2011年江苏省东台市小肠结肠炎耶尔森菌监测分析

目的 了解江苏省东台市腹泻患者和不同宿主粪便小肠结肠炎耶尔森菌感染及其毒力基因携带情况。 方法 从所检标本中分离小肠结肠炎耶尔森菌,同时用聚合酶链反应方法检测分离菌株5种毒力基因携带状况。 结果 15种样品795份标本中,小肠结肠炎耶尔森菌检出率为3.77%,猪粪中的检出率最高,达22.50%;犬粪次之,为8.05%。猪粪中携带ail、 ystA、yadA 和virF 4种毒力基因的致病菌株检出率最高,均达12.50%;犬粪次之,分别为4.60%、4.60%、4.60%和4.60%。 结论 猪、犬是东台市小肠结肠炎耶尔森菌的重要携带者。     

Specific detection of plasmid bearing Yersinia isolates by PCR

A total of 210 isolates belonging to 9 different species of the genus Yersinia (Y.) was investigated with three different PCR assays targeting two plasmoidal genes, the Yersinia adhesin gene (yadA) and the V-antigen gene. The yadA PCR assay described in 1995 by Blais and Phillipe, targeting a Y. enterocolitica specific gene region and a newly designed assay targeting the gene region functionally responsible for autoagglutination, were compared. Both assays identified the same Y. enterocolitica strains. To exclude the possibility that false negative results were obtained due to mutations that had occurred in parallel in both gene regions, a third PCR assay by Neubauer et al. (2000) targeting a conserved region of the V-antigen gene was used as control. Again, DNA of the same Y. enterocolitica strains was amplified. In contrast to the yadA PCR assay described by Blais and Phillipe, the newly established yadA and the V-antigen PCR assays amplified DNA from Y. pseudotuberculosis strains. Therefore, by using the PCR technique as a molecular tool spontaneous mutations could be excluded as the cause of anomalous reactions in PCR assays targeting genes of the Yersinia virulence plasmid. Based on these results, it can be assumed that all presumptive pathogenic Yersinia isolates can be identified on the basis of PCR analysis. These molecular assays may also produce fewer false positive reactions in comparison to phenotypic tests such as the autoagglutination test which depend heavily on the handler's experience. It has to be stressed that the PCR assays used in this study have not been evaluated for routine use. Therefore, standardization of the PCR methodology including sample preparation, primer target sequences and PCR reagents is needed for the reliable and safe diagnosis of pathogenic Yersinia spp. in future.     

Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.     

Characterization of defective beta-lactamase genes in Yersinia enterocolitica

OBJECTIVES: To study at the molecular level the heterogeneity of expression of the two chromosomal beta-lactamases, BlaA and BlaB, in Yersinia enterocolitica strains isolated from clinical samples. METHODS: MIC determination by the agar dilution method and beta-lactamase assays was performed to determine the resistance level conferred by these enzymes. DNA cloning, PCR and direct sequencing were used to detect the presence of mutations. RESULTS: The blaA allele from strain IP97 (blaA97) was found to carry a deletion of 51 bp which entirely abolished its beta-lactamase activity. Both the ampR gene and the promoter region of strain Y56 were shown to be functional by a gene swapping experiment. The blaB allele from strain Y56 was found to carry two point mutations, only one of them resulting in a change in the amino acid sequence of the protein. This single amino acid change created a practically inactive BlaB or AmpC cephalosporinase in Y. enterocolitica Y56. CONCLUSIONS: The lack of activity observed in the beta-lactamases of some Y. enterocolitica isolates was due to the presence of point mutations or small deletions in the corresponding genes.     

