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1.
The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.  相似文献   

2.

Background  

Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.  相似文献   

3.
Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.  相似文献   

4.
The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg μl−1. This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.  相似文献   

5.
Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.  相似文献   

6.
A rapid and sensitive 8-h PCR assay has been developed for detection of Salmonella serovars in seafood. A total of 110 fresh and raw seafood samples were analysed for the presence of Salmonella using different enrichment periods prior to PCR assay. Seafood samples included in this study were fish, shrimps, mussels, crabs, edible oysters, and clams, collected from local fish markets in Cochin (India). The assay was performed with a Salmonella-specific 284 bp invA gene amplicon. Specificity and sensitivity of the assay were ascertained with seafoods spiked with viable Salmonella cells to a level of 106 to 2 CFU per 25 g. Detection efficiency of the assay increased with increasing enrichment period for seafood, and 33.6% of seafood samples were found positive for Salmonella by 8-h PCR assay. Detection limit for the 8-h PCR assay showed visible 284 bp amplicon from seafood homogenates spiked with 2 CFU per 25 g. Seafood samples spiked with different Salmonella serovars, namely Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis, Salmonella mbandka, Salmonella bareilly, and Salmonella weltevreden, were detected, confirming this technique would be ideal for detection of the Salmonella serovars prevalent in seafood. This study also covered inhibition by the seafood matrix and the detection limit for dead Salmonella cells during the PCR assay. There was no visible inhibition of this Salmonella PCR assay by seafood matrices. The detection limit for dead Salmonella cells by 8-h PCR assay was 2 × 103 CFU per 25 g seafood. The data indicated that dead cells of Salmonella in naturally contaminated seafood samples do not interfere with the assay resulting in false positives.  相似文献   

7.
Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.  相似文献   

8.
Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.  相似文献   

9.
Fifteen strains of Salmonella were isolated from children with clinically diagnosed diarrhoea aged below 3 years old, who had been admitted to K7 ward, Pediatric Institute, Kuala Lumpur Hospital. The isolates were tested for their susceptibility to a range of antimicrobial agents, and typed by serological tests and randomly amplified polymorphic DNA (RAPD) fingerprinting. All the strains had a similar pattern of antimicrobial susceptibility, where they were susceptible to a wide range of antimicrobial agents. The serological test has typed them into three serovars, which were identified as Salmonella enterica ser. Akanji, Salmonella enterica ser. Hindmarch and Salmonella enterica ser. Richmond. In contrast, the RAPD fingerprinting classed them into two major clusters, cluster 1 consisting of 12 strains of Salmonella and cluster 2 consisting of three strains of Salmonella.  相似文献   

10.
We report sequencing of the O antigen encoded by the rfb gene cluster of Salmonella enterica serotype Jangwani (O17) and Salmonella serotype Cerro (O18). We developed serogroup O17- and O18-specific PCR assays based on rfb gene targets and found them to be sensitive and specific for rapid identification of Salmonella serogroups O17 and O18.  相似文献   

11.
Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N6(GTG)4, N6(CAC)4, N6(CGG)4, N6(CCG)4 and N6(CTG)4, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N6(GTG)4 primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.  相似文献   

12.
【目的】研究鼠伤寒沙门菌致病岛1(SPI-1)内部的假定调控蛋白STM14_3514的功能及其作用机制。【方法】以鼠伤寒沙门菌模式菌株ATCC 14028为亲本株,构建了STM14_3514基因的缺失突变体及互补菌株,通过小鼠实验、细胞侵袭实验、Western blot及实时荧光定量PCR(q RT-PCR)等实验技术,深入研究了STM14_3514基因对鼠伤寒沙门菌致病过程的影响。【结果】STM14_3514突变提高了细菌对小鼠的致病能力,突变体在小鼠肠道、肝和脾中的定殖能力均增强;细胞实验揭示,突变体致病力提升主要由于STM14_3514突变能显著增强细菌对上皮细胞的侵袭力(2倍,P0.05)。q RT-PCR及Western blot分析表明,STM14_3514显著抑制SPI-1内部主要调控因子hil A及侵袭相关基因的表达。此外,STM14_3514对hil A的抑制由Hil C介导。【结论】STM14_3514是鼠伤寒沙门菌SPI-1内部的负调控因子,能通过Hil C抑制hil A及SPI-1其他入侵基因的表达,该基因的生物学意义可能与细菌进入细胞后对SPI-1的负调控相关。  相似文献   

13.
A total of 748 Salmonella strains (97 serovars) isolated from human (291), animal (119), environmental (141), food (102) and animal feed (95) sources were examined for resistance to 9 antimicrobial agents. Most of the human isolates were from hospitalized patients (282). An overall resistance rate of 98.8% was determined with 100% for human and environmental isolates. Resistance to sulfadiazine (87.7%) was most common, followed by streptomycin (61.2%), ampicillin (39%) and trimethoprim-sulphamethoxazole (37.9%). Fifty one different resistance patterns were identified with Su (164 strains), Su-Sm (122) and Su-Sm-Tc-Cm-Km-Ap-Nx-Gm-Tm (95) predominating, the latter occurring only in human isolates. Multiple resistance was most frequently found among human isolates, particularly in S. derby and S. typhimurium strains. The relationship between antibiotic resistance, serovar and source of isolation of the Salmonella strains is discussed.  相似文献   

