Enteritidis Isolates Research Articles

Occurrence of ciprofloxacin resistance, plasmid-mediated quinolone resistance genes and virulence factors in Salmonella enterica serovar Enteritidis isolated from broilers in Thailand isolated from broilers in Thailand

Salmonella Enteritidis is one of the most common serovars associated with gastroenteritis in humans. Fluoroquinolone resistance in non-typhoidal Salmonella including S. Enteritidis has increased globally and is considered as a threat to public health. In this study, we aimed to investigate the occurrence of ciprofloxacin resistance and plasmid-mediated quinolone resistance (PMQR) genes, and examine virulence gene profiles of 69 S. Enteritidis isolates recovered from 46 boot swab and 23 intestinal samples collected from 69 commercial intensive broiler farms in Thailand. Ciprofloxacin susceptibility of these isolates was determined using microbroth dilution method. PCR was used to detect 5 common PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA) and 12 important virulence genes (agfA, invA, spaN, prgH, sitC, ssaQ, mgtC, sopB, sifA, tolC, cdtB and spvC). All S. Enteritidis showed reduced susceptibility to ciprofloxacin, with the MIC values of 0.125-0.50 µg/mL. However, these isolates did not carry PMQR genes investigated. The same virulence profile was observed among 69 S. Enteritidis isolates in which 11 virulence genes, except cdtB, were detected. The presence of virulence genes identified in invasive salmonellosis in the S. Enteritidis isolates with reduced susceptibility to ciprofloxacin could be of public health concerns. Our findings underline the need for constant monitoring of ciprofloxacin-resistant S. Enteritidis in poultry production chain to reduce public health risk.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Phenotypic and genotypic characterization of antimicrobial resistance in commonly isolated Salmonella serovars from chickens

Salmonellosis caused by Salmonella agents is the second most common zoonotic infection in humans. In recent years, Salmonella's increasing antimicrobial resistance (AMR) has been a concern. The major transmission route of Salmonella is consumption of contaminated poultry products. Therefore, monitoring of antimicrobial resistance in chicken-originated Salmonella is critically important. This study investigated AMR in four commonly isolated Salmonella serovars from chickens, namely Salmonella Enteritidis (S. Enteritidis), Salmonella Infantis (S. Infantis), Salmonella Kentucky (S. Kentucky), and Salmonella Typhimurium (S. Typhimurium). A total of 133 isolates were examined by phenotypic and genotypic AMR characterization. Resistance to 14 different antimicrobials and eight resistance genes were investigated in all isolates. The AMR test indicated that there was no resistant isolate to all antimicrobials while 14.3% were susceptible to all antimicrobials. The highest resistance was to sulfonamides (57.1%), nalidixic acid (48.1%), and tetracycline (39.1%). The highest susceptibilities were to cefotaxime (86.5%), cefoxitin (92.5%), ceftazidime (78.2%), and ceftriaxone (97%). S. Infantis and S. Kentucky isolates had higher resistance to all antimicrobials than S. Enteritidis and S. Typhimurium isolates. Significantly high multidrug-resistance (MDR) was detected in 50.4% of all isolates, although MDR prevalence varied widely between serovars: 78.7% of all S. Infantis isolates were MDR whereas only 18.8% of S. Enteritidis isolates were MDR. The most prevalent resistance genes were tetA (35.2%) and sul1 (31.6%), with 12.5% and 3.1% of S. Enteritidis isolates being positive for tetA and sul1, respectively, whereas 17.4% and 8.7% of S. Typhimurium isolates were positive. These rather low prevalence rates are probably due to effective monitoring of these serovars by control programs in Türkiye. The nondetection of mcr1 and mcr2 can be explained by the rare use of colistin in chicken flocks in Türkiye. The obtained findings emphasize the importance of AMR monitoring for Salmonella and the risks of chicken-originated isolates to humans.

