invA Gene Research Articles

Implication of bats in the transmission of zoonotic strains of Salmonella in Benin

Bats play a very important role in the transmission of zoonosis, including Salmonella. Salmonella are responsible for salmonellosis, which is a major public health concern. They are the cause of many hospitalizations and deaths worldwide. The objective of this study was to contribute to the im-provement of the control of zoonotic strains of Salmonella in Benin. To do so, a collection of 400 bats was made and after slaughter and dissection, bacteriological analyses were made on the gut to isolate and identify the different strains of Salmonella carried by these bats. The resistance profile and the presence of specific virulence gene such invA, spvR, spvC and stn were studied. The strain Salmonella Typhimurium ATCC 14028 was used as a positive control. Of the 400 bats slaughtered, 14 isolate of Salmonella spp were identified by API gallery. Therefore, the prevalence of Salmonella strains in bats was 3.5%. Salmonella spp strains isolated showed total re-sistance to amoxicillin, clavulanic acid and also to first and second genera-tions of cephalosporins. The stn and invA genes have been found in the DNA of all strains of Salmonella isolated. The consumption of bats being a com-mon practice in Benin, the risk of virulent Salmonella strains transmission must be taken very seriously and people must be sensitized to this in order to slow down the risk of infection.

Antimicrobial Drug-Resistant Salmonella in Urban Cats: Is There an Actual Risk to Public Health?

The present study was undertaken to investigate the presence of Salmonella spp. in the faeces of client-owned cats in urban areas and to evaluate the risk that is posed to public health. Fresh faecal samples were collected directly from the rectums from 53 diarrhoeic and 32 non-diarrhoeic cats. The samples were individually screened for the presence of Salmonella spp. using standard methods and, in the case of positive findings, the resulting typical colonies were then biochemically confirmed using the VITEK®2 automated system. Subsequently, all of the Salmonella spp. isolates were molecularly tested for the presence of the invA gene. All of the isolates were serotyped using the slide agglutination technique according to the White–Kauffmann–Le Minor scheme. The phenotypic antimicrobial susceptibility profile of the isolated strains was obtained from the VITEK®2 system using specific cards from the Gram-negative bacteria. A total of 16 of the samples (18.82%) tested positive for Salmonella spp. according to conventional and molecular testing methods. Serotyping of the Salmonella isolates showed the presence of three serotypes, namely S. enteritidis (n = 9; 56.3%), S. typhimurium (n = 4; 25%), and S. kentucky (n = 3; 18.8%). All of the tested strains showed strong resistance towards cefazolin, cefepime, ceftazidime, and ceftriaxone. Additionally, resistance (listed in descending order of strength) was observed to trimethoprim/sulfamethoxazole (11/16; 68.8%), ampicillin (10/16; 62.5%), ampicillin/sulbactam (9/16; 56.3%), gentamicin (9/16; 56.3%), nitrofurantoin (8/16; 50.0%), and amikacin (5/16; 31.3%). No resistance was expressed against ciprofloxacin, ertapenem, imipenem, levofloxacin, piperacillin/tazobactam, and tobramycin. The results of this study highlight a substantial public health issue and medical concern, especially in vulnerable people, such as children, the elderly, and immunocompromised individuals.

RAPD Fingerprinting and Genetic Diversity of Salmonella Spp. Isolated from Broiler and Layer Flocks in Karbala, Iraq.

