Simultaneous detection of Salmonella pathogenicity island 2 and its antibiotic resistance genes from seafood (original) (raw)
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Frontiers in Microbiology, 2019
Gastrointestinal illnesses continue to be a global public health risk. Exposure to foodborne Salmonella directly or indirectly through consumption of ready-to-eat seafood can be an important route of infection to humans. This study was designed to estimate the population cell density, prevalence, virulence gene signatures, and antibiotic resistance of Salmonella serovars from ready-to-eat shrimps. Ready-to-eat (RTE) shrimp samples were obtained from different open markets in Delta and Edo States, Nigeria from November 2016 to October 2017. We employed classical and polymerase chain reaction (PCR) approaches. The mean Salmonella species enumerated from the RTE shrimps ranged from −0.301 to 5.434 log 10 cfu/g with 210/1440 (14.58%) of the RTE shrimp samples harbored Salmonella species. After biochemical and PCR approach, the identified isolates were Salmonella Enteritidis 11(24.4%), Salmonella Typhimurium 14 (31.1%) and other Salmonella spp. 20 (44.4%). All Salmonella species recovered were resistant to penicillin and erythromycin with 100% sensitivity to cefotaxime, cephalothin, colistin, and polymyxin B. Findings on the multidrug-resistant (MDR) profile showed that a total of 9/14 (64.3%) of Salmonella Enteritidis were resistant to 5 antibiotics which belongs to 3 different groups of antimicrobials with a multiple antibiotic-resistant (MAR) index of 0.21; while 3/11 (27.3%) of Salmonella Typhimurium were resistant to 11 antibiotics which belongs to 7 different groups of antimicrobials with a MAR index of 0.46. Virulence genes (spiA, sipB, invA, sifA, fljB, and sefA) and resistance genes (class 1 and II integrase, sul2, catB3, flor, tmp, bla TEM , strB, dfr1, and tetC) were also detected in some of the Salmonella species with variable percentage. This study indicates that ready-to-eat shrimps are probable reservoirs harboring Salmonella strains. The identified Salmonella isolates which exhibited virulence determinants and antibiotic-resistant coupled with high MAR index constitute a consumer health risk to the communities.
Food Microbiology, 2011
A total of 39 Salmonella enterica serovar Saintpaul strains from imported seafood, pepper and from environmental and clinical samples were analyzed for the presence of virulence genes, antibiotic resistance, plasmid and plasmid replicon types. Pulsed-field gel electrophoresis (PFGE) fingerprinting using the XbaI restriction enzyme and plasmid profiling were performed to assess genetic diversity. None of the isolates showed resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Seventeen virulence genes were screened for by PCR. All strains were positive for 14 genes (spiA, sifA, invA, spaN, sopE, sipB, iroN, msgA, pagC, orgA, prgH, lpfC, sitC, and tolC) and negative for three genes (spvB, pefA, and cdtB). Twelve strains, including six from clinical samples and six from seafood, carried one or more plasmids. Large plasmids, sized greater than 50 kb were detected in one clinical and three food isolates. One plasmid was able to be typed as IncI1 by PCR-based replicon typing. There were 25 distinct PFGE-XbaI patterns, clustered to two groups. Cluster A, with 68.5% similarity mainly consists of clinical isolates, while Cluster C, with 67.6% similarity, mainly consisted of shrimp isolates from India. Our findings indicated the genetic diversity of S. Saintpaul in clinical samples, imported seafood, and the environment and that this serotype possesses several virulent genes and plasmids which can cause salmonellosis.
An eight-hour PCR-based technique for detection of Salmonella serovars in seafood
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 10 6 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 · 10 3 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.
Applied and Environmental Microbiology, 2009
A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.
Pathogens
The quantity of seafood imported and produced by domestic aquaculture farming has increased. Recently, it has been reported that multidrug-resistant (MDR) Salmonella Typhimurium may be associated with seafood. However, information is limited to the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium recovered from imported and domestic seafood. This study investigated the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium isolated from domestic and imported catfish, shrimp, and tilapia. A total of 127 isolates were tested for the presence of multidrug-resistance (MDR), virulence genes (invA, pagC, spvC, spvR), and genetic diversity using the Sensititre micro-broth dilution method, PCR, and pulsed-field gel electrophoresis (PFGE), respectively. All isolates were uniformly susceptible to six (amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, imipenem, nitrofurantoin, and trimethoprim/sulfamethoxazole) of the 1...
Molecular Characterization of Salmonella enterica and detection of antimicrobial resistance genes
Salmonella Typhimurium is an important nontyphoidal Salmonella serovar associated with foodborne diseases in many parts of the world. This organism is the major causative agent of nontyphoidal salmonellosis in Malaysia. We aimed to investigate the genetic profiles of the strains isolated from clinical, zoonotic, and dietary sources in Malaysia using multilocus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). By focusing on the 5 common variable number tandem repeat (VNTR) loci, we found that PFGE (D ＝ 0.99) was more discriminative than MLVA (D ＝ 0.76). The low MLVA score might be because of a lack of VNTR loci STTR6 (81.0z) and STTR10pl (76.2z). Both subtyping methods suggested that our S. Typhimurium strains were largely endemic with limited genetic variation. Furthermore, we observed that biphasic S. Typhimurium strains were dominant (99z) and multidrug resistance was prevalent (50z) within our sample pool. The most frequently observed phenotypes were resistance to compound sulfonamides (49z), tetracycline (51z), and streptomycin (52z). In this study, we documented the genetic relationship, antimicrobial resistance characteristics, and flagellar-phase dominance among S. Typhimurium strains found in Malaysia.