1367. Resistance to Ceftriaxone and Ciprofloxacin in Nontyphoidal Salmonella from Humans and Relatedness to Isolates from Animal Sources

Abstract Background Nontyphoidal Salmonella (NTS) is a leading cause of foodborne bacterial infections. Annually, ∼200,000 antimicrobial-resistant (AMR) NTS illnesses occur in the United States. NTS infections resistant to ceftriaxone and ciprofloxacin, drugs used for the treatment of severe infections, are a growing concern. Methods We identified patient NTS isolates submitted to the Pennsylvania Bureau of Laboratories by two hospitals from the same health system during 2018–2020. All were tested by broth microdilution for susceptibility to antibiotics tracked by the National Antimicrobial Resistance Monitoring System and analyzed by whole-genome sequencing. We identified resistance mechanisms and plasmids with ResFinder and PlasmidFinder, respectively, and compared each isolate in selected serotypes against the PulseNet database to identify genetic relatedness within ≤5 allele differences to non-human sequences uploaded during 2017–2021. Results Of 164 human NTS analyzed for susceptibility, 28 (17%) had decreased susceptibility to ciprofloxacin (DSC) [MIC ≥0.12 µg/mL] while 6 (3.7%) were ceftriaxone-resistant. AMR varied by serotype; 8 (50%) S. Infantis and 16 (42%) Enteritidis had DSC, representing 86% of all isolates with DSC (Figure). Six ceftriaxone-resistant isolates had genes that confer resistance to third-generation cephalosporins including blaCMY−2 in 1 Dublin and 2 Typhimurium and blaCTX-M-65 in 3 Infantis isolates. The 3 Infantis isolates also had a mutation in gyrA that results in DSC, plus a known transmissible plasmid IncFIB(pN55391) linked to multiple resistance genes. An Infantis isolate was related to a chicken meat isolate and a Hadar isolate was related to isolates from 3 animals and 3 meat sources. One Dublin isolate was related to 2 isolates from beef in Pennsylvania. Figure.Percent of antimicrobial resistance in nontyphoidal Salmonella (NTS) clinical isolates (n=164) submitted to public health authorities by two hospital systems in Pennsylvania, 2018–2020. Multidrug resistance (MDR) was defined as resistance to ≥3 of the 9 antimicrobial classes tested for NTS clinical isolates. Resistance to ceftriaxone (AXO), a third-generation cephalosporin preferred for treating severe infections in children, was over 5% and 19% in serotypes S. Typhimurium and S. Infantis, respectively. Half (8/16) of Infantis isolates and 42% (16/38) of Enteritidis isolates had decreased susceptibility to ciprofloxacin (DSC). Conclusion Ceftriaxone resistance and DSC in NTS from patients in Pennsylvania varied by serotype and some isolates harbored blaCMY−2 and blaCTX-M-65 genes. Dissemination of mechanisms that confer resistance to ceftriaxone and DSC is concerning as is the genetic relatedness of isolates from human and animal sources. Our study highlights the need for enhanced One Health AMR monitoring combined with a review of animal production and food processing practices. Disclosures All Authors: No reported disclosures.

Occurrence of virulence factors and carbapenemase genes in Salmonella enterica serovar Enteritidis isolated from chicken meat and egg samples in Iraq

