fljB Genes Research Articles

Development and application of a competitive ELISA for the detection of antibodies against Salmonella Abortusequi in equids.

The high abortion rate associated with Salmonella Abortusequi (S. Abortusequi) infection in equids has re-emerged over the past 10 years and has caused serious economic losses to China. Our previous studies showed that the flagellin FljB gene could distinguish S. Abortusequi from most Salmonella serotypes. In this study, the flagellin antigen was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) that could be used to detect both horse and donkey serum samples using a monoclonal antibody (MAb) that was found to bind to FljB. A cELISA was established using the purified MAb coating of the plate and incubation of the mixture of horseradish peroxidase (HRP)-conjugated FljB antigen with the undiluted serum sample. The performance of the cELISA and the tube agglutination test (TAT) assay was compared with respect to sensitivity and specificity, by testing a panel containing 660 S. Abortusequi-positive and 515 S. Abortusequi-negative serum samples, all of which had been characterized by Western blotting. Receiver operator characteristic (ROC) analyses were performed to determine the cutoff value and estimate the detection specificity (Sp) and sensitivity (Se). ROC analysis showed that the area under the ROC curve (AUC) values of cELISA [AUC = 0.9941; 95% confidence interval (CI), 0.9898-0.9984] were higher than those of TAT (AUC = 0.7705; 95% Cl, 0.7437-0.7972). A cutoff value of 39.5% was selected with Sp and Se values of 100 (95% Cl, 99.26-100.00) and 97.58 (95% Cl, 96.10-98.50), respectively. The cELISA has excellent futures compared with TAT, such as shortened detection time, no need for pre-treatment of sera, and easy interpretation of the results, and is more suitable for disease surveillance.

Prevalence of Salmonella Typhimurium and Salmonella Enteritidis isolated from poultry meat: virulence and antimicrobial-resistant genes

Salmonellosis, a zoonotic disease, is one of the leading causes of foodborne illness worldwide. It is responsible for most infections caused by consumption of contaminated food. In recent years, a significant increase in the resistance of these bacteria to common antibiotics has been observed, posing a serious threat to global public health. The aim of this study was to investigate the prevalence of virulent antibiotic-resistant Salmonella spp. strains in Iranian poultry markets. A total of 440 chicken meat samples were randomly selected from meat supply and distribution facilities in Shahrekord and tested for bacteriological contamination. After culturing and isolating the strains, identification was performed using the classical bacteriological method and PCR. To determine antibiotic resistance, a disc diffusion test was performed according to the recommendations of the French Society of Microbiology. PCR was used to detect resistance and virulence genes. Only 9% of the samples were positive for Salmonella. These were Salmonella typhimurium isolates. All Salmonella typhimurium serotypes tested positive for the rfbJ, fljB, invA and fliC genes. Resistance to TET, cotrimoxazole, NA, NIT, piperacillin/tazobactam and other antibiotics was found in 26 (72.2%), 24 (66.7%), 22 (61.1%) and 21 (58.3%) isolates, respectively. The sul1, sul2 and sul3 genes were present in 20, 12 and 4 of 24 cotrimoxazole-resistant bacteria, respectively. Chloramphenicol resistance was found in six isolates, but more isolates tested positive for the floR and cat two genes. In contrast, 2 (33%) of the cat three genes, 3 (50%) of the cmlA genes and 2 (34%) of the cmlB genes were all positive. The results of this investigation showed that Salmonella typhimurium is the most common serotype of the bacterium. This means that most of the antibiotics commonly used in the livestock and poultry industries are ineffective against most Salmonella isolates, which is important for public health.

Potential of a novel flagellin epitope as a broad-spectrum vaccine candidate against enteric fever.

