡源奇异变形杆菌的分离鉴定及毒力分析

【背景】奇异变形杆菌(Proteus mirabilis)是一种机会致病菌,广泛存在于周围环境中,常导致动物和人类感染。【目的】探究吉林省长春地区某养鸡场病鸡死亡原因,为疾病防控提供参考。【方法】从病死青年鸡脏器分离到病原菌TSA-1,通过革兰氏染色、生化试验和16S rRNA基因序列鉴定,并进行药敏试验、毒力基因检测、细胞毒性和黏附性试验、大蜡螟攻毒试验研究。【结果】分离株TSA-1在镜下呈短杆状、球状的革兰氏阴性菌,生化特性与奇异变形杆菌相一致,16S rRNA基因序列比对显示与奇异变形杆菌相似度为100%;药敏试验结果显示分离株TSA-1对氨苄西林、四环素、卡那霉素、头孢唑林等14种药物耐药,对环丙沙星、恩诺沙星等7种药物敏感;分离株还具有较强的生物被膜形成能力,携带ireA、ucaA、pmfA、atfA、ptA、zapA、hpmA和flhC这8种毒力基因,并对巨噬细胞RAW264.7表现出细胞毒性,而且对Caco-2细胞具有很强的黏附作用;同时对大蜡螟幼虫表现出比禽致病性大肠杆菌(avian pathogenic Escherichia coliO78,APEC-O78)更强的致死作用。【结论】从病死鸡脏器分离得到的奇异变形杆菌TSA-1具有多重耐药性,并携带多种毒力基因,对细胞和大蜡螟幼虫表现出强毒性作用,说明该菌是引起病鸡死亡的主要致病菌之一,应引起重视。     

Characterization of a novel unique restriction-modification system from Yersinia enterocolitica O:8 1B

Genetic manipulations with enteropathogenic Yersinia enterocolitica O:8 are complicated by the presence of an efficient PstI-like YenI restriction-modification (R-M) system. We have characterized the YenI R-M system in Y. enterocolitica O:8, biotype 1B. A 5039 bp DNA fragment of the pSAK2 recombinant plasmid carrying the yenI locus was used to determine the nucleotide sequence. DNA sequence analysis identified a single 2481 bp open reading frame (ORF) that encodes an 826 amino acid large polypeptide having an apparent molecular mass of 93 kDa. The N-terminal part of the YenI ORF has 45 and 40% identity to PstI and BsuI methyltransferases (MTases), respectively; while the C-terminal part depicts 55 and 45% identity to endonucleases (ENases) of both isoschyzomeric enzymes. The yenI gene was cloned into pT7-5 plasmid and has been shown to encode a single polypeptide of expected molecular mass. A specific recognition sequence, typical to the type II R-M systems and single peptide organization, typical to type IV R-M systems, make YenI unique among known restriction-modification systems. We have constructed a truncated recombinant variant of YenI enzyme, which conserved only MTase activity, and that can be applied to YenI methylation of the DNA to be transformed into Y. enterocolitica O:8 biotype 1B strains.     

Structural genesvirC1 andvirC2 of the host range determiningvirC operon are determinants of virulence inAgrobacterium tumefaciens

The host range determiningvir C operon ofAgrobacterium tumefaciens is known to consist of two open rea’ding frames designatedvirC1 andvirC2. Earlier work that employed insertional mutations invirC1 andvirC2 established the role of thevirC2 component in the determination of virulence. In this work a plasmid with an internal deletion invirCl was constructed. This deletion derivative restored virulence to bacteria carrying a mutation in thevirC2 region but not to bacteria carrying avirC1 mutation. This evidence establishes that bothvirC1 andvirC2 are required for efficient host plant transformation byAgrobacterium tumefaciens.     

Nucleotide sequence of a new class A beta-lactamase gene from the chromosome of Yersinia enterocolitica: implications for the evolution of class A beta-lactamases

Summary The nucleotide sequence has been determined of a 1400 by fragment from the chromosome of Yersinia enterocolitica containing the gene for -lactamase I. An ORF of 882 by was identified, which could code for a polypeptide of 294 amino acids, closely related to other -lactamases of molecular class A. Amino acids 1–30 could constitute a signal peptide. The mature protein would be 264 amino acids long with a calculated pI of 6.2. Alignment of the amino acid sequence of the class A -lactamases suggested the existence of two subgroups in the same class, and this is discussed in the context of the evolution of the enzymes.This sequence will appear in the EMBL Data Library under the accession number X57074     

Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 μM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.     

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red

We determined and analyzed the Shigella flexneri serotype 5 (pSF5) and S. dysenteriae serotype 1 (pSD1) virulence plasmid genomes. The total length of pSF5 is 136513 bp, including 165 open reading frames (ORFs). Of these ORFs, 133 were identified and 32 of those had no significant homology to proteins with known functions. The length of pSD1 is 182545 bp, including 224 ORFs, of which we identified 181. The remaining 43 ORFs were not significantly homologous to proteins with known functions. The insertion sequence (IS) elements are 53787 bp in pSF5, and 49616 bp in pSD1, which represents 39.4% and 27.1% of the genome, respectively. There are 22 IS element types in pSF5 and pSD1, among which we report ISEc8 and ISSbo6 for the first time in the Shigella virulence plasmid. Compared to pCP301, there are a large number of deleted genes and gene inversions in both pSF5 and pSD1. The ipa-mxi-spa locus in pSF5 is completely absent, and the genes related to the O-antigen biosynthesis are partially missing. In contrast, the above genes in pSD1 are integral, with the exception of virF. The whole genome analysis of the two plasmids shows that the loss of genes related to gene invasion or regulation also obliterates the ability of pPF5 and pSD1 to bind Congo red (Crb). Whether these genes determine the Crb function requires continued investigation. These authors contributed equally to this work.     

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.     

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.     

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.     

Distribution of some virulence related-properties of Vibrioalginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibriocholerae virulence genes

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibrio cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.     

24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

[目的]旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估.[方法]利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化,挑选可疑菌落进行微生物质谱鉴定,进一步通过16S rRNA基因测序进行鉴定,16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析;同时,进行所有...