BackgroundFood-borne infections mainly due to Salmonella enterica serovar Enteritidis (S. Enteritidis) are major concerns worldwide. S. Enteritidis isolates may serve as reservoirs for spreading antimicrobial drug resistance genes including carbapenemases. This study aimed to screen the occurrence of virulence factors, carbapenemases, and antibiotic resistance genes in S. Enteritidis isolated from chicken meat and eggs in Iraq.ResultsIn total, 1000 non-duplicated chicken meat and 1000 egg samples were collected during 2019–2020. Presumptive S. Enteritidis isolates were initially identified by standard bacteriology tests and then were confirmed using polymerase chain reaction (PCR). Carbapenem resistance was detected using the disk diffusion method. Virulence and carbapenemase genes were screened using the PCR method. In total, 100 (5.0%) S. Enteritidis isolates were identified from 2000 samples collected using phenotypic and molecular methods. These isolates were identified from 4.9% chicken meat (n = 49/1000) and 5.1% egg (n = 51/1000) samples, respectively. The most and the least susceptibility was found to gentamicin and ceftazidime antibiotics, respectively. The prevalence of different virulence factors were as follows: phoP/Q (40.0%), traT (30.0%), stn (22.0%), slyA (11.0%), and sopB (9.0%). Among 20 carbapenem-resistant S. Enteritidis isolates, the most predominant carbapenemase gene was blaIMP (35.0%, n = 7), followed by blaOXA−48−like (25.0%, n = 5), and blaNDM (10.0%, n = 2), while the blaKPC and blaVIM genes were not detected. The coexistence of blaIMP, blaOXA−48−like, and blaNDM genes was determined in two isolates. The prevalence of different antibiotic resistance genes were as follows: tetA (87.1%), tetB (87.1%), dfrA1 (77.6%), and sul1 (83.6%).ConclusionConsidering the existence of carbapenem-resistant S. Enteritidis harboring different virulence and antibiotic resistance genes in chicken meat and egg samples, adherence to proper hygienic conditions should be considered.

Comparing Multiple Locus Variable-Number Tandem Repeat Analyses with Whole-Genome Sequencing as Typing Method for Salmonella Enteritidis Surveillance in The Netherlands, January 2019 to March 2020.

ABSTRACTIn the Netherlands, whole-genome sequencing (WGS) was implemented as routine typing tool for Salmonella Enteritidis isolates in 2019. Multiple locus variable-number tandem repeat analyses (MLVA) was performed in parallel. The objective was to determine the concordance of MLVA and WGS as typing methods for S. Enteritidis isolates. We included S. Enteritidis isolates from patients that were subtyped using MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as part of the national laboratory surveillance of Salmonella during January 2019 to March 2020. The concordance of clustering based on MLVA and cgMLST, with a distance of ≤5 alleles, was assessed using the Fowlkes-Mallows (FM) index, and their discriminatory power using Simpson’s diversity index. Of 439 isolates in total, 404 (92%) were typed as 32 clusters based on MLVA, with a median size of 4 isolates (range:2 to 141 isolates). Based on cgMLST, 313 (71%) isolates were typed as 48 clusters, with a median size of 3 isolates (range:2 to 39 isolates). The FM index was 0.34 on a scale from 0 to 1, where a higher value indicates greater similarity between the typing methods. The Simpson’s diversity index of MLVA and cgMLST was 0.860 and 0.974, respectively. The median cgMLST distance between isolates with the same MLVA type was 27 alleles (interquartile range [IQR]:17 to 34 alleles), and 2 alleles within cgMLST clusters (IQR:1-5 alleles). This study shows the higher discriminatory power of WGS over MLVA and a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates.IMPORTANCE Salmonella is the most frequently reported agent causing foodborne outbreaks and the second most common zoonoses in the European Union. The incidence of the most dominant serotype Enteritidis has increased in recent years. To differentiate between Salmonella isolates, traditional typing methods such as pulsed-field gel electrophoresis (PFGE) and multiple locus variable-number tandem repeat analyses (MLVA) are increasingly replaced with whole-genome sequencing (WGS). This study compared MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as typing tools for S. Enteritidis isolates that were collected as part of the national Salmonella surveillance in the Netherlands. We found a higher discriminatory power of WGS-based cgMLST over MLVA, as well as a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates. This is especially relevant for cluster delineation in outbreak investigations and confirmation of the outbreak source in trace-back investigations.

Genome-Based Assessment of Antimicrobial Resistance and Virulence Potential of Isolates of Non-Pullorum/Gallinarum Salmonella Serovars Recovered from Dead Poultry in China.