Relentless emergence of antibiotic resistant Salmonella strains, coupled with the drawbacks associated with currently available vaccines against enteric fever, warrants an urgent need to look for new vaccine candidates. Out of the multiple virulence factors harbored by Salmonella, flagella are regarded as one of the most important targets of innate as well as adaptive immune response. Individual Salmonella serotypes alternate between expression of two different antigenic forms encoded by fliC and fljB genes, respectively thereby employing this as a strategy to escape the host immune response. In the present study, using various immunoinformatic approaches, a flagellin epitope, present in both antigenic forms of typhoidal Salmonellae has been targeted. Following B-cell epitope and B-cell derived T-cell epitope prediction and interaction studies with major histocompatibility complexes using molecular docking, a peptide epitope was selected. Further, it was screened for its presence in majority of typhoidal serovars along with other useful attributes, in silico. Thereafter, safety studies were performed with the synthesized peptide. Subsequently, immunization studies were carried out using S. Typhi as well as S. Paratyphi A induced murine peritonitis model. Active immunization with peptide-BSA conjugate resulted in 75% and 80% mice survival following lethal challenge with S. Typhi and S. Paratyphi A respectively, along with a significant IgG antibody titer, thereby highlighting its immunogenic potential. Reduced bacterial burden in vital organs along with improved histoarchitecture and cytokine levels further substantiated the protective efficacy of the proposed candidate. Passive immunization studies with the candidate verified the protective efficacy of the generated antibodies against lethal challenge of bacteria in mice. Given the endemic nature of enteric fever and the antigenic variability observed in Salmonella serotypes, present study highlights the importance of using a vaccine candidate, which, along with generating a strong immune response, also exhibits a broad coverage against both, S. Typhi as well as S. Paratyphi A strains.

Development and application of a multiplex PCR method to differentiate Salmonella enterica serovar Typhimurium from its monophasic variants in pig farms

Salmonella enterica serovar Typhimurium monophasic variants (Salmonella 4,[5],12:i:-) has increased dramatically, causing human salmonellosis and colonization in pigs. With a difference to S. Typhimurium, the monophasic variants of S. Typhimurium lose the gene cassettes encoding the second phase flagellin. To establish a rapid method to detect and differentiate the two serotypes, we analyzed the published 679 genomes of S. Typhimurium and its monophasic variants and found that no Salmonella 4,[5],12:i:- strains carry both fljB and hin genes. Therefore, we established a novel multiplex PCR method using the fljB-hin region and mdh gene as target sequences to detect and differentiate both serotypes. This method can be used to specifically detect both serotypes with a detection limit for DNA concentration at 10 pg/μL. In addition, the PCR assay successfully differentiated 36 S. Typhimurium isolates from 62 isolates of monophasic variants preserved in our laboratory from 2009 to 2017, which corresponds to the whole-genome-based serotyping results. Application of the multiplex PCR method to 60 fecal samples from a pig farm identified 11.7% (7/60) of S. Typhimurium monophasic variants, which is consistent with the whole-genome-based serotyping results. The multiplex PCR assay is a rapid and precise method for the detection of S. Typhimurium monophasic variants from samples across food production chains.

Genomic Characterization of Salmonella Typhimurium Isolated from Guinea Pigs with Salmonellosis in Lima, Peru.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is one of the most important foodborne pathogens that infect humans globally. The gastrointestinal tracts of animals like pigs, poultry or cattle are the main reservoirs of Salmonella serotypes. Guinea pig meat is an important protein source for Andean countries, but this animal is commonly infected by S. Typhimurium, producing high mortality rates and generating economic losses. Despite its impact on human health, food security, and economy, there is no genomic information about the S. Typhimurium responsible for the guinea pig infections in Peru. Here, we sequence and characterize 11 S. Typhimurium genomes isolated from guinea pigs from four farms in Lima-Peru. We were able to identify two genetic clusters (HC100_9460 and HC100_9757) distinguishable at the H100 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing (HierCC-cgMLST) scheme with an average of 608 SNPs of distance. All sequences belonged to sequence type 19 (ST19) and HC100_9460 isolates were typed in silico as monophasic variants (1,4,[5],12:i:-) lacking the fljA and fljB genes. Phylogenomic analysis showed that human isolates from Peru were located within the same genetic clusters as guinea pig isolates, suggesting that these lineages can infect both hosts. We identified a genetic antimicrobial resistance cassette carrying the ant(3)-Ia, dfrA15, qacE, and sul1 genes associated with transposons TnAs3 and IS21 within an IncI1 plasmid in one guinea pig isolate, while antimicrobial resistance genes (ARGs) for β-lactam (blaCTX-M-65) and colistin (mcr-1) resistance were detected in Peruvian human-derived isolates. The presence of a virulence plasmid highly similar to the pSLT plasmid (LT2 reference strain) containing the spvRABCD operon was found in all guinea pig isolates. Finally, seven phage sequences (STGP_Φ1 to STGP_Φ7) were identified in guinea pig isolates, distributed according to the genetic lineage (H50 clusters level) and forming part of the specific gene content of each cluster. This study presents, for the first time, the genomic characteristics of S. Typhimurium isolated from guinea pigs in South America, showing particular diversity and genetic elements (plasmids and prophages) that require special attention and also broader studies in different periods of time and locations to determine their impact on human health.