Salmonellosis in poultry is one of the most significant bacterial infections causing mortality, reduced production, and serious economic losses. This study aimed to study the molecular diversity among Salmonella isolates and investigate the epidemiological spread of these bacteria in broiler and layer chicken flocks in five different farms in Karbala, Iraq, using random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). In total, 217 cloac a swabs were collected from the farms, out of which 129 and 88 swabs were taken from broiler and layer chickens. The samples were screened by PCR for S. enterica subsp. enterica using primers specific for the invA gene. Afterward, RAPD-PCR with uniplex or multiplex octamer primers was applied to genotype the isolates. The incidence rate of Salmonella infections in broilers and layers was estimated to be 27.9% and 12.5%. The uniplex primers P2 and P3, along with the multiplex primers yielded discriminatory patterns. Moreover, the RAPD typing showed a diverse range of banding patterns of Salmonella spp. Dendrograms created through GelJ software revealed various Salmonella genotypes in broilers and layers. The RAPD-PCR could be used as an accurate and fast tool to identify genetic relatedness among Salmonella spp. The obtained results would assist researchers in epidemiological studies and controlling salmonellosis in poultry fields.

Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR

Manila Bay contributes significantly to the Philippines’ economy through industry, trade, tourism, and agriculture. However, microbiological contamination in the bay is putting it at risk in the present. Pathogen contamination of the water poses a serious threat to food safety, implicating public health. To address these concerns, the present study examined the prevalence of Salmonella in six different aquaculture commodities, Perna viridis (mussel), Crassostrea iridalei (oyster), Scylla serrata (crab), Penaeus spp. (shrimp), Oreochromis niloticus (tilapia), and Chanos chanos (milkfish), as well as in environmental water from growing areas. A tandem approach of culture-based and real-time PCR methods for the isolation and identification of Salmonella was carried out. To accurately identify the isolates, a real-time PCR Taqman assay based on the invA gene was used. Among the fishery resources examined, no positive samples were observed from C. chanos and O. niloticus. In addition, Salmonella was found in twenty (20) samples, representing 16.26% of all aquaculture commodities collected. Furthermore, Salmonella was found in 13.58% of the 81 environmental water samples examined. P. viridis had the highest prevalence of 55.55% out of 18 samples examined. Samples contaminated with Salmonella failed to meet the regulatory limits set by BFAR FAO 210 series 2001 and EC No. 2073/2005. In addition, it was observed that the sample matrix had a significant impact on the presence of Salmonella (p < 0.05). However, the spatial and temporal distribution of Salmonella did not vary greatly (p > 0.05). This study underscores the importance of imposing strict policies by regulatory bodies to prevent diseases, thus avoiding severe health implications.

Whole genome sequencing and protein structure analyses of target genes for the detection of Salmonella

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and β-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11–6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10–100 PFU for the MNV-1 and 1–10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice.

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae. In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

Molecular Detection and Antibiotic Sensitivity of Salmonella Species Isolated from Goat Feces in Sylhet District of Bangladesh

The present study aimed at the molecular detection of Salmonella species from feces of goats and the characterization of the isolated Salmonella by biochemical and antimicrobial sensitivity techniques. A total of 220 goat feces samples were collected, of which 27 (12.27%) were positive for Salmonella by conventional culture methods and 20 (9.09%) by biochemical and PCR techniques. The prevalence was higher in goats under one year of age (20%), compared to older animals aged one to two years (7.8%) and more than two years of age (4.7%), respectively. Moreover, the prevalence of diarrheic goats was significantly higher (38.46%) than healthy animals (2.76%). DNA was extracted from Salmonella strains and amplified by PCR using the specific primers of Salmonella invasion gene (invA gene). The antibiotic sensitivity test indicated that Ciprofloxacin (100 percent sensitivity), Gentamycin (100 percent sensitivity), and Neomycin (100 percent sensitivity) were the most effective antibiotics for the majority of Salmonella isolates. On the other hand, Salmonella isolates were found to have substantially high resistance to Erythromycin (100%), Amoxicillin (100%), Trimethoprim-Sulfamethoxazole (81.48%), Streptomycin (62.96%), and Tetracycline (55.56 percent). Since the rate of Salmonella carriers was relatively high, eating goat meat could increase the risk of foodborne salmonellosis.

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia).