Veterinary World
Background and Aim: All serotypes of Salmonella enterica are considered potentially pathogenic. However, the non-typhoidal Salmonella (NTS) serotypes vary considerably in terms of pathogenicity and the severity of infections. Although diverse serotypes of NTS have been reported from tropical seafood, their sources, physiological characteristics, and virulence potentials are not well understood. This study aimed to compare the physiological characteristics of selected serovars of Salmonella from seafood and investigate possible variations in the distribution of known genes within the pathogenicity islands. Materials and Methods: A series of biochemical tests, including carbohydrate fermentation and amino acid decarboxylation tests were performed to physiologically compare the isolates. The genetic characterization with respect to putative virulence genes was done by screening for genes associated with Salmonella pathogenicity island (SPI) I– V, as well as the toxin- and prophage-asso...
Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens
Veterinary Microbiology, 2008
Aims: The aim of this research was to develop multiplex PCR assay that could simultaneously detect Salmonella genus, Salmonella subsp. I, Salm. Enteritidis, Heidelberg and Typhimurium because these Salmonella serovars are the most common isolates associated with poultry products. Methods and Results: Five primers were utilized to establish multiplex PCR and applied to Salmonella isolates from chickens and farm environments. These isolates were identified as Salmonella subsp. I and 16 of 66 isolates were classified as Salm. Enteritidis, while Heidelberg or Typhimurium was not detected. We also spiked three Salmonella strains on chicken breast meat to evaluate the specificity and sensitivity of multiplex PCR as well as qPCR to optimize quantification of Salmonella in these samples. The optimized multiplex PCR and qPCR could detect approx. 2Á2 CFU of Salmonella per gram after 18 h enrichment. Conclusions: The multiplex PCR and qPCR would provide rapid and consistent results. Also, these techniques would be useful for the detection and quantification of Salmonella in contaminated poultry, foods and environmental samples. Significance and Impact of the Study: The strategy for the rapid detection of Salmonella serovars in poultry is needed to further reduce the incidence of salmonellosis in humans. The optimized multiplex PCR will be useful to detect prevalent Salmonella serovars in poultry products.
Characterization of Multiple-Antimicrobial-Resistant Salmonella Serovars Isolated from Retail Meats
Applied and Environmental Microbiology, 2004
A total of 133 Salmonella isolates recovered from retail meats purchased in the United States and the People's Republic of China were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and horizontal transfer of characterized antimicrobial resistance determinants via conjugation. Seventy-three (82%) of these Salmonella isolates were resistant to at least one antimicrobial agent. Resistance to the following antibiotics was common among the United States isolates: tetracycline (68% of the isolates were resistant), streptomycin (61%), sulfamethoxazole (42%), and ampicillin (29%). Eight Salmonella isolates (6%) were resistant to ceftriaxone. Fourteen isolates (11%) from the People's Republic of China were resistant to nalidixic acid and displayed decreased susceptibility to ciprofloxacin. A total of 19 different antimicrobial resistance genes were identified in 30 multidrug-resistant Salmonella isolates. The bla CMY-2 gene, encoding a class A AmpC -lactamase, was detected in all 10 Salmonella isolates resistant to extended-spectrum -lactams. Resistance to ampicillin was most often associated with a TEM-1 family -lactamase gene. -IVa, were commonly present in the Salmonella isolates. Sixteen (54%) of 30 Salmonella isolates tested had integrons ranging in size from 0.75 to 2.7 kb. Conjugation studies demonstrated that there was plasmid-mediated transfer of genes encoding CMY-2 and TEM-1-like -lactamases. These data indicate that Salmonella isolates recovered from retail raw meats are commonly resistant to multiple antimicrobials, including those used for treating salmonellosis, such as ceftriaxone. Genes conferring antimicrobial resistance in Salmonella are often carried on integrons and plasmids and could be transmitted through conjugation. These mobile DNA elements have likely played an important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.
Veterinary World, 2020
Background and Aim: Salmonella spp. are one of the most important food-borne pathogens in the world, emerging as a major public health concern. Moreover, multidrug-resistant (MDR) strains have been isolated from salmonellosis outbreaks, which compromise its treatment success. This study was conducted to characterize the phenotypic and genotypic antibiotic resistance profile of Salmonella strains isolated from broilers and humans from the regions of Tolima and Santander (Colombia). Materials and Methods: Salmonella spp. strains (n=49) were confirmed through molecular detection by amplification of the invA gene. Phenotypic antibiotic resistance was determined by the automated method and the agar diffusion method, and the presence of resistance genes was evaluated by PCR. Genotypic characterization was conducted using the enterobacterial repetitive intergenic consensus (ERIC)-PCR method, from which a dendrogram was generated and the possible phylogenetic relationships were established. Results: Salmonella isolates were classified as MDR strains exhibiting resistance to four antibiotic classes, penicillins, aminoglycosides, sulfonamides, and cephalosporins, and the human strains were resistant to gentamicin. At the genotypic level, the isolates contained the genes blaCMY2, blaCTX-M, blaPSE-1, blaTEM, aadA1, srtB, dfrA1, sul2, and floR. The genotyping results obtained by ERIC-PCR allowed the grouping of strains according to the source of isolation. Conclusion: The Salmonella spp. strains exhibited resistance to multiple antibiotics, as well as multiple genes associated with them, and the ERIC-PCR method was a technique that was helpful in generating clusters with biological significance.