Identification of a Recently Dominant Sublineage in Salmonella 4,[5],12:i:- Sequence Type 34 Isolated From Food Animals in Japan

Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) and its monophasic variant (Salmonella 4,[5],12:i:-) are among the most frequently isolated clones from both humans and animals worldwide. Our previous study demonstrated that Salmonella Typhimurium/4,[5],12:i:- strains isolated in Japan could be classified into nine clades and that clade 9 consisted of ST34 strains. In Japan, ST34/clade 9 was first found in the 1990s and has become predominant among food animals in recent years. In the present study, we analyzed the whole genome-based phylogenetic relationships and temporal information of 214 Salmonella Typhimurium/4,[5],12:i:- ST34/clade 9 strains isolated from 1998 to 2017 in Japan. The 214 strains were classified into two sublineages: the newly identified clade 9–2 diverged from clade 9 in the early 2000s and has predominated in recent years. Clonally expanding subclades in clades 9–1 or 9–2 lacked Gifsy-1 or HP1 prophages, respectively, and some strains in these subclades acquired plasmids encoding antimicrobial resistance genes. Additional genome reduction around the fljB gene encoding the phase 2-H antigen was generated by an IS26-mediated deletion adjacent to the transposon in clade 9–2. Although most of the clade 9 strains were isolated from cattle in Japan, the clonally expanding subclades in clade 9–2 (i.e., all and 24% strains of subclades 9–2a and 9–2b, respectively) were isolated from swine. The spread of clade 9 in recent years among food animals in Japan was responsible for the emergence of multiple host-adapted sublineages involving the clonally expanding subclades generated by mobile genetic element-mediated microevolution.

Environmental persistence and virulence of Salmonella spp. Isolated from a poultry slaughterhouse.

Salmonella spp. is responsible for severe foodborne disease, and is one of the main agents involved in foodborne outbreaks worldwide. Contamination occurs mainly as a result of poultry and egg consumption since they can carry some serotypes pathogenic to humans. The aim of the study was to evaluate the persistence and pathogenic potential of Salmonella spp. (n=40) isolated from poultry slaughterhouse mats, using adhesion and invasion assays, antimicrobial susceptibility by disc diffusion, and biofilm production as phenotypic tests and genotypic analyses. Polystyrene mats presented 3.2 times greater chance of isolating Salmonella than canvas mats. Besides, we observed resistance to tetracycline (17.5%), ampicillin (10%), cefotaxime (7.5%), trimethoprim-sulfamethoxazole (5%), and chloramphenicol (2.5%). All strains possessed the invA, sipB, sipD, ssaR, sifA, sitC, iroN, tolC, flgK, fljB, and flgL genes. The genes sopB and sipA were both present in 92.5% of the isolates, while sopD and spvB were observed in 90% and 32.5% of strains, respectively. All strains adhered to and invaded HeLa cells. Regarding biofilm production, 31 (77.5%) strains were able to produce biofilm on polystyrene microplates. Using PFGE, we detected the persistence of clones in the environment for up to 18 fromthe 20weeks. The ability of these strains to produce a biofilm and thus persist in the environment and disperse through contact surfaces in the processing plant favors the contamination of food, aggravated by the pathogenic potential of these isolates demonstrated by their adhesion capacity, invasion and resistance to various antibiotic agents.

