Virulence and Antibiotic Resistance Patterns of Extended-Spectrum Beta-Lactamase-Producing Salmonella enterica serovar Heidelberg Isolated from Broiler Chickens and Poultry Workers: A Potential Hazard.

The current study investigated the emergence of multidrug-resistance (MDR), extended-spectrum beta-lactamase (ESBL)-producing Salmonella enterica serovar Heidelberg in broiler chickens and workers in poultry farms. A total of 33 S. Heidelberg isolates were recovered; 24 from the broiler cloacal swabs and 9 from the farm workers. All the S. Heidelberg isolates were tested for susceptibility to 11 antimicrobial agents and for the presence of resistance and virulence genes. MDR strains were found in 95.8% (23/24) and 88.8% (8/9) of the broiler and human isolates, respectively. Among the MDR strains, 66.6% of the broiler isolates and 55.5% of the human isolates were ESBL producing. The majority of broiler isolates showed resistance to ampicillin (100%) and ceftriaxone (91.6%), followed by ceftazidime and imipenem, (87.5%) and (75%). The resistance rate of the human isolates to those antibiotics were lower than the broiler isolates; ampicillin (88.8%), ceftriaxone (66.6%), ceftazidime (77.7%), and imipenem (66.6%). The resistance determinant genes found among the isolated strains was blaSHV-1, blaTEM-1, blaCMY-2, blaOXA-1, blaCMY-M2, blaPSE-1, and ampC. The most detected ESBL genes for broiler and human isolates were ampC (63.7%) and blaSHV-1 (56.6%), followed by blaCMY-M2 (48.5%), blaTEM-1 (39.4%), and blaOXA-1 (27.3%); whereas blaCMY-2 and blaPSE-1 were not detected. The finding of chromosomal and plasmid virulence genes revealed that the invA (100%), stn, sipC, and rck (72.8%), spvC (66.7%), ssr (63.6%), sopB (54.6%), and hilA and sipA (3.0%), while pefA and ssaR were absent. An elevated rate of MDR Salmonella Heidelberg in chickens is of potential great health risk. This signifies the role of the food of animal origin as a reservoir of MDR Salmonella that can affect the human health.

Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain.

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.

Overview of antimicrobial resistance and virulence factors in Salmonella spp. isolated in the last two decades from chicken in Brazil.

Foodborne infections caused by Salmonella have been linked to a variety of poultry products. The aim of this study was to compare the molecular profile of virulence genes considering different serotypes of Salmonella, isolates were from chicken breast sampled during the last two decades (1999 to 2010 and 2011 to 2018). The resistance to antimicrobials was also evaluated, establishing a comparative epidemiological parameter on the pathogenic potential on this bacterium over time. We tested 238 Salmonella isolates, and 18 different serotypes were observed. These being S. Enteritidis (42.3%, 58/137) and S. Ohio (28.3%, 36/137), the most frequent in the first decade; and S. Heidelberg (25.7%, 26/101) and S. Typhimurium (21.8%, 22/101), in the second. We found four (1.68%) multidrug resistant isolates from the first decade and 28 (11.76%) in the second. All extended spectrum beta-lactamase (ESBL) positive isolates belonged to the S. Heidelberg serotype, and were also detected in the second decade. Considering the nine different antimicrobial classes tested, an increase in the number of resistant isolates was observed over time: from five classes with resistant isolates in the first decade to eight classes in the second, with cefotaxime being the antimicrobial with the highest number of resistant isolates in both decades. All isolates (100%) presented the invA, sitC and tolC genes. In sequence, the most frequent genes were flgL (99.6%), sopB (98.3%), flgK (97.9%), fljB (96.6%), sipA (94.9%), sipB (88.6%), sifA (86.4%), sipD (66.1%), ssaR (51.3%), sopD (37.3%) and spvB (34.3%) was the least frequent; and 13 isolates showing all 14 virulence genes investigated. The ability of these isolates to resist certain antimicrobials, and to express genes encoding virulence factors, reinforce their marked pathogenic potential; while the possibility to trigger diseases in humans through the food chain is a serious public health threat through.

Virulence genes identification and characterization revealed the presence of the Yersinia High Pathogenicity Island (HPI) in Salmonella from Brazil.

Salmonella spp. is one of the major agents of foodborne disease worldwide, and its virulence genes are responsible for the main pathogenic mechanisms of this micro-organism. The whole-genome sequencing (WGS) of pathogens has become a lower-cost and more accessible genotyping tool providing many gene analysis possibilities. This study provided an in silico investigation of 129 virulence genes, including plasmidial and bacteriophage genes from Brazilian strains' public Salmonella genomes. The frequency analysis of the four most sequenced serovars and a temporal analysis over the past four decades was also performed. The NCBI sequence reads archive (SRA) database comprised 1077 Salmonella public whole-genome sequences of strains isolated in Brazil between 1968 and 2018. Among the 1077 genomes, 775 passed in Salmonella in silico Typing (SISTR) quality control, which also identified 41 different serovars in which the four most prevalent were S. Enteritidis, S. Typhimurium, S. Dublin, and S. Heidelberg. Among these, S. Heidelberg presented the most distinct virulence profile, besides presenting Yersinia High Pathogenicity Island (HPI), rare and first reported in Salmonella from Brazil. The genes mgtC, csgC, ssaI and ssaS were the most prevalent within the 775 genomes with more than 99% prevalence. On the other hand, the less frequent genes were astA, iucBCD, tptC and shdA, with less than 1% frequency. All of the plasmids and bacteriophages virulence genes presented a decreasing trend between the 2000 s and 2010 s decades, except for the phage gene grvA, which increased in this period. This study provides insights into Salmonella virulence genes distribution in Brazil using freely available bioinformatics tools. This approach could guide in vivo and in vitro studies besides being an interesting method for the investigation and surveillance of Salmonella virulence. Moreover, here we propose the genes mgtC, csgC, ssaI and ssaS as additional targets for PCR identification of Salmonella in Brazil due to their very high frequency in the studied genomes.