14.
Members of Salmonella enterica are important foodborne pathogens of significant public health concern worldwide. This study aimed to determine a range of virulence genes among typhoidal (S. typhi) and non-typhoidal (S. enteritidis) strains isolated from different geographical regions and different years. A total of 87 S. typhi and 94 S. enteritidis strains were tested for presence of 22 virulence genes by employing multiplex PCR and the genetic relatedness of these strains was further characterized by REP-PCR. In S. typhi, invA, prgH, sifA, spiC, sopB, iroN, sitC, misL, pipD, cdtB, and orfL were present in all the strains, while sopE, agfC, agfA, sefC, mgtC, and sefD were present in 98.8, 97.7, 90.8, 87.4, 87.4 and 17.2 %, of the strains, respectively. No lpfA, lpfC, pefA, spvB, or spvC was detected. Meanwhile, in S. enteritidis, 15 genes, agfA, agfC, invA, lpfA, lpfC, sefD, prgH, spiC, sopB, sopE, iroN, sitC, misL, pipD, and orfL were found in all S. enteritidis strains 100 %, followed by sifA and spvC 98.9 %, pefA, spvB and mgtC 97.8 %, and sefC 90.4 %. cdtB was absent from all S. enteritidis strains tested. REP-PCR subtyped S. typhi strains into 18 REP-types and concurred with the virulotyping results in grouping the strains, while in S. enteritidis, REP-PCR subtyped the strains into eight profiles and they were poorly distinguishable between human and animal origins. The study showed that S. typhi and S. enteritidis contain a range of virulence factors associated with pathogenesis. Virulotyping is a rapid screening method to identify and profile virulence genes in Salmonella strains, and improve an understanding of potential risk for human and animal infections.  相似文献   

15.
In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR technology in tandem with an adapted two-step enrichment protocol were assessed and validated against a reference culture method, ISO 6579, 2004. The DNA and RNA real-time PCR assays amplified a 270 bp region of the hilA gene of Salmonella enterica serovars, and incorporated an internal amplification control (IAC) which was co-amplified with the hilA gene to monitor potential PCR inhibitors and ensure successful amplification. The inclusivity and exclusivity of the hilA primer set was examined for both the DNA and RNA methods and detected the 30 S. enterica serovars but not the 30 non-salmonellae strains. The inoculation of meat carcass swabs with five different S. enterica serovars at five different inocula, indicated both PCR methods were able to detect between 1 and 10 CFU per carcass swab. The real-time DNA PCR assay performed as well as the traditional cultural method in detecting Salmonella sp. in artificially contaminated salad, chocolate, fish and cheese samples. The relative accuracy, relative sensitivity and relative specificity of the DNA PCR real-time method were determined to be 98.5, 98.1 and 100%, respectively. The DNA method was further validated in a collaborative inter-laboratory trial according to ISO 16140, 2003. The validated methods provide an accurate means for the rapid detection and tracking of S. enterica serovars giving equivalent results to the standard method within three days, thus providing an alternative testing method to the reference microbiological method. The real-time PCR methodology not only offers significant time-saving advantages compared to traditional methods, it can also be applied to a wide range of samples types.  相似文献   

16.
The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C1. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.  相似文献   

17.
In seawater, enteric bacteria evolve toward a stressed state that is difficult to identify because of major alterations of their phenotype. In this study, we incubated four reference strains of S. enterica serovar Typhimurium in seawater microcosms for 10 months and studied the modifications of their main phenotypic characters. All of the strains lost some key characters used for traditional identification of the Salmonella genus. They became able to produce acetoin, and tryptophane deaminase activity became positive, but they lost the capacity to use rhamnose. We were able to show some modifications of the level of enzymatic profile as well as in their antibiotic susceptibility. The atypical cells of S. enterica serovar Typhimurium were identified by polymerase chain reaction (PCR) methods using the internal transcribed spacer region, and they were confirmed by multiplex PCR after the simultaneous amplification of the phoP, Hin, and H-li genes.  相似文献   

18.
Salmonella enterica and its serovars have been associated with pathogen contamination of tomatoes with numerous outbreaks of salmonellosis. To improve food safety, pathogen control is of immediate concern. The aim of this research was to assess the populations of natural microflora (aerobic mesophilic bacteria, lactic acid bacteria, yeasts and moulds and Pseudomonas species) on tomatoes, and evaluate the efficacy of Pseudomonas fluorescens (Pf) and Pseudomonas chlororaphis (Pc) for inactivation of Salmonella on tomatoes. Microflora were determined on sanitised and unsanitised produce and enumerated on Plate Count Agar, de Man, Rogosa and Sharpe medium, Potato Dextrose Agar and Pseudomonas Agar F media. The efficacy of Pc and Pf for inactivation of S. enterica serovars Montevideo, Typhimurium and Poona was determined on spot-inoculated tomato stem scars. The effects of storage time on bacterial populations were also investigated. On unsanitised tomatoes, lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria and yeasts and moulds ranged from 3.31–4.84, 3.93–4.77, 4.09–4.80 and 3.83–4.67 log CFU/g of produce, respectively. The microflora were similar at 0 and 24 storage hours on sanitised produce. The suppression of Salmonella Montevideo by P. chlororaphis and P. fluorescens on tomatoes ranged from 0.51 to 2.00 log CFU/g of produce. On Salmonella Montevideo and S. Typhimurium, the suppressive effects ranged from 0.51 to 0.95 and 0.46 to 2.00 log CFU/g of produce, respectively. The pathogen suppressive effects may be attributed to competition ability of Pseudomonas relative to Salmonella strains. Pseudomonas strains may be effective against Salmonella strains as a post-harvest application, but strain synergy is required to optimise pathogen reductions.  相似文献   

19.
In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.  相似文献   

20.
Herpetofaunal Salmonella enterica serovars have not been fully examined in any U.S. region. Thirty-three Salmonella serovars were isolated from 156 samples from 34 species, all within Indiana County, Pennsylvania. Results suggest that herpetofaunas could potentially pose a threat to humans. Further understanding of Salmonella in herpetofaunas may prevent future human cases.  相似文献   

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