Abstract 4782: STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment

Abstract We have engineered a tumor-targeted, microbial immunotherapy platform called STACT (Salmonella Typhimurium (Attenuated) and Checkpoint Therapy). The platform strain has been engineered to increase tumor-specific enrichment, reduce immunosuppressive inflammation and enhance tolerability. Upon phagocytosis by tumor-resident immune cells, the microbe delivers plasmid DNA, which can either encode inhibitory microRNAs to knockdown immune targets of interest, or encode immuno-modulatory cDNA expression cassettes, alone or in specific combinations. The STACT platform strain is over 10,000-fold attenuated for virulence due to disruptions in the msbB and purI genes, that result in the detoxification of the surface LPS and purine/adenosine auxotrophy, respectively. The STACT platform strain required addition of exogenous adenosine or purines at concentrations found in the tumor micro-environment, but not in healthy tissue for replication in vitro. The STACT platform strain was unable to replicate significantly in infected macrophage cell lines, but was able to colonize tumors in mice and deliver functional plasmids. This strain was further engineered by precise genome deletions of the fljB and fliC genes, encoding the bacterial flagellar subunits that are strong TLR5 agonists and induce inflammasome-mediated pyroptosis in macrophages. Deletion of the flagellin genes prevented bacterial cell motility but did not affect tumor-specific colonization after IV administration, and tumor enrichment was observed at levels over 100,000 times greater than in spleens. A plasmid maintenance system was engineered by deletion of the chromosomal asd gene and complementation of the asd gene on a copy-number optimized plasmid to ensure plasmid maintenance in vivo. The asd system allowed for significantly improved plasmid retention in vivo without antibiotic selection. Furthermore, the vector incorporates immunostimulatory CpG motifs into the strain to help promote a TLR9-mediated adaptive immune response to tumor antigens. A library of inhibitory RNAi’s against a set of immuno-modulatory targets, including TREX1, PD-L1, and TGF-beta were screened for optimal knockdown of gene expression by qPCR and western blot. Pairwise combinatorial knockdown of specific targets was also observed in human cells. IV administration of STACT encoding TGF-beta RNAi demonstrated significant tumor growth inhibition in a subcutaneous tumor model. STACT is a highly attenuated microbial immunotherapy platform engineered to deliver immunomodulatory molecules to phagocytic cells in the tumor microenvironment after systemic administration. This platform can be engineered to knock down combinations of immune checkpoints or express immunostimulatory genes in a single therapeutic modality. Citation Format: Chris S. Rae, Marina Besprozvannaya, John Faulhaber, Anastasia M. Makarova, Beverly King, Laura Hix Glickman, Christopher D. Thanos, Justin Skoble. STACT: A novel tumor-targeting, systemically-administered delivery platform capable of targeting intractable pathways and precise immunomodulation of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4782.

Detection and characterization of multidrug-resistant Salmonella enterica serovar Infantis as an emerging threat in poultry farms of Faisalabad, Pakistan.

The aim of this study was the molecular identification of Salmonella enterica serovar Infantis (S. Infantis) isolated from poultry samples and their antimicrobial resistance and virulence profiling. A total of 149 isolates, belonging to genus Salmonella, originally isolated from 340 suspected poultry post mortem specimens reported by us earlier were preliminary identified as Salmonella by biochemical methods and confirmed by polymerase chain reaction targeting genus-specific gene invA. Targeting serovar-specific gene fragment (fljB) resulted in confirmation of 54 isolates as S. Infantis which were further confirmed by sequencing of 16S RNA and fljB genes. Swimming and swarming motilities were detected in 98·1 and 11·1% isolates respectively. Phenotypic disc diffusion assay against 23 antimicrobial agents showed the highest resistance against pefloxacin (PEF) (94·4%), chloramphenicol (83·3%) and imipenem (77·7%) while 5·3% isolates showed extended-spectrum β-lactamase production. Fifty-nine genes reported for antimicrobial resistance and 12 for conferring virulence were targeted. The most prevalent resistance gene for aminoglycosides was aadA (42·3%), for quinolone resistance determining region parE (62·5%), for penicillin's Int1 (62·9%), for chloramphenicol cat3 (66·1%) and for beta-lactams blaTEM -1 (44·4%). Among efflux pump coding genes, armA showed highest (74·2%) prevalence and for virulence, a high prevalence of SopE (89·2%) showed the zoonotic potential of the isolates. The activity of efflux pumps was detected through Ethidium Bromide-agar method. Poultry could act as reservoirs of multidrug resistance Salmonella. We firstly report the prevalence and molecular characterization of virulence/drug resistance in S. Infantis from this region and the results may contribute to designing precisely targeted therapy. This study has also highlighted the possible emergence of S. Infantis with zoonotic potential.

Salmonella Typhimurium in Iran: Contribution of molecular and IS200 PCR methods in variants detection.