ABSTRACTParatyphoid avian salmonellosis is considered one of the leading causes of poultry death, resulting in significant economic losses to poultry industries worldwide. In China, especially in Shandong province, the leading producer of poultry products, several recurrent outbreaks of avian salmonellosis have been reported during the last decade where the precise causal agent remains unknown. Moreover, the establishment of earlier and more accurate recognition of pathogens is a key factor to prevent the further dissemination of resistant and/or hypervirulent clones. Here, we aim to use whole-genome sequencing combined with in silico toolkits to provide the genomic features of the antimicrobial resistance and virulence potential of 105 regionally representative non-Pullorum/Gallinarum Salmonella isolates recovered from dead poultry between 2008 and 2019 in Shandong, China. Additionally, phenotypic susceptibility to a panel of 15 antibiotics representing 11 classes was assessed by the broth microdilution method. In this study, we identified eight serovars and nine multilocus sequence typing (MLST) types, with Salmonella enterica serovar Enteritidis sequence type 11 (ST11) being the most prevalent (84/105; 80%). Based on their phenotypic antimicrobial resistance, 77.14% of the isolates were defined as multidrug resistant (≥3 antimicrobial classes), with the detection of one S. Enteritidis isolate that was resistant to the 11 classes. The highest rates of resistance were observed against nalidixic acid (97.14%) and ciprofloxacin (91.43%), followed by ampicillin (71.43%), streptomycin (64.77%), and tetracycline (60%). Genomic characterization revealed the presence of 41 resistance genes, with an alarmingly high prevalence of blaTEM-1B (60%), in addition to genomic mutations affecting the DNA gyrase (gyrA) and DNA topoisomerase IV (parC) genes, conferring resistance to quinolones. The prediction of plasmid replicons detected 14 types, with a dominance of IncFIB(S)_1 and IncFII(S)_1 (87.62% for both), while the IncX1 plasmid type was considered the key carrier of antimicrobial resistance determinants. Moreover, we report the detection of critical virulence genes, including cdtB, rck, sodCI, pef, and spv, in addition to the typical determinants for Salmonella pathogenicity island 1 (SPI-1) and SPI-2. Furthermore, phylogenomic analysis revealed the detection of three intra-farm and five inter-farm transmission events. Overall, the detection of Salmonella isolates presenting high antimicrobial resistance and harboring different critical virulence genes is of major concern, which requires the urgent implementation of effective strategies to mitigate non-Pullorum/Gallinarum avian salmonellosis.IMPORTANCE Avian salmonellosis is one of the leading global causes of poultry death, resulting in substantial economic losses in China (constituting 9% of overall financial losses). In Shandong province, a top poultry producer (30% of the overall production in China, with 15% being exported to the world), extensive outbreaks of avian salmonellosis have been reported in the past decade where the causal agents or exact types remain rarely addressed. From approximately 2008 to 2019, over 2,000 Salmonella strains were isolated and identified from dead poultry during routine surveillance of 95 poultry farms covering all 17 cities in Shandong. Approximately 1,500 isolates were confirmed to be of non-Pullorum/Gallinarum Salmonella serovars. There is an urgent need to understand the mechanisms behind the implication of zoonotic Salmonella serovars in systemic infections of poultry. Here, we analyzed populations of clinically relevant isolates of non-Pullorum/Gallinarum Salmonella causing chicken death in China by a whole-genome sequencing approach and determined that antimicrobial-resistant Salmonella Enteritidis remained the major cause in the past decades.

Genetic Profiling of Salmonella enteritidis Strains Isolated in the Stavropol Region in 2016–2019