Comparison of multiplex PCR,PCR-ELISA and fluorogenic 5' nuclease PCR assays for detection of plasmid-bearing virulent Yersinia enterocolitica in swine feces

Swine are implicated as the principal animal reservoir for plasmid-bearing Yersinia enterocolitica (YEP(+)) strains that are pathogenic to humans. To evaluate the utility of the PCR for detection of YEP(+) strains in naturally-contaminated pig feces, samples were first enriched in Irgasan ticarcillin potassium chlorate broth for 48 h at 25 degrees C and then tested by multiplex PCR, PCR-ELISA, and fluorogenic 5' nuclease PCR assays. Three different primer sets for amplification of the ail gene sequences were used in these three assays. Three out of 50 (6%) samples were positive for YEP(+) strains using the multiplex PCR targeting the chromosomal ail (170 bp) and plasmid virF (591 bp) genes. Two of the 3 samples positive by the multiplex PCR were also positive by the PCR-ELISA method using primers targeting the ail gene (425 bp). In contrast, the fluorogenic 5' nuclease PCR assay failed to detect an ail gene sequence (118 bp) in any of the 50 samples. These results indicate that the multiplex PCR was the most reliable and sensitive assay for detecting YEP(+) strains in feces among the three assays evaluated.     

Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization

A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.     

天津市蓟县小肠结肠炎耶尔森菌的带菌动物调查

目的 了解天津市蓟县动物宿主小肠结肠炎耶尔森菌的带菌情况,做好小肠结肠炎耶尔森菌感染的预防与控制工作.方法 2006年5～6月份在全县范围内开展易携带病原菌的猪、牛、羊、鸡等家禽家畜的监测工作.用冷增菌培养法进行分离培养.结果 从猪粪便中检出4株小肠结肠炎耶尔森菌,经聚合酶链反应(PCR)鉴定,其中3株菌株带有ail、ystA、yadA、virF 4种毒力基因,同时还具有O:3血清型特异性的rfbC基因,1株菌株带有ystB一种毒力基因.结论 猪为天津市该菌的动物宿主之一,猪粪可能成为食品污染的来源.     

Identification of invasive Yersinia species using oligonucleotide probes.

Oligonucleotide probes directed to the inv and ail invasion genes of Yersinia species were used to analyse yersiniae and non-yersiniae isolates by colony hybridization. The INV-3 probe, targeted to the inv gene of Yersinia pseudotuberculosis, hybridized with all 48 HeLa cell-invasive Y. pseudotuberculosis isolates examined; the PF-13 probe, specific for the ail gene of Yersinia enterocolitica, identified all invasive strains (36 of 52) of Y. enterocolitica tested. Neither probe hybridized with non-yersinia isolates or other Yersinia species. Southern analyses of restriction enzyme-digested genomic DNA confirmed the specificity of both probes. INV-3 hybridized with a 4.5 kilobase (kb) Bam HI fragment known to carry the inv gene in Y. pseudotuberculosis. PF-13 was specific for a 1.2 kb Cla I-Ava I fragment in Y. enterocolitica that carried the ail locus. Reactivity with either probe correlated closely with the ability of Y. pseudotuberculosis and Y. enterocolitica isolates to invade HeLa cells.     

Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica.

The nucleotide sequence of a 3.1-kb region from the chromosome of the Yersinia enterocolitica O:5b strain IP97 containing the gene for an inducible chromosomal cephalosporinase has been determined. The cephalosporinase gene was homologous to other enterobacterial chromosomal cephalosporinase genes, and it was accompanied by a gene homologous to the regulatory ampR gene. The arrangement of genes in the Y. enterocolitica ampCR unit was identical to that in the Enterobacter cloacae and Citrobacter freundii ampCR units.     

Tandem amplification of a 28-kilobase region from the Yersinia enterocolitica chromosome containing the blaA gene

Most Yersinia enterocolitica strains are resistant to beta-lactam antibiotics due to the production of one or two chromosomally encoded beta-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A beta-lactamase. To select mutants with increased levels of resistance to beta-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 micro g of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.     