Salmonella Typhimurium, a zoonotic pathogen, is regarded as a major health and economic concern worldwide. Recently, monophasic variants of this serovar have been significantly associated with human gastroenteritis outbreaks globally, making its accurate identification essential for epidemiological and control purposes. We have identified and analyzed 150 S. Typhimurium from 884 Salmonella genus isolated from humans, domestic animals, poultry, food items and abattoirs origins. The Salmonella isolates were obtained from Iranian National Veterinary Reference Laboratories of 9 provinces during 2007–2016, and from five hospitals in Tehran in 2015. The isolates were evaluated biochemically, serologically, and by PCR amplification of invA, mdh, STM4492, fliC, fljA, fljB, hin genes, IS200 and DT104. invA and mdh genes were used to confirm the S. Typhimurium serotype, fliC and fljB genes for determination of monophasic variants and amplification of IS200 to discriminate the monophasic variants from the closely related serotypes. We identified 78.6% (118/150) as classical S. Typhimurium (fliC, fljB and IS200 positive), 12.6% (19/150) were IS200 negative from all isolates. DT104 is another marker for S.Typhimurium serovar typing. Contrary to EFSA guidelines 20.6% (19/29) of human isolates that lacked IS200 insertion sequence, were confirmed as S.Typhimurium. Compared to the North American/European isolates the low prevalence of fljB negative 6% (9/150) and the high abundance of fliC negative 23.3% (35/150) isolates also were indicative of a different regional atypical population. Studies have shown that the prevalence of monophasic (fljB-) S. Typhimurium worldwide is promoted by the Swine industry. Thus, one reason for this high number of different atypical strains could be inhibition of swine breeding system (house hold and industry) in Iran. These results demonstrate a need for a modified identifying protocol to overcome the regional differences.

Distribution of virulence factors, determinants of antibiotic resistance and molecular fingerprinting of Salmonella species isolated from cattle and beef samples: suggestive evidence of animal-to-meat contamination

In this study, three hundred presumptive Salmonella strains isolated from cattle faeces and raw beef samples were subjected to both preliminary and confirmatory tests specific for Salmonella. PCR assays revealed that 100%, 20% and 26.7% of the isolates were positive for 16S rRNA, fliC and fljB gene fragments, respectively. Large proportions (62.4 to 94.3%) of these isolates were multiple antibiotic resistant (MAR) strains that were resistant to three or more antibiotics belonging to different classes. MAR phenotypes Ab1, Ab2, Ab3, Ab7, Ab8, Ab9, Ab26 and Ab27 were dominant among the isolates. Cluster analysis of antibiotic inhibition zone diameter data revealed two major clusters (clusters 1 and 2), and each cluster contained two sub-clusters (1A, 1B, 2A and 2B). PCR data revealed that 27.1% and 30.7% of the isolates possessed the spvC and invA virulent genes, respectively. There was a significant correlation between the possession of MAR phenotypes and virulent gene determinants. Analysis of restriction fragment length polymorphism (RFLP) of 16S rRNA gene fragments using EcoRI and HaeIII showed that large proportions of isolates from beef and cattle faeces produced similar genetic fingerprints. From these results, it is suggested that Salmonella species in cattle are transmitted to beef and, therefore, the consumption of undercooked beef could pose severe health complications on consumers. These findings provide baseline data that could be of great epidemiological importance and, thus, the need to utilise more sensitive typing tools in determining the genetic relatedness of isolates from different sources.

Rapid molecular identification and differentiation of common Salmonella serovars isolated from poultry, domestic animals and foodstuff using multiplex PCR assay.

Salmonella is widely distributed throughout the world and can be found in poultry industry, animal breeding centers, food and feedstuffs of all geographical regions. This study was conducted to determine and identify Salmonella serovars isolated from poultry, calves and foodstuffs (poultry and animals products such as egg and meat). A total of one hundred isolates of Salmonella serovars including Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Infantis, Salmonella Gallinarum and Salmonella Pullorum consecutively were subjected to the conventional culture, biochemical and serological assays. The utility of molecular multiplex PCR was investigated to identify and differentiate among five Salmonella serovars which were identified according to the presence of rfbJ, fljB, invA, and fliC genes in S. Typhimurium, sefA, invA and spv genes in Salmonella Enteritidis, fljB, fliC and invA genes in Salmonella Infantis, hut and slgC genes in both Salmonella Gallinarum and Salmonella Pullorum and speC gene specifically in Salmonella Gallinarum. Biochemical assays and serotyping are complicated to directly differentiate between Salmonella Gallinarum and Salmonella Pullorum because of their antigenic similarity. According to the results, Multiplex PCR can be considered as simple, rapid, accurate and useful test to identify and differentiate among Salmonella serovars.