Introduction: Salmonella enteritidis is the most common etiologic agent of salmonellosis in humans in the Stavropol Region, which is in line with the global trend. In this regard, the definition of a serovar provides little information in the course of an epidemiological investigation and necessitates subspecific typing of S. enteritidis isolates. Objective: MLVA typing of S. enteritidis strains isolated in the Stavropol Region in 2016–2019 and the analysis of the genetic structure of the local Salmonella population. Materials and methods: We studied 122 strains of S. enteritidis isolated in 2016–2019 from stool samples of patients with acute intestinal infections in the city of Stavropol and the Caucasian Mineral Waters (CMW) of the Stavropol Region. Multi Locus Variable Number Tandem Repeat (MLVA) typing was performed based on five variable loci. The size of amplified loci was determined by capillary electrophoresis. Results: The studied strains were distinguished by high genetic heterogeneity and belonged to 25 MLVA genotypes. S. enteritidis strains belonging to 24 MLVA types were identified on the territory of Stavropol. The dominant genetic variant in the city was 3-10-5-4-1, which included 40 Salmonella strains (44.4%) isolated in 2016–2019. In some years, the proportion of other genetic variants increased. In the CMW area, strains belonging to seven MLVA genotypes were isolated, most of which belonged to the profile 3-10-5-4-1, while a change in the dominant variant of S. Enteritidis was registered annually. Prevalent regional MLVA types are widespread in the world and have a significant epidemic potential. Conclusions: We obtained new data on S. enteritidis MLVA genotypes in the Stavropol Region, established dominant genetic variants, and noted differences in the ratio of circulating MLVA genotypes of S. enteritidis in 2016–2019. Our findings necessitate constant molecular genetic monitoring to assess real-time dynamic changes in the genetic structure of the Salmonella population. They can also be used in investigations of possible cases (outbreaks) of salmonellosis to determine their source and routes of transmission.

Research Note: Persistent Salmonella problems in slaughterhouses related to clones linked to poultry companies

Salmonellosis remains one of the main foodborne zoonoses in Europe, with poultry products as the main source of human infections. The slaughterhouse has been identified as a potential source for Salmonella contamination of poultry meat. Despite the mandatory programme of the EU, there are companies with persistent Salmonella that are unable to remove the bacteria from their processing environment, compromising the entire production line. In this context, an intensive sampling study was conducted to investigate a slaughterhouse with persistent Salmonella problems, establishing the genetic relationship among Salmonella strains isolated during the slaughter process. A total of 36 broiler flocks were sampled during processing at the slaughterhouse. Salmonella was identified based on ISO 6579-1:2017 (Annex D), serotyped by Kauffman-White-Le-Minor technique, and the genetic relationship was assessed with ERIC-PCR followed by PFGE. The outcomes showed that 69.4% of the batches sampled carried Salmonella upon arrival at the slaughterhouse and that 46.3% of the different samples from carcasses were contaminated with Salmonella. The two serovars isolated at the different steps in the slaughterhouse were Enteritidis (98.2%) and Kentucky (1.8%). Pulsed-field gel electrophoresis analysis revealed a low genetic diversity, with all S. Enteritidis isolates showing a nearly identical pulsotype (similarity >85%) and S. Kentucky strains showed the same XbaI PFGE profile (95.0% genetic similarity). The results of this study showed a high genetic relationship among isolates recovered from carcasses and environmental samples in the slaughterhouse from both Salmonella-positive and Salmonella-free flocks. Salmonella strains re-circulated across to poultry flocks and re-entered the slaughterhouse to survive on the processing line. Thus, it is necessary to implement molecular diagnosis methods in time at the field level to determine the Salmonella epidemiology of the flock, to make rapid decisions for the control of Salmonella and prevent entry into the slaughterhouse environment.

Characterisation of Salmonella Enteritidis ST11 and ST1925 Associated with Human Intestinal and Extra-Intestinal Infections in Singapore.

Salmonella Enteritidis is a major foodborne pathogen worldwide. In this study, a total of 276 S. enteritidis isolates, collected between 2016 and 2017 from human, food and farm/slaughterhouse samples, were studied to enhance the understanding of the epidemiology of human salmonellosis in Singapore. Results showed all 276 isolates belonged either to ST1925 (70.3%) or ST11 (29.7%), with ST11 being significantly more frequent in extra-intestinal isolates and chicken isolates. Food isolates, most of which were from poultry, showed the highest prevalence of resistance (33–37%) against beta-lactams or beta-lactams/beta-lactamase inhibitor combination (ampicillin, piperacillin and ampicillin/sulbactam). The analysis showed the detection of genes associated with resistance to aminoglycoside genes (99.6%), tetracycline (55.1%), and beta-lactams (14.9%) of all isolates. Nine types of plasmids were found in 266 isolates; the most common incompatibility group profiles were IncFIB(S)-IncFII(S)-IncX1 (72.2%) and IncFIB(S)-IncFII(S) (15.8%). Most plasmid harbouring isolates from chicken (63.6%, 14/22) and from human (73.8%, 175/237) shared the same plasmid profile (IncFIB(S)-IncFII(S)-IncX1). SNP analysis showed clustering of several isolates from poultry food products and human isolates, suggesting phylogenetic relatedness among these isolates. Lastly, this study provides important epidemiological insights on the application of phenotypic and next-generation sequencing (NGS) tools for improved food safety and public health surveillance and outbreak investigation of S. enteritidis.