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C. jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

Development of an artificial zinc finger – Luciferase fusion protein for the rapid detection of Salmonella typhimurium

A novel artificial Zinc finger – luciferase fusion protein was successfully developed for rapid detection of Salmonella typhimurium, a worldwide-distributed foodborne pathogen. The designed Zinc finger (ZF) protein bound specifically to a 12 bp region of the Salmonella spp invasion gene invA. While the luciferase from Gaussia princeps called Gaussia luciferase (Gluc) was for the first time fused with the artificial ZF domain to improve the detection sensitivity. The fusion protein successfully recognized and bound to the synthesized invA dsDNA with high specificity and sensitivity. The detection limit was as low as 10 fmol of dsNDA. Then, the bacteria PCR products were subsequently used to assess the zinc finger – luciferase fusion protein. The final results indicated that the ZF-Gluc fusion protein system could detect S. typhimurium as low as 1 CFU/mL in 2 h after the PCR. Therefore, this study provided us with a novel artificial zinc finger fusion protein and an efficient method to accomplish the rapid detection of the major foodborne pathogen S. typhimurium. In addition, the specific artificial ZF proteins that bund to particular dsDNA sequences could be easily designed, the ZF-Gluc might has broad application prospects in the field of rapid pathogenic bacteria detection.

Rapid and sensitive detection of Salmonella in milk based on hybridization chain reaction and graphene oxide fluorescence platform

Salmonella is a foodborne pathogen that has contributed to numerous food safety accidents worldwide, making it necessary to detect contamination at an early stage. A pair of specific primers based on the invA gene of Salmonella was designed for PCR. Target double-stranded DNA (dsDNA) from PCR was purified and denatured at high temperature to obtain target single-stranded DNA (ssDNA). Two carboxyfluorescein-labeled hairpin probes (H1-FAM and H2-FAM) were designed with complementary portions to the ssDNA sequence so that binding could trigger H1-FAM and H2-FAM hybridization chain reaction (HCR) to produce a long dsDNA complex. In this study, graphene oxide (GO) was used in the development of a homogeneous fluorescence detection platform for Salmonella. Using this HCR-GO assay platform, Salmonella detection was completed in 3.5 h. Salmonella was reliably and specifically detected with a limit of detection (LOD) of 4.2 × 101 cfu/mL in pure culture. Moreover, this new HCR-GO assay platform was successfully applied to the detection of Salmonella in artificially contaminated milk with a LOD of 4.2 × 102 cfu/mL.

Detection and differentiation of Salmonella Enteritidis and Salmonella Typhimurium by multiplex quantitative PCR from different poultry matrices

ABSTRACT 1. The aim of this study was to develop a multiplex quantitative polymerase chain reaction (qPCR) based molecular diagnostic kit for rapid diagnosis of Salmonella enterica serovar Enteritidis and S. enterica serovar Typhimurium serotypes, which are frequently isolated worldwide from poultry samples. 2. Detection and discrimination of S. Enteritidis and S. Typhimurium were performed by targeting the sdf and the STM4492 (putative cytoplasmic protein) gene, respectively. The invA (invasion protein) gene was used to detect Salmonella spp. as a target gene, since it is considered a standard. In this study, a total of 200 bacterial strains (178 Salmonella spp. strains and 22 other genera) were used to test the specificity and sensitivity of the developed kit. The limit of detection (LOD) of the assays was determined to be 100–101 cfu/25 g from chicken meat samples artificially contaminated by litter and 100–101 cfu/ml for cloacal swab samples. 3. The multiplex qPCR results were 100% compatible with conventional serotyping results while the specificity and sensitivity values were 100%. These findings indicated that the newly developed multiplex qPCR technique can provide an alternative method to conventional serotyping of S. Enteritidis and S. Typhimurium in laboratories lacking adequate infrastructure.

Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.

Multilocus sequence typing of multidrug-resistant Salmonella strains circulating in poultry farms of Bangladesh.