Circulation and etiological characterization of Salmonella enterica serotype in human in Guangdong province, 2007-2016

Objective: To understand the circulation, drug resistance and molecular characteristics of Salmonella1, 4, [5], 12: i:- in human in Guangdong province. Methods:Salmonella1, 4, [5], 12: i:- isolated from diarrhea patients in Guangdong during 2007-2016 were detected for drug resistance, genes and PFGE characteristics. Results: A total of 2 960 strains Salmonella1, 4, [5], 12: i: - were isolated from human diarrhea cases during this period. The positive rates of the isolation increased year by year. The male to female ratio of the infection cases was 1.58∶1, and the infection mainly occurred in infants and young children. Except imipenem, Salmonella1, 4, [5], 12: i: - was resistant to other 17 antibiotics to some extent. The drug resistant rates to ceftazidime, cefotaxime and ciprofloxacin increased from 2011 to 2016. Multi-drug resistance was serious, for example, the multi-drug resistant strains with ASSuT accounted for 70.62% (435/616) and the multi-drug resistant strains with ACSuGSTTm accounted for 27.11% (167/616). The lack of fljA, fljB and hin genes, as well as the retaining of iroB, STM2740, STM2757 genes, resulted in the unable expression of FljBenx gene with 8 different defection profiles. There were 934 different PFGE patterns observed in 2 347 strains, which displayed a relatively large fingerprint polymorphism. The major PFGE pattern was JPXX01. GD0226, which was found in 97 strains, accounting for 4.13% (97/2 347). The PFGE patterns in 168 Salmonella1, 4, [5], 12: i: - strains were consistent with that of Salmonella typhimurium. Conclusions:Salmonella1,4,[5], 12: i: - strains has become the major serotype of Salmonella that cause diarrhea in human in Guangdong. The multi-drug resistance of Salmonella1,4, [5], 12: i: - was serious, and since the defection of fljA, fljB and hin genes, the expression of FljBenx protein failed. The PFGE results were diverse, which displayed polymorphism in inheritance.

Heterogeneity of Multidrug-Resistant Salmonella enterica Isolates with Increasing Frequency of Resistance to Ciprofloxacin During a 4-Year Period in Iran.

The study was conducted to assess the trend of antimicrobial resistance among Salmonella enterica strains during a period of 4 years and to compare the effectiveness of three genotyping methods, including flagellin polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and pulsed-field gel electrophoresis (PFGE), and to determine the most efficient method for S. enterica genotyping. About 50 S. enterica isolates were isolated from 5,064 stool samples. All of the isolates harbored fliC gene, 29 of which (58%) showed diphasic characteristic with a fliC+/fljB+ genotype. Simpson diversity index (Di) for RFLP analysis of fliC and fljB genes was calculated as 0.71 and 0.82, respectively. Strains were differentiated into 40 ERIC genotypes and 27 pulsotypes. All isolates with identical pulsotypes belonged to common serogroups which depict their correlation in a good manner. The Di calculated for ERIC-PCR and PFGE analysis was 0.99 and 0.94, respectively. The frequency of multidrug resistance (MDR) was dramatically increased from 25% in 2009-2010 to 63% in 2011-2012 with the emergence of 10% ciprofloxacin resistance in the latter period. About 44% increase in MDR phenotype among S. enterica isolates during a 4-year period and concomitant appearance of ciprofloxacin resistance is a traumatic situation for health professionals in Iran. PFGE profiles offered a satisfactory discriminatory power, which was positively correlated with S. enterica serogrouping. The current study marks the superiority of PFGE method over other conventional molecular techniques in epidemiological investigations; however, their limitations need to be addressed for further refinement.

Genotypic and phenotypic characterization of Salmonella enterica subsp. enterica serovar Typhimurium monophasic variants isolated in Thailand and Japan.