Incidence of invasive non-typhoidal Salmonella in Blantyre, Malawi between January 2011-December 2019.

Background: The Malawi-Liverpool Wellcome Trust Clinical Research Programme (MLW) has undertaken sentinel surveillance of bloodstream infection and meningitis at Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi for 20 years. Previously, three epidemics of Salmonella bloodstream infection have been identified. Here we provide updated surveillance data on invasive non-typhoidal Salmonella disease from 2011 - 2019. Methods: Surveillance data describing trends in invasive non-typhoidal Salmonella disease and associated antimicrobial susceptibility profiles are presented for the period January 2011 - December 2019. Results: Between January 2011-December 2019, 128,588 blood cultures and 40,769 cerebrospinal fluid cultures were processed at MLW. Overall, 1.00% of these were positive for S. Typhimurium, 0.10% for S. Enteritidis, and 0.05% positive for other Salmonella species. Estimated minimum incidence of invasive non-typhoidal Salmonella (iNTS) disease decreased from 21/100,000 per year in 2011 to 7/100,000 per year in 2019. Over this period, 26 confirmed cases of Salmonella meningitis were recorded (88.5% S. Typhimurium). Between 2011-2019 there was a substantial decrease in proportion of S. Typhimurium (78.5% to 27.7%) and S. Enteritidis (31.8% in 2011 to 0%) that were multidrug-resistant. Resistance to fluoroquinolones and third-generationgeneration cephalosporins (3GC) remained uncommon, however 3GC increased amongst Salmonella spp. and S. Typhimurium in the latter part of the period. Conclusions: The total number of iNTS bloodstream infections decreased between 2011-2019. Although the number multidrug resistance (MDR) S. Typhimurium and S. Enteritidis isolates has fallen, the number of MDR isolates of other Salmonella spp. has increased, including 3GC isolates.

Virulence Factors and Antimicrobial Resistance in Salmonella Species Isolated from Retail Beef in Selected KwaZulu-Natal Municipality Areas, South Africa

Salmonellosis and antimicrobial resistance caused by non-typhoidal Salmonella are public health concerns. This study aimed at determining prevalence, serovars, virulence factors and antimicrobial resistance of Salmonella from beef products. Four-hundred beef samples from 25 retail outlets in KwaZulu-Natal, South Africa were analyzed for Salmonella using standard methods, confirmation with matrix-assisted laser desorption ionization–time of flight and serotyping according to the White–Kauffmann–Le Minor scheme. The Kirby Bauer disk diffusion method was used to determine antimicrobial resistance against Cefotaxime, Kanamycin, Ampicillin, Amoxicillin, Trimethoprim Sulfamethoxazole, Ciprofloxacin, Chloramphenicol, Gentamicin Cefoxitin and Tetracycline. A polymerase chain reaction was performed to detect invA, agfA, lpfA, hilA, sivH, sefA, sopE, and spvC virulence genes. Salmonella was observed in 1.25% (5/400) of the samples. Four serovars (Enteritidis, Hadar, Heidelberg, Stanley) were identified. Almost all Salmonella were susceptible to all antimicrobials except S. Enteritidis isolate that was resistant to Tetracycline, Ampicillin and Amoxicillin. All Salmonella isolates carried at least two virulence factors. The findings indicate low Salmonella prevalence in meat from selected KZN retail beef; however, routine surveillance to monitor risk associated with virulence factors is required to mitigate potential outbreaks. The resistant S. Enteritidis highlights a need to routinely monitor antimicrobial resistance in order to enhance human health.

CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.

Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.

Antimicrobial resistance genes, virulence markers and prophage sequences in Salmonella enterica serovar Enteritidis isolated in Tunisia using whole genome sequencing.

Salmonella Enteritidis causes a major public health problem in the world. Whole genome sequencing can give us a lot of information not only about the phylogenetic relatedness of these bacteria but also in antimicrobial resistance and virulence gene predictions. In this study, we analyzed the whole genome data of 45 S. Enteritidis isolates recovered in Tunisia from different origins, human, animal, and foodborne samples. Two major lineages (A and B) were detected based on 802 SNPs differences. Among these SNPs, 493 missense SNPs were identified. A total of 349 orthologue genes mutated by one or two missense SNPs were classified in 22 functional groups with the prevalence of carbohydrate transport and metabolism group. A good correlation between genotypic antibiotic resistance profiles and phenotypic analysis were observed. Only resistant isolates carried the respective molecular resistant determinants. The investigation of virulence markers showed the distribution of 11 Salmonella pathogenicity islands (SPI) out of 23 previously described. The SPI-1 and SPI-2 genes encoding type III secretion systems were highly conserved in all isolates except one. In addition, the virulence plasmid genes were present in all isolates except two. We showed the presence of two fimbrial operons sef and ste previously considered to be specific for typhoidal Salmonella. Our collection of S. Enteritidis reveal a diversity among prophage profiles. SNPs analysis showed that missense mutations identified in fimbriae and in SPI-1 and SPI-2 genes were mostly detected in lineage B. In conclusion, WGS is a powerful application to study functional genomic determinants of S. Enteritidis such as antimicrobial resistance genes, virulence markers and prophage sequences. Further studies are needed to predict the impact of the missenses SNPs that can affect the protein functions associated with pathogenicity.

Occurrence of Salmonella spp. in backyard poultry in Bosnia and Herzegovina.

Infected poultry is one of the most important reservoirs of Salmonella. The investigation presented here was conducted to examine the occurrence of Salmonella in fecal samples among selected flocks of backyard poultry in Bosnia and Herzegovina (B&H). Isolation and identification of Salmonella was performed in accordance with BAS EN ISO 6579/AMD 1:2007. When genus Salmonella was confirmed, the determination of the antigenic formula of Salmonella isolates was performed in accordance with BAS CEN ISO/TR 6579-3:2015. After that, Salmonella serotypes were subjected to antibiotic susceptibility testing using EUVSEC sensititre microtiter plates impregnated with different concentrations of antibiotics. At the end, real-time PCR was used to detect extended spectrum β-lactamases (ESBL) and carbapeneamase encoding genes (bla TEM, bla SHV, bla CTX-M, bla CMY, bla KPC, bla NDM, bla OXA-48, bla VIM and bla GES). Salmonella spp. was detected in pooled feces from four backyards, housed by chickens only. Three isolates were confirmed by slide agglutination as serotype Enteritidis and one as serotype Typhimurium. Antibiotic susceptibility testing by microdilution did not reveal phenotypical resistance among these four isolates. Real-time PCR used to detect ESBL and carbapeneamase encoding genes revealed the bla TEM gene in one S. Enteritidis isolate. Data presented in this study provide further evidence on the circulation of different Salmonella serotypes in backyard poultry in B&H. These findings emphasize the potential role of backyard poultry in the epidemiology of salmonellosis and the risks it poses for keepers, consumers, and general public health.

Invasive atypical non-typhoidal Salmonella serovars in The Gambia.