Salmonella is one of the most important foodborne zoonotic pathogens, and becoming multidrug-resistant (MDR), which represents a serious public health concern worldwide. This study aimed to identify the circulating MDR strains of Salmonella through cutting edge molecular techniques including gene specific PCR, RAPD-PCR, ribosomal gene sequencing, and multilocus sequence types (MLST) in the poultry industry of Bangladesh. Two hundred Salmonella isolates were retrieved from 154 samples comprising droppings (n = 60), cloacal swabs (n = 60), feeds (n = 14), feeding water (n = 14), and handler's swab (n = 6) from 14 commercial layer farms of Bangladesh. The isolates were confirmed as Salmonella through invA gene specific PCR, and further genotyping was done by RAPD-PCR, and 16S rRNA sequencing. The isolates were distributed into 18 different genotypes according to RAPD typing. The phylogenetic analysis identified three diverging phylogroups such as S. enterica Litchfield, S. enterica Enteritidis and S. enterica Kentucky with 11, 8, and 6 strains, respectively. The in vitro antibiogram profiling the Salmonella isolates through disc diffusion method using 13 commercially available antibiotics revealed highest resistance against doxycycline (91.5%) followed by tetracycline and ampicillin (86.0%, in each), and 72.0% isolates as MDR, being resistant to ≥ 5 antibiotics. The MLST typing was carried out based on the PCR amplification of seven housekeeping genes (aroC, hisD, hemD, purE, secA, thrA, and dnaN). MLST typing also revealed three sequence types (STs) such as ST11, ST198, and ST214 in these isolates, and eBURST analysis showed ST11 as the founder genotype. The three STs were highly resistant to tetracyclines and quinolone group of antibiotics, and all of the isolates harboring S. enterica Litchfield showed the highest resistance. Circulating common MLSTs with MDR properties in different farms confirmed the possibility of a common route of intra-farm transmission. We report for the first time of the association serovar Litchfield (ST11) in avian salmonellosis with MDR properties which is an urgent public health concern in Bangladesh.

Induction of a Viable but Nonculturable State, Thermal and Sanitizer Tolerance, and Gene Expression Correlation with Desiccation-Adapted Biofilm and Planktonic Salmonella in Powdered Infant Formula

This study was conducted to investigate the effects of the physiological state, desiccation adaptation, and storage of powdered infant formula on Salmonella cell survival and expression of desiccation stress-related genes. Powdered infant formula was inoculated with Salmonella Typhimurium in the biofilm state on beads and in the planktonic state on nitrocellulose filters and stored at 25°C for up to 270 days. Both 5-cyano-2,3-ditoyl tetrazolium chloride flow cytometry and xylose lysine deoxycholate agar plate counts revealed that biofilm-forming Salmonella cells tended to enter the viable but nonculturable (VBNC) state (P < 0.05). The population reduction of all desiccation-adapted Salmonella Typhimurium decreased significantly in both physiological states after exposure to mild heat (60°C) compared with nonadapted control cells (P < 0.05). Salmonella cells were cross-protected from heat in both physiological states, but cross-protection against hydrogen peroxide was induced for only planktonic Salmonella cells. The reverse transcription quantitative PCR results revealed that the rpoS gene in biofilm Salmonella cells on all desiccation adaption days and in planktonic Salmonella cells on day 7 of dry storage was significantly upregulated (P < 0.05). The rpoE, grpE, and invA genes in Salmonella cells in both physiological states were significantly down-regulated (P < 0.05). Physiological state and storage time might affect expression of these genes. Prior exposure to adverse conditions, including low water activity, and the physiological state impacted Salmonella survival, and its ability to enter the VBNC state and gene expression.

Antimicrobial resistance patterns, virulence gene profiles, and genetic diversity of Salmonella enterica serotype Enteritidis isolated from patients with gastroenteritis in various Iranian cities.