Monophasic variants of Salmonella enterica serovar Typhimurium isolated in Thailand and Japan were characterized to elucidate the genetic basis of the monophasic phenotype, genetic relatedness, and antimicrobial resistance. A total of 20 Salmonella isolates agglutinated with anti-O4 and anti-H:i serum and not agglutinated with either anti-H:1 or anti-H:2 serum were identified as monophasic variants of Salmonella serovar Typhimurium because they harbored IS200, specific to this serovar, and lacked the fljB gene. An allele-specific PCR-based genotyping method that detects a clade-specific single nucleotide polymorphism indicated that seven swine isolates and one human isolate from Thailand were grouped into clade 1; five isolates from layer chicken houses and layer chicken feces from Japan were grouped into clade 8, together with two Salmonella serovar Typhimurium isolates from chicken houses in Japan; and five isolates from swine feces from Thailand and two isolates from layer chicken feces from Japan were grouped into clade 9. Multilocus sequencing typing demonstrated that sequence type (ST) 34 isolates were solely grouped into clade 9. Clade 1 and 8 isolates were assigned as ST19. Pulsed-field gel electrophoresis revealed multiple types within each of the clades. The presence of antimicrobial resistance genes and plasmid replicon type, of the clade 1 and 9 isolates were comparable to those reported for epidemic strains of monophasic variants. Our results suggest that monitoring monophasic variants of serovar Typhimurium is important for understanding of the spread of these variants in Thailand and Japan.

Comprehensive Analysis Reveals Two Distinct Evolution Patterns of Salmonella Flagellin Gene Clusters.

Salmonella is one of the primary causes of foodborne disease, especially Salmonella enterica subsp. enterica (I) which has caused ~99% of clinical salmonellosis cases for humans and domestic mammals. The flagella genes, fliC and fljB, which encode the Salmonella phase 1 and phase 2 antigens respectively, are considered as the Salmonella serotype determinant genes, and contribute to the virulence of Salmonella. However, the evolution of the two flagellin genes is still not well-understood. In this study, the fliC and fljB gene clusters were analyzed among 205 S. enterica subspecies I genomes. The dataset covered 87 different serovars of S. enterica subsp. enterica and included 9 genomes (six serovars) of four other Salmonella subspecies. Based on a pan-genome definition and flanked gene linkages, the fliC and fljB gene clusters were identified in 207 (91 serovars) and 138 (61 serovars) genomes, respectively. A phylogenetic tree constructed based on SNPs (Single Nucleotide Polymorphisms) of core genes were used to reflect the essential evolutionary relationships among various serovars. Congruence analysis was performed among the core genome and each gene of fliC and fljB gene clusters, with only fliA and fliS showing congruence to Salmonella core genome. Congruence was also observed among fliB, fliC/fljB, and fliD genes, and their phylogeny revealed a division into two major groups, which strongly corresponded to monophasic and biphasic serovars. Besides, homologous recombination events referring fliB, fliC, and fliD were found to have mainly occurred within each group. These results suggested two distinct evolutionary patterns of Salmonella flagellin gene clusters. Further insight on the evolutionary implication of the two patterns and a framework for phase variation mechanism are needed to be further processed.

Identification of Salmonella H antigens by xTAG technology

Objective To develop a microsphere-based suspension array for simultaneous detection and identification of Salmonella H antigens by using Luminex xTAG technology and to evaluate its capability in serotyping Salmonella strains. Methods The fliC and fljB genes, encoding the H antigen of Salmonella, were selected as the target genes. Universal upstream primers were designed based on the highly conserved regions of fliC and fljB genes, and the corresponding specific reverse primers were designed based on the variable regions. While synthesizing, the 5′ end of each upstream primer was labeled with biotin and the 5′ end of each specific reverse primer was modified with its certain TAG sequence. After amplified and labeled with biotin and TAG sequence, the PCR products of specimens were hybridized with the mixture of various MagPlex-xTAG™ microspheres. Each set of microspheres contained its unique anti-TAG sequences. The results of hybridization were analyzed by using Luminex MagPix reader system and the median fluorescence intensity (MFI) was reported. The H antigens of 145 Salmonella strains were identified with this developed xTAG suspension array, and the results were compared with those obtained by using traditional serum agglutination test. Results The PCR products of different H antigens ranged from 94 bp to 245 bp and could be identified by hybridizing with MagPlex-xTAG™ microspheres. There was no cross-reaction between different H antigens or with DNAs derived from Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus and Shigella flexneri. Compared with the traditional serum agglutination test, the sensitivity and specificity of the xTAG suspension array in the identification of H antigens of 145 Salmonella strains were 95.1% and 100%, respectively. Conclusion The developed xTAG suspension array was a specific, accurate and effective method for simultaneous detection and identification of 31 H antigens of common Salmonella serovars strains. It could be used for determining the H antigens of more than 90 Salmonella strains within 5 hours. Key words: Salmonella; H antigen; xTAG technology