聚合酶链反应法和分离培养法筛检小肠结肠炎耶尔森菌方法的比较研究

目的 比较针对小肠结肠炎耶尔森菌的两种不同检测方法,即ail基因联合foxA基因的聚合酶链反应(polymerase chain reaction,PCR)检测和分离培养法的差异。 方法 从生猪咽拭子和回盲部肠内容物标本中提取细菌基因组DNA并进行小肠结肠炎耶尔森菌保守基因foxA和粘附侵袭位点基因ail的PCR检测;菌株分离培养按常规方法进行。将两种方法的检出结果进行统计分析,以判定哪种更有优势。 结果 700份标本中,小肠结肠炎耶尔森菌特异性基因PCR检测阳性402份,阳性率57.43%;小肠结肠炎耶尔森菌分离培养阳性278份,阳性率39.71%。在PCR检测阳性的402份标本中,分离小肠结肠炎耶尔森菌271株,分离阳性率为67.41%;PCR检测阴性的298份标本中,分离小肠结肠炎耶尔森菌7株,分离阳性率仅为2.35%。 结论 本次实验证实,检测来源于生猪标本中的小肠结肠炎耶尔森菌,采用foxA和ail基因联合的PCR检测法,检出率明显高于小肠结肠炎耶尔森菌分离培养法(P0.05)。     

Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica

Streptogramins are polypeptide antibiotics inhibiting protein synthesis by the prokaryotic ribosome. Gram-positive organisms are susceptible to streptogramins, while most gram-negative bacteria are intrinsically resistant. We have found a genomic fragment from a Yersinia enterocolitica isolate with an open reading frame coding for a polypeptide similar to the virginiamycin acetyltransferases found in various plasmids from gram-positive bacteria. The susceptible Escherichia coli strain DB10 was transformed to resistance to the type A streptogramins and to mixed (A + B) streptogramins upon introduction of a plasmid containing that gene. In addition, we showed streptogramin acetylating activity in vitro dependent on the presence of the Y. enterocolitica sat gene. Southern blot hybridization experiments showed that the sat gene was present in all the Y. enterocolitica isolates examined. These data together show that the gene in the Y. enterocolitica chromosome encoded an active streptogramin acetyltransferase. The deduced sequence of the Y. enterocolitica Sat protein was close to those of sat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin.     

Sequence analysis of cfxA2-like beta-lactamases in Prevotella species

Sixty-two strains of oral (32) and non-oral (30) Prevotella producing beta-lactamases were screened for cfxA by PCR, using an intragenic primer pair. All 62 were cfxA/cfxA2 positive. Fourteen of these strains, representing seven pigmented and seven non-pigmented Prevotella species were submitted to further PCR with specific primers that amplified the whole beta-lactamase structural gene (966 bp). After cloning and sequencing, the deduced amino acid sequences were compared with that of Bacteroides vulgatus CfxA beta-lactamase. All 14 sequences possessed the E272K substitution characteristic of CfxA2. CfxA sensu stricto was not observed in the present series. G83D, F/V189L, W193L and D239Y substitutions were observed more than once, without species specificity. This sequence analysis indicates that most oral and non-oral beta-lactamase-producing Prevotella isolates from French patients produce variants of the CfxA enzyme.     

Sepsis associated with transfusion of red cells contaminated with Yersinia enterocolitica

Between April 1987 and May 1989, the Centers for Disease Control investigated seven cases of transfusion-associated Yersinia enterocolitica sepsis; four were caused by organisms of serotype O:3, and one each was caused by organisms of serotype O:1,2,3; O:5,27; and O:20. All seven recipients developed septic shock after receiving units of red cells (RBCs) contaminated with Y. enterocolitica; five recipients died. The cases occurred in seven states and were unrelated. There was no evidence for contamination of the RBC units during processing. Six of the seven donors had serologic evidence of recent Y. enterocolitica infection, and it is hypothesized that these donors had asymptomatic bacteremia when they donated the implicated blood. Four of the seven donors reported gastrointestinal illness in the 4 weeks before blood donation, and one donor became ill on the day he donated blood. Y. enterocolitica grows well at 4 degrees C and in the presence of dextrose and iron. If blood is contaminated at the time of collection, storage of the RBCs at 4 degrees C provides an ideal environment for bacterial growth and endotoxin production. These cases demonstrate the need for careful evaluation of patients with transfusion reactions for possible sepsis and suggest a need to screen prospective blood donors for mild gastrointestinal illness, including those illnesses not requiring physician evaluation or medication.     

