Genome Analysis and Multiplex PCR Method for the Molecular Detection of Coresistance to Cephalosporins and Fosfomycin in Salmonella enterica Serovar Heidelberg

Abstract

Heidelberg is among the top three Salmonella enterica serovars associated with human foodborne illness in Canada. Traditional culture and antimicrobial susceptibility testing techniques can be time-consuming to identify Salmonella Heidelberg resistant to cephalosporins and fosfomycin. Rapid and accurate detection of such antibiotic-resistant Salmonella Heidelberg isolates is essential to adopt appropriate control measures. In this study, 15 Salmonella Heidelberg strains isolated from feces of Canadian broiler chickens were characterized by whole genome sequencing. Salmonella Heidelberg genomes had an average coverage of greater than 80-fold, an average of 4,761 protein-coding genes, and all belonged to multilocus sequence type ST15. Genome sequences were compared with genomes in the National Center for Biotechnology Information Pathogen Detection database (www.ncbi.nlm.nih.gov/pathogens/), including human outbreak isolates. The Canadian broiler isolates clustered with chicken isolates from the United States and an equine clinical isolate from Ontario, Canada. In agreement with their antimicrobial resistance phenotypes, several chromosomally encoded specific antimicrobial resistance genes including fosA7 and multidrug resistance efflux pump determinants were detected. An AmpC-like β-lactamase gene, blaCMY-2, linked with a quaternary ammonium compound resistance gene, sugE, on a replicon type IncI1 plasmid was detected in all 15 broiler Salmonella Heidelberg isolates. Of the 205,031 published Salmonella genomes screened in silico, 4,954 (2.4%) contained blaCMY-2, 8,143 (4.0%) contained fosA7, and 919 (0.4%) contained both resistance genes. The combination of both resistance genes (fosA7 and blaCMY-2) was detected in 64% of the Heidelberg genomes and in a small proportion of various other serovars. A PCR method was developed to detect Salmonella Heidelberg in pure culture and chicken feces based on specific primers targeting genes conferring fosfomycin (fosA7) and third-generation cephalosporin (blaCMY-2) resistance as well as the Salmonella-specific invA gene and the universal 16S rRNA genes. The PCR assay was specific and sensitive for blaCMY-2 and fosA7 harboring Salmonella Heidelberg. However, some other Salmonella serovars containing these two resistance genes could also be detected by the developed PCR method.

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

“…The presence of the fosA7 gene was identified via WGS, co-existing with bla CTX−M (strain H06) and bla CMY−2 (strain H18). There are other reports that identified fosfA7/bla CMY−2 in SH (Rehman et al, 2019) and fosA3/bla CMY−2 in other enterobacteria (Zhao et al, 2015). This shows that these genes can be co-selected and transported in plasmids IncFIIa and F33: A-: B-for bla CTX−M (Hou et al, 2012;Cunha et al, 2017) and HD0149 for bla CMY−2 blaCMY-2 (Zhao et al, 2015), intensifying the horizontal transfer of genes and MDR profile in SH (Rehman et al, 2017) and other enterobacteria (Zhao et al, 2015).…”

“…75% of them belong to serovar SH. In addition, studies show that this gene easily integrates with Salmonella chromosomal DNA (Keefer et al, 2019;Rehman et al, 2019). Fosfomycin is used to treat systemic diseases caused by members of the Enterobacteriaceae family with advanced resistance to antimicrobial drugs, including ESBL producers (Hou et al, 2012).…”

“…https://www.ncbi.nlm.nih.gov/pathogens/ all, 58.2% are environmental and chicken-isolated samples, while only 6.2% are described as clinical isolates(Rehman et al, 2019).…”

Extended-Spectrum-β-Lactamase- and AmpC-Producing Escherichia coli in Domestic Dogs: Spread, Characterisation and Associated Risk Factors

“…The detection of resistance genes was performed through a multiplex PCR using specific primers [51] for bla CTX-M , bla TEM and bla SHV and according to Rehman et al [52] for bla CMY . The PCR amplification was carried out using DreamTaq Green PCR master mix (Thermo scientific, Waltham, MA, USA).…”

Spread and Molecular Characteristics of Enterobacteriaceae Carrying fosA -Like Genes from Farms in China

“…Previous research discovered that fosA7 is mainly found on the chromosomes of Salmonella from various sources (human, cattle, sheep, and environment) ( 13 ). Subsequently, this gene was detected in different countries ( 14 – 16 ). In 2020, a study reported that the FosA identified in Escherichia coli differed from FosA7 protein, which was first reported in Salmonella , and its encoding gene was named fosA7.5 Q86E ( 17 ).…”