Invasive non-typhoidal Salmonella (iNTS) disease continues to be a significant public health problem in sub-Saharan Africa. Common clinical misdiagnosis, antimicrobial resistance, high case fatality and lack of a vaccine make iNTS a priority for global health research. Using whole genome sequence analysis of 164 invasive Salmonella isolates obtained through population-based surveillance between 2008 and 2016, we conducted genomic analysis of the serovars causing invasive Salmonella diseases in rural Gambia. The incidence of iNTS varied over time. The proportion of atypical serovars causing disease increased over time from 40 to 65 % compared to the typical serovars Enteritidis and Typhimurium that decreased from 30 to 12 %. Overall iNTS case fatality was 10%, but case fatality associated with atypical iNTS alone was 10 %. Genetic virulence factors were identified in 14/70 (20 %) typical serovars and 45/68 (66 %) of the atypical serovars and were associated with: invasion, proliferation and/or translocation (Clade A); and host colonization and immune modulation (Clade G). Among Enteritidis isolates, 33/40 were resistant to four or more of the antimicrobials tested, except ciprofloxacin, to which all isolates were susceptible. Resistance was low in Typhimurium isolates, but all 16 isolates were resistant to gentamicin. The increase in incidence and proportion of iNTS disease caused by atypical serovars is concerning. The increased proportion of atypical serovars and the high associated case fatality may be related to acquisition of specific genetic virulence factors. These factors may provide a selective advantage to the atypical serovars. Investigations should be conducted elsewhere in Africa to identify potential changes in the distribution of iNTS serovars and the extent of these virulence elements.

Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan.

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

Virulence Comparison of Salmonella enterica Subsp. enterica Isolates from Chicken and Whole Genome Analysis of the High Virulent Strain S. Enteritidis 211.

Background: Salmonella enterica is one of the common pathogens in both humans and animals that causes salmonellosis and threatens public health all over the world. Methods and Results: Here we determined the virulence phenotypes of nine Salmonella enterica subsp. enterica (S. enterica) isolates in vitro and in vivo, including pathogenicity to chicken, cell infection, biofilm formation and virulence gene expressions. S. Enteritidis 211 (SE211) was highly pathogenic with notable virulence features among the nine isolates. The combination of multiple virulence genes contributed to the conferring of the high virulence in SE211. Importantly, many mobile genetic elements (MGEs) were found in the genome sequence of SE211, including a virulence plasmid, genomic islands, and prophage regions. The MGEs and CRISPR-Cas system might function synergistically for gene transfer and immune defense. In addition, the neighbor joining tree and the minimum spanning tree were constructed in this study. Conclusions: This study provided both the virulence phenotypes and genomic features, which might contribute to the understanding of bacterial virulence mechanisms in Salmonella enterica subsp. enterica. The first completed genomic sequence for the high virulent S. Enteritidis isolate SE211 and the comparative genomics and phylogenetic analyses provided a preliminary understanding of S. enterica genetics and laid the foundation for further study.

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.

Antibiotic Resistance and Plasmid Profile Analysis of Salmonella Enteritidis Isolated in Siberia and the Far East of Russia between 1990 and 2017.

Salmonella is one of the major causes of foodborne disease outbreaks globally. Specifically, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the major causes of zoonotic Salmonella infection in humans worldwide. In this study, we present data on antimicrobial resistance (AMR) and plasmid profiles of S. Enteritidis strains isolated from patients, food, and the environment in Siberia and the Far East of Russia obtained during Salmonella monitoring between 1990 and 2017. A total of 345 S. Enteritidis isolates were tested by the disk diffusion method with a set of 15 antibiotics using EUCAST breakpoints v. 10 and by plasmid profile analysis using the alkaline lysis method. The results have shown a substantial decrease in susceptibility to aminoglycosides and quinolones during the study period. No significant differences were found in the susceptibility of strains between regions as well as in the its correlation with different plasmid types of the pathogen. Several S. Enteritidis strains were found to be resistant to ampicillin, kanamycin, tetracycline, chloramphenicol, and cephalosporins. All tested S. Enteritidis strains were susceptible only to imipenem. In this study, we observed a relatively low level of AMR in S. Enteritidis strains isolated in Siberia and the Far East of Russia. Nevertheless, it is important to continue the molecular genetic monitoring and AMR surveillance of S. Enteritidis to track further increases in AMR using conventional phenotypic susceptibility testing and by introducing whole-genome sequencing to identify AMR mechanisms.