Objective(s):This study aimed to evaluate antibiotic resistance profiles and presence of virulence genes among Salmonella enterica serovar Enteritidis (S. Enteritidis) isolated from patients with gastroenteritis in various regions of Iran. Moreover, genetic relatedness among the strains was assessed by pulsed-field gel electrophoresis (PFGE).Materials and Methods:From April through September 2017, 59 Salmonella strains were isolated from 2116 stool samples. Of these strains, 27 S. Enteritidis were recovered. These strains were subjected to disk diffusion tests, polymerase chain reaction (PCR) for detection of virulence genes (invA, hilA, pefA, rck, stn, ssrA, ssaR, sefA, spvC, sipA, sipC, sopB, sopE, and sopE2), and PFGE.Results:High prevalence of resistance towards cefuroxime (n = 20, 74.1%) and ciprofloxacin (n = 13, 48.2%) were demonstrated. All tested strains possessed invA, hilA, sefA, sipA, sopB, and sopE. The least prevalent virulence gene was rck (n = 6; 22.2%). Based on combinations of virulence genes, 12 virulotypes were observed. The most common virulotype was VP2 (n = 12; 44.4%), harboring all of the virulence genes except for rck. PFGE typing showed only two distinct fingerprints among tested strains. Each fingerprint had completely different virulotypes. Notably, VP4 (harboring all genes except for rck and spvC) was only presented in pulsotype A, while VP2 was confined to pulsotype B.Conclusion: S. Enteritidis strains were derived from a limited number of clones, suggesting that it is highly homogenous. Future works should consider combinations of other genotyping methods together with larger sample sizes from more diverse sources.

Prevalence and Antimicrobial Resistance of Salmonella spp. in Aquacultured Nile Tilapia (Oreochromis niloticus) Commercialized in Federal District, Brazil.

This study aimed to assess Salmonella spp. prevalence in aquaculture Nile tilapia commercialized in the Federal District, Brazil, and determine the antimicrobial resistance profile of the isolates. Fifty-seven Salmonella spp. strains were isolated from 101 samples of fresh tilapia fillets collected in the Federal District, Brazil. These isolates were subjected to antimicrobial susceptibility testing by the Kirby-Bauer disk diffusion method and analyzed for the presence of blaCTX, tetB, sul2, and floR resistance genes. The Salmonella spp. prevalence in fresh tilapia fillets was 45.5%; that is, 46 of 101 samples were positive for the InvA gene. The antimicrobial resistance profile showed high resistance rates for amoxicillin/clavulanic acid (87.7%), tetracycline (82.5%), sulfonamide (57.9%), and chloramphenicol (26.3%). Additionally, 56.1% of Salmonella spp. isolates were multidrug-resistant (MDR) isolates. The beta-lactam-resistant gene blaCTX was identified in 66.7% of isolates, the tetracycline resistance gene tetA in 54.4%, and the chloramphenicol resistance gene floR in 50.9%, while the sulfonamide resistance gene sul2 was present in 49.1%. The results revealed that tilapia fillets were highly contaminated with MDR Salmonella. These Salmonella spp. strains carried multiple antimicrobial resistance genes, which might facilitate their dissemination to consumers along the production chain. Hence, there is an evident need to control Salmonella in fish production systems to ensure public health.

Critical occurrence of verotoxgenic E.coli and non-typhoidal salmonella in some heat treated dairy products.

