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.

Salmonella Heidelberg resistant to ceftiofur and disinfectants routinely used in poultry / Salmonella Heidelberg resistente ao ceftiofur e desinfetantes rotineiramente utilizados na avicultura

Bacteria of the genus Salmonella may infect humans and domestic animals, causing a serious public health problem worldwide. Nowadays, Salmonella enterica serovar Heidelberg (SH) is among the top three serovars isolated from people with salmonellosis and it is present in the poultry production chain. Moreover, it seems to be more invasive than other serotypes that cause enteritis. The overall status of the antimicrobial resistant of Brazilian strains of SH is still unknow. The bacterium may use similar mechanisms of resistance to antibotics, as well as disinfectants such as the efflux system and enzymatic degradation of chemical compounds. Thus, the objective of this study was to identify the Minimal Inhibitory Concentration (MIC) for ceftiofur of SH isolated from different materials of poultry origin, as well as to verify the relation between antibiotic resistance and disinfectant resistance. In addition, the screening efflux system was performed, using ethidium bromide to determine the presence of this mechanism of resistance. MIC results indicated high levels of SH resistance to ceftiofur, indicating the need for alternative drugs to treat salmonellosis. The concentration of ceftiofur needed to eliminate SHresistant isolates were 32 times higher than the therapeutic dose. Regarding disinfectants, most of the disinfectants tested were efficient to eliminate SH, however one isolate was resistant to glutaraldehyde-quaternary ammonia. All isolates were negatives in the screening efflux system, which suggest a different mechanism of resistance. It is possible to conclude that SH shows a real threat to poultry production, and caution should be taken when choosing the right antibiotic and disinfectant against this serovar.(AU)

Bactérias do gênero Salmonella podem infectar o homem e animais domésticos, causando sérios problemas de saúde pública no mundo todo. Hoje, a Salmonella enterica serovar Heidelberg (SH) está entre os três sorovares mais comumente isolados de pessoas com salmonelose, e presente na cadeia produtiva avícola. Além disso, SH parece ser mais invasiva que outros sorovares que causam enterite.O perfil geral de resistência aos antimicrobianos de cepas brasileiras de SH ainda é desconhecido. Esta bactéria parece usar mecanismos de resistência aos antibióticos similares aos usados para desinfetantes,tais como a bomba de efluxo e a degradação enzimática de compostos químicos. Por isso, o objetivo deste trabalho foi de identificar a Concentração Mínima Inibitória (CMI) para o ceftiofur de isolados de SH oriundas de produtos avícolas diversos, bem como de verificar a relação entre resistência a antibióticos e desinfetantes. Além disso, o sistema de bomba de efluxo foi realizado usando o brometo de etídio para determinar se esse era o mecanismo de resistência utilizado. Os resultados do MIC indicaram altos níveis de resistência para ceftiofur, demonstrando a necessidade de formas alternativas para o tratamento da salmonelose. Desinfetantes routineiramente utilizados pela indústria avícola foram eficientes no combate da SH, porém um isolado mostrou-se resistente ao desinfetante glutaraldeído associado à amônia quaternária. Todos os isolados foram negativos para o sistema de bomba de efluxo,sugerindo que há outro mecanismo de resistência envolvido. É possível concluir que os isolados brasileiros de SH representam uma ameaça para a avicultura, e cautela deveria ser tomada na hora da escolha do antibiótico e do desinfetante a ser utilizado contra este sorovar.(AU)

Salmonella Heidelberg resistant to ceftiofur and disinfectants routinely used in poultry / Salmonella Heidelberg resistente ao ceftiofur e desinfetantes rotineiramente utilizados na avicultura

Bacteria of the genus Salmonella may infect humans and domestic animals, causing a serious public health problem worldwide. Nowadays, Salmonella enterica serovar Heidelberg (SH) is among the top three serovars isolated from people with salmonellosis and it is present in the poultry production chain. Moreover, it seems to be more invasive than other serotypes that cause enteritis. The overall status of the antimicrobial resistant of Brazilian strains of SH is still unknow. The bacterium may use similar mechanisms of resistance to antibotics, as well as disinfectants such as the efflux system and enzymatic degradation of chemical compounds. Thus, the objective of this study was to identify the Minimal Inhibitory Concentration (MIC) for ceftiofur of SH isolated from different materials of poultry origin, as well as to verify the relation between antibiotic resistance and disinfectant resistance. In addition, the screening efflux system was performed, using ethidium bromide to determine the presence of this mechanism of resistance. MIC results indicated high levels of SH resistance to ceftiofur, indicating the need for alternative drugs to treat salmonellosis. The concentration of ceftiofur needed to eliminate SHresistant isolates were 32 times higher than the therapeutic dose. Regarding disinfectants, most of the disinfectants tested were efficient to eliminate SH, however one isolate was resistant to glutaraldehyde-quaternary ammonia. All isolates were negatives in the screening efflux system, which suggest a different mechanism of resistance. It is possible to conclude that SH shows a real threat to poultry production, and caution should be taken when choosing the right antibiotic and disinfectant against this serovar.

Bactérias do gênero Salmonella podem infectar o homem e animais domésticos, causando sérios problemas de saúde pública no mundo todo. Hoje, a Salmonella enterica serovar Heidelberg (SH) está entre os três sorovares mais comumente isolados de pessoas com salmonelose, e presente na cadeia produtiva avícola. Além disso, SH parece ser mais invasiva que outros sorovares que causam enterite.O perfil geral de resistência aos antimicrobianos de cepas brasileiras de SH ainda é desconhecido. Esta bactéria parece usar mecanismos de resistência aos antibióticos similares aos usados para desinfetantes,tais como a bomba de efluxo e a degradação enzimática de compostos químicos. Por isso, o objetivo deste trabalho foi de identificar a Concentração Mínima Inibitória (CMI) para o ceftiofur de isolados de SH oriundas de produtos avícolas diversos, bem como de verificar a relação entre resistência a antibióticos e desinfetantes. Além disso, o sistema de bomba de efluxo foi realizado usando o brometo de etídio para determinar se esse era o mecanismo de resistência utilizado. Os resultados do MIC indicaram altos níveis de resistência para ceftiofur, demonstrando a necessidade de formas alternativas para o tratamento da salmonelose. Desinfetantes routineiramente utilizados pela indústria avícola foram eficientes no combate da SH, porém um isolado mostrou-se resistente ao desinfetante glutaraldeído associado à amônia quaternária. Todos os isolados foram negativos para o sistema de bomba de efluxo,sugerindo que há outro mecanismo de resistência envolvido. É possível concluir que os isolados brasileiros de SH representam uma ameaça para a avicultura, e cautela deveria ser tomada na hora da escolha do antibiótico e do desinfetante a ser utilizado contra este sorovar.

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.