云南部分鼠疫疫源地中小肠结肠炎耶尔森菌的分离及鉴定

目的 了解云南部分鼠疫疫源地中小肠结肠炎耶尔森菌的分布及病原学特征。方法 2012年5月至2014年4月采集剑川、梁河两县鼠类盲肠,对其进行培养,通过菌落聚合酶链反应初筛小肠结肠炎耶尔森菌,通过生化鉴定确定菌株并作毒力基因检测。结果 915份标本中分离到小肠结肠炎耶尔森菌35株,总检出率为3.83%,分离株包含1株致病株、34株非致病株。35株小肠结肠炎耶尔森菌全部为生物1A型,1株致病株为1A/O:9生物血清型,毒力基因为(ail+、ystA-、ystB-、yadA-、virF-、rbfc-),非致病株血清为O:5、O:8及未分型,毒力基因为(ail-、ystA-、ystB+、yadA-、virF-、rbfc-)。 结论 小肠结肠炎耶尔森菌在剑川野鼠鼠疫疫源地鼠间流行,分离菌株血清型别较为多样,分离到的致病株缺乏典型毒力基因;小肠结肠炎耶尔森菌在梁河家鼠鼠疫疫源地尚未发现鼠间的流行。     

Detection of OXA-4 beta-lactamase in Pseudomonas aeruginosa isolates by genetic methods

In Pseudomonas aeruginosa, resistance to cefclidin is usually associated with resistance to another third-generation cephalosporin, ceftazidime. In this study we analysed 22 isolates of P. aeruginosa, collected at Showa University Fujigaoka Hospital between 1992 and 1993, which were resistant to cefclidin but susceptible to ceftazidime. All polymerase chain reaction (PCR) products amplified by a primer pair covering the full-length gene of OXA-4 (also OXA-1) precursor beta-lactamase were 0.84 kb in length. The isoelectric points of the beta-lactamases produced by these isolates were typical of the OXA-4 type of beta-lactamase (pl 7.5) rather than the OXA-1 type (pl 7.4). All PCR products at 216 bp were amplified by the primer pair covering the A928-->T point mutation, which corresponds to the Asp48-->Val amino acid substitution of OXA-1 beta-lactamase to form OXA-4 beta-lactamase. These single-strand conformation polymorphism (SSCP) patterns are typical of the OXA-4 gene, rather than the OXA-1 gene, demonstrating that these enzymes can be classified by SSCP analyses based on the PCR method. Although OXA-4 beta-lactamase is generally plasmid-mediated, the chromosomal DNA of these isolates, but not their plasmids, hybridized with the OXA-4 gene amplified by the PCR method. Based on these results, we suspected that the plasmids encoding OXA-4 beta-lactamase had been spontaneously cured, or that the gene had been deleted from the plasmid. The distribution of P. aeruginosa producing OXA-4 beta-lactamase amongst hospital wards and clinical specimens demonstrated that the OXA-4 enzyme in this collection period was representative of hospital P. aeruginosa.     