Pathogenic strains of E.coli and Salmonella are common causes of foodborne illness and have been frequently isolated from inadequately heat-treated milk products in Mansoura city. The current study was performed to explore the prevalence of E.coli and Salmonella spp. in heattreated milk products intended for consumption in Mansoura university hospitals and hostels, as well as, to investigate their serotypes and virulence potential. Seventyfive samples of heat-treated milk products (Soft cheese, yoghurt, and processed cheese, 25 of each) were randomly gathered and directed to further investigation using conventional and molecular microbiology. Result revealed that 3(12%) of soft cheese samples harbored E.coli O146:H21, O26:H11 and O128:H2 serotypes and 2(8%) of yoghurt samples were contaminated with O128:H2 and O121:H7 serotypes while 3(12%) of processed cheese samples were positive for non-typhoidal Salmonella (NTS) serovars (Salmonella Typhimurium, Salmonella Infantis and Salmonella Essen). Virulence gene profiling reported that all E.coli isolates harbored eaeA gene and only E.coli O26:H11 and O121:H7 encoded stx2 (verotoxin) gene. Further, all Salmonella isolates harbored invA and stn genes, while only Salmonella Typhimurium and Salmonella Infantis encoded spvC gene. This study confirmed the existence of highly pathogenic verotoxogenic E.coli (VTEC) and NTS in investigated milk products which could be hazardous for public health and resident in Mansoura hospitals and hostels. Hence, the implementation of good hygienic practices together with hazard analysis, and risk-based preventive control measures are rigorously required in the process of HACCP plan to eliminate the risk of contamination that may occur during the manufacturing process.

Molecular Characterization and Survive Abilities of Salmonella Heidelberg Strains of Poultry Origin in Brazil.

The aim of the study was to evaluate the genotypic and phenotypic characteristics of 20 strains of S. Heidelberg (SH) isolated from broilers produced in southern Brazil. The similarity and presence of genetic determinants linked to virulence, antimicrobial resistance, biofilm formation, and in silico-predicted metabolic interactions revealed this serovar as a threat to public health. The presence of the ompC, invA, sodC, avrA, lpfA, and agfA genes was detected in 100% of the strains and the luxS gene in 70% of them. None of the strains carries the blaSHV, mcr-1, qnrA, qnrB, and qnrS genes. All strains showed a multidrug-resistant profile to at least three non-β-lactam drugs, which include colistin, sulfamethoxazole, and tetracycline. Resistance to penicillin, ceftriaxone (90%), meropenem (25%), and cefoxitin (25%) were associated with the presence of blaCTX–M and blaCMY–2 genes. Biofilm formation reached a mature stage at 25 and 37°C, especially with chicken juice (CJ) addition. The sodium hypochlorite 1% was the least efficient in controlling the sessile cells. Genomic analysis of two strains identified more than 100 virulence genes and the presence of resistance to 24 classes of antibiotics correlated to phenotypic tests. Protein-protein interaction (PPI) prediction shows two metabolic pathways correlation with biofilm formation. Virulence, resistance, and biofilm determinants must be constant monitoring in SH, due to the possibility of occurring infections extremely difficult to cure and due risk of the maintenance of the bacterium in production environments.

Antibiotic Susceptibility of Salmonella spp Isolated from Fresh Leafy Vegetables Samples by Using Culture and Polymerase Chain Reaction Methods

Background: Microbial contamination continues to be one of the leading risks to food safety. Contaminated leafy green vegetables are the primary cause of infection among children, elderly, and immunocompromised people. The purposes of this work were to isolate and identify of Salmonella spp. in fresh leafy vegetables collected from Jeddah Central Market, Jeddah district, western area, kingdom of Saudi Arabia, estimated of the number and percentage of isolated Salmonella spp and determined the antimicrobial susceptibility of the isolated Salmonella spp.&#x0D; Methods: Five-hundred samples were examined for the presence of Salmonella spp, by using standard microbiological and biochemical tests. Further, detection of Salmonella spp. was done by PCR with the primers targeting invA gene, a key factor for entry of Salmonella into epithelial cells. Susceptibility of the isolated Salmonella spp was done toward thirteen different antibiotics.&#x0D; Results: The percentage of isolation of Salmonella spp was 1.2 % (06/500). It was isolated as (0.40%, 02/500) from Basil, (0.20%, 01/500) from Spinach, Rocket, Parsley and Chards. Two isolates (2/6, 33.3%) showed positive Salmonella invA gene (244 bp). All isolated Salmonella showed resistance to Cephalexin (30 µg/disc), Metronidazole (5 µg/disc) and Methicillin (5 µg/disc).