Study on Salmonella serotyping by use of Microsphere-based Liquid Array method

To understand the effect of serotyping on Salmonella isolates, by use of Microsphere-based Liquid Array method, among diarrhea patients, in Guangdong. Salmonella isolated from humans in Guangdong province were serotyped on the Microsphere-based Liquid Array platform with SSA kit. A total of 4 942 Salmonella strains with 189 serotypes, were identified in Guangdong province in 2010-2014. The top 100 serotypes accounted for 98.08% (4 847/4 942) of all the strains. 98% of the top 100 species serotypes could completely be serotyped with SSA kit. In order to detect O antigen among 198 isolates with SSA kit, 181 strains were carrying the O antigen, with the coincidence rate as 100%. However, under the SSA, 98.32% (528/537) of the H antigen could be detected and were consistent with the traditional serum agglutination test. The coincidence rate of fljB gene was 93.09% (175/188), with false negative rate and false positive rate of fljB gene as 7.35% (9/134) and 7.41% (4/54) respectively. The coincidence rate of sdf gene and Vi gene were 100%. 11 out of the 12 Salmonella strains could not be serotyped under the traditional methods but were successfully serotyped by the molecular serotyping method. Using the SSA kit, more than 96% of the anthropogenic Salmonella strains could be serotyped in Guangdong province. Comparing with the traditional methods, the coincidence rate of serotyping appeared over 98% . Under the Microsphere-based Liquid Array techniques, the molecular serotyping method appeared faster and more accurate on Salmonella serotyping than those traditional methods.

Use of <i>invA</i> Gene Specific PCR Analysis for the Detection of Virulent <i>Salmonella</i> Species in Beef Products in the North West Province, South Africa

A total of 32 beef samples were collected from shops around the North West Province, South Africa and analyzed for the presence of Salmonella species. A total of 96 presumptive isolates were screened for characteristics of Salmonella. All (100%) of the isolates were Gram negative rods and catalase positive. However, only a small proportion (25%) utilized citrate as a sole carbon source while 78% of the isolates fermented the sugars glucose, lactose and sucrose. Gas was produced by 34.4% while only 3.1% of these isolates produced hydrogen sulphide gas. Despite the fact that a small proportion (12.5%) of these isolates were identified as Salmonella species by the API 20E test, a much larger proportion agglutinated with Salmonella Poly A-S antiserum (53.1%) when compared to the Salmonella Poly O antiserum. To avoid bias, all the 96 isolates were screened for the Salmonella specific invA gene through PCR analysis and only 10 (10.4%) isolates were positively identified. Moreover, none of the isolates possessed the fliC flagella gene while a small proportion 11(11.5%) were positive for the fljB gene fragments. The presence of these pathogenic Salmonella species in raw meat indicates the health risk that these food products can cause to consumers, especially if consumed undercooked.

Characterization of the EmergingSalmonella4,[5],12:i:- in Danish Animal Production

The monophasic Salmonella variant with the antigenic formula Salmonella 4,[5],12:i:- has emerged in the last decade as one of the main serotypes related to human salmonellosis. In the present study, a collection of 94 isolates of the S. 4,12:i:- and S. 4,5,12:i:- coming from Danish farm animals, swine (86), cattle (7), and poultry (1), with well-defined identification was further typed by polymerase chain reaction serotyping, phage typing, and molecular typing (polymerase chain reaction and multilocus variable-number tandem-repeat analysis [MLVA]). Moreover, the determination of antimicrobial resistance pattern of each isolate was tested. In 68 of the isolates the fljB gene was absent (i.e., they were true monophasic strains), whereas in 26 isolates, the gene was present despite the fact that the isolates did not express it. The results clustered the isolates in three main pulse-types. The predominant cluster was compatible with the previously described pattern STYMXB.0131. All the isolates included in this cluster lacked the fljB gene, and all the isolates except one belonged to phage type DT 193 with the AMP-STR-SMX-TET resistance pattern. MLVA analysis divided the clusters in several MLVA profiles previously reported by other studies. Finally, antimicrobial resistance and multiresistance was frequent, although no resistance was detected in critical compounds: fluoroquinolones and cephalosporins. The present study demonstrates the presence of monophasic Salmonella Typhimurium-like strains in Danish food animal production with well-characterized clones that are described by previous studies, demonstrating the emergence and spread of this serotype in Denmark.