Genomic Characterization of Extended-Spectrum Cephalosporin-Resistant Salmonella enterica in the Colombian Poultry Chain.

Salmonella enterica serovars have been isolated from Colombian broilers and broiler meat. The aim of this study was to investigate the diversity of ESBL/pAmpC genes in extended-spectrum cephalosporin resistant Salmonella enterica and the phylogeny of ESBL/pAmpC-carrying Salmonella using Whole Genome Sequencing (WGS). A total of 260 cefotaxime resistant Salmonella isolates, obtained between 2008 and 2013 from broiler farms, slaughterhouses and retail, were included. Isolates were screened by PCR for ESBL/pAmpC genes. Gene and plasmid subtyping and strain Multi Locus Sequence Typing was performed in silico for a selection of fully sequenced isolates. Core-genome-based analyses were performed per ST encountered. bla CMY-2-like was carried in 168 isolates, 52 carried bla CTX-M-2 group, 7 bla SHV, 5 a combination of bla CMY-2-like-bla SHV and 3 a combination of bla CMY-2-like-bla CTX-M-2 group. In 25 isolates no ESBL/pAmpC genes that were screened for were found. WGS characterization of 36 selected strains showed plasmid-encoded bla CMY-2 in 21, bla CTX-M-165 in 11 and bla SHV-12 in 7 strains. These genes were mostly carried on IncI1/ST12, IncQ1, and IncI1/ST231 plasmids, respectively. Finally, 17 strains belonged to S. Heidelberg ST15, 16 to S. Paratyphi B variant Java ST28, 1 to S. Enteritidis ST11, 1 to S. Kentucky ST152 and 1 to S. Albany ST292. Phylogenetic comparisons with publicly available genomes showed separate clustering of Colombian S. Heidelberg and S. Paratyphi B var. Java. In conclusion, resistance to extended-spectrum cephalosporins in Salmonella from Colombian poultry is mainly encoded by bla CMY-2 and bla CTX-M-165 genes. These genes are mostly associated with IncI1/ST12 and IncQ1 plasmids, respectively. Evolutionary divergence is observed between Colombian S. Heidelberg and S. Paratyphi B var. Java and those from other countries.

Evaluation of recombinant Salmonella vaccines to provide cross-serovar and cross-serogroup protection.

Historically, Salmonella vaccines have been either live attenuated or killed bacterin vaccines that fail to offer cross-serogroup protection, which limits risk mitigation and protection of consumers. Subunit recombinant vaccines which possess highly conserved antigens offer potential to provide cross-serogroup protection, and the ability to express immune-enhancing molecules that promote recognition by the immune system. Six Salmonella subunit vaccine candidates were developed in either attenuated S. Enteritidis (SE) or S. Typhimurium (ST) that cell-surface express antigenic epitopes of high mobility group box 1 immune-enhancing sequence (H), peptidoglycan associated lipoprotein (P), and Omp18 protein Cj0113 (C) in different pattern arrangements for evaluation against S. Heidelberg (SH) challenge in broilers. In exp. 1, chicks were orally vaccinated with SE-CPH, SE-HCP, SE-CHP, ST-CPH, ST-HCP, or ST-CHP at 1 × 107 cfu/chick, or saline on d 1 and d 14. On d 17 all birds were challenged with 6 × 106 cfu/chick SH, and ceca collected on d 23 and d 28. On d 23 only SE-CPH reduced (P < 0.05) SH recovery at 0.34 ± 0.23 log10 cfu when compared to control at 1.19 ± 0.26 log10 cfu. On d 28, SE-CPH and ST-HCP reduced SH recovery at 0.40 ± 0.40 and 0.51 ± 0.26 log10 cfu, respectively in comparison to control at 1.36 ± 0.23 log10 cfu. For exp. 2, chicks were orally vaccinated with 1 × 108 cfu/chick SE-CPH, SE-HCP, SE-CHP or saline on d 1. At d 7 all chicks were orally challenged with 7 × 106 cfu/chick SH and ceca collected on d 28 and d 35. SE-CPH reduced (P < 0.05) SH recovery on d 28 when compared to control (6.16 ± 0.13 vs. 4.71 ± 0.55 log10 cfu). In exp 3, chicks were vaccinated by spray in a commercial vaccination cabinet with SE-CPH vaccination, 1.6 × 107 cfu/chick, or saline. Birds were challenged on d 14 with 3 × 107 cfu/chick SH and ceca collected on d 18 and d 25. SE-CPH reduced SH recovery (P < 0.05) on d 18, 2.75 ± 0.05 log10 cfu, and d 25, 1.89 ± 0.43 log10 cfu, as compared to control chickens at 5.6 ± 0.37 (d 18) and 3.98 ± 0.5 log10 cfu (d 25). The results of these experiments suggest that cross-serogroup protection is possible using these SE and ST-vectored subunit vaccines.