Erg11基因突变与白念珠菌对唑类药物耐药性的初步研究

目的探讨Erg11基因突变与白念珠菌对唑类药物耐药的关系,以初步了解白念珠菌对唑类药物耐药的机制。方法从临床分离耐唑类药物的白念珠菌,通过PCR扩增Erg11基因并测序,与敏感白念珠菌进行对照,以确定是否发生基因突变。结果2株唑类药物敏感的白念珠菌中,通过Erg11基因测序,未发现有义突变;6株耐唑类药物的白念珠菌中,Erg11基因存在E266D、Y257H、K259E、A114S、F487L突变,其中A114S、Y257H、K259E、F487L为新发现的突变位点。结论白念珠菌对唑类药物耐药与Erg11基因的突变有关,通过Erg11基因突变,可引起Erg11P的蛋白结构发生改变,最终可导致唑类药物与Erg11P亲和力下降而产生耐药。     

Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples

The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were designed to target the nuclease (nuc) and the attachment invasion locus (ail) genes of S. aureus and Y. enterocolitica, respectively and used in PCR. A combination of organic solvents, detergents and alkali in the DNA extraction method permitted a detection limit of 10 cfu ml(-1) milk for both the pathogens. When equal numbers of S. aureus and Y. enterocolitica were spiked in milk samples, the individual detection limit was determined to be 10(3) cfu ml(-1) milk. Simultaneous amplification of 482 and 359 bp fragments of the nuc and ail genes was obtained using the primer pairs in a single reaction. Multiplex PCR enabled the detection of 10(4) cfu ml(-1) milk of S. aureus and Y. enterocolitica without any pre-enrichment step. A combination of conventional isolation technique and PCR using DNA extracted by the proposed method was used to test raw milk samples for possible contamination with S. aureus and Y. enterocolitica. The presence of S. aureus in the tested samples was indicated by both the methods while Y. enterocolitica could not be detected in any of the samples. The template DNA extraction method developed in this study is rapid, sensitive and avoids interference from potential PCR inhibitors and demonstrates the potential of detecting multiple pathogens in milk samples without any enrichment.     

Modeling the growth of Yersinia enterocolitica in donated blood

BACKGROUND: Sepsis and death subsequent to the transfusion of blood containing Yersinia enterocolitica is an increasing problem. The organisms probably originate from bacteremia in the donor and can subsequently multiply at low temperature. STUDY DESIGN AND METHODS: Reported here are experiments with a strain of Y. enterocolitica associated with a case of transfusion-associated bacteremia. RESULTS: It was found that the rapid early killing of Y. enterocolitica injected into donated blood does not require viable phagocytes and can be explained by complement-mediated killing. Complement resistance in Y. enterocolitica is known to be plasmid-coded. It is expressed at 37 degrees C, but not at 20 degrees C, and is favored by calcium-deficient culture media. Y. enterocolitica organisms induced to express complement resistance were still killed in donated blood, though the initial rate was slower. Such organisms multiplied in plasma at 37 degrees C, but were killed after 6 hours of incubation at 20 degrees C, presumably because complement resistance genes are switched off at this temperature. CONCLUSION: This experiment is thought to reflect the natural history of Y. enterocolitica contamination of blood, in which complement-resistant organisms in the donor blood encounter lower temperatures after donation. These observations suggest that the practice of plasma depletion may have contributed to the increased incidence of mortality due to Y. enterocolitica contamination of donated blood.     

Various applications of direct PCR using blood samples

Samples of blood or other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), meaning that isolation of DNA, involving multiple labor-intensive steps, is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances. Using this reagent cocktail, DNA from various targets can be efficiently amplified directly from various forms of blood samples without DNA isolation. 1. DNA sequences within the beta-globin gene could be amplified directly from human blood samples treated with various anticoagulants. Either fresh blood or blood samples stored frozen for up to 4 years could be used for PCR. 2. DNA sequences of up to 2056 bp within the beta-globin gene could be amplified directly from human blood samples. 3. Human chromosomal and mitochondrial DNA from different individuals could be amplified directly from blood samples. 4. Low titers of hepatitis B virus could be amplified directly from human blood samples. 5. DNA could be amplified directly from various target sequences using dried blood in a PCR tube or on a filter paper. 6. Transgenes could be detected directly in blood samples from transgenic mice.