Clostridium perfringens is the second-leading cause of bacterial foodborne illness in the United States. The Wadsworth Center (WC) at the New York State Department of Health enumerates infectious dose from primary patient and food samples and until recently, identified C. perfringens to the species level only. We investigated whether whole-genome sequence-based subtyping could benefit epidemiological investigations of this pathogen, as it has with other enteric organisms. We retrospectively sequenced 76 patient and food samples received between May 2010-February 2020, including 52 samples linked epidemiologically to 13 outbreaks and 24 sporadic samples not linked to other samples. Phylogenetic trees were built using two web-based platforms; National Centers for Biotechnology Information Pathogen Detection (NCBI-PD) and GalaxyTrakr (a Galaxy instance supported by the GenomeTrakr initiative). For GalaxyTrakr analyses single nucleotide polymorphism (SNP) matrices and maximum likelihood (ML) trees were generated using 3 different reference genomes. Across the four separate analyses phylogenetic clustering was generally concordant with epidemiologically-identified outbreaks. SNP diversity among phylogenetically-linked samples in an outbreak ranged from 0-20 SNPs, excepting one outbreak ranging from 4-62 SNPs. Importantly, four of the 13 outbreaks harbored one or more samples that were phylogenetic outliers, and for two outbreaks, no samples were closely related. Three specimens were found harboring two distinct genotypes. For samples below CDC enumeration dose threshold, phylogenetic clustering was robust and linked patient and/or food samples. We concluded that WGS phylogenetic clusters are: 1) largely concordant with epidemiologically-defined outbreaks, irrespective of analysis platform or reference genome we employed; 2) have limited pairwise SNP diversity, allowing phylogenetic clusters to be distinguished from sporadic cases; 3) can aid in epidemiological investigations by identifying outlier and polyclonal samples.

Salmonella enterica serovar 4,5,12:i:- (S. 4,5,12:i:-), a monophasic variant of Salmonella Typhimurium (STm) lacking the phase 2 flagellin encoding genes fljAB, has become increasingly prevalent worldwide. The increasing trends in multidrug resistant (MDR) S. 4,5,12:i:- prevalence also pose an important global health threat. Though many reports have characterized phenotypic and genotypic drug resistance of this serovar, few studies have characterized antimicrobial resistance of this serovar in Thailand. In this study, 108 S. 4,5,12:i:- isolates from various sources in Thailand and four international S. 4,5,12:i:- isolates were screened using polymerase chain reaction (PCR) to detect the presence of five target regions which are associated with antimicrobial resistant (AMR) genes, in the genomic region that contained fljAB genes in STm. We determined AMR phenotypes of all isolates by Kirby-Bauer disk diffusion method. Whole genome sequencing (WGS) was performed on 53 representative isolates (based on differences in the pulsed filed gel electrophoresis profiles, the sources of isolate, and the PCR and AMR patterns) to characterize the genetic basis of AMR phenotype and to identify the location of AMR determinants. Based on PCR screening, nine PCR profiles showing distinct deletion patterns of the five target regions have been observed. Approximately 76% of isolates (or 85 of 112 isolates), all of which were Thai isolates, contained five target regions inserted between STM2759 and iroB gene. A total of 21 phenotypic AMR patterns were identified with the predominant AmpST resistant phenotype [i.e., 84% (or 94 of 112) tested positive for resistance to ampicillin, streptomycin, and tetracycline], and 89% (or 100 of 112) were found to be MDR (defined here as resistant to at least three classes of tested antimicrobials). Using WGS data, a total of 24 genotypic AMR determinants belonging to seven different antimicrobial groups were found. AMR determinants (i.e., blaTEM–1, strB-A, sul2, and tetB, conferring resistance to ampicillin, streptomycin, sulfonamides, and tetracycline, respectively) were found to be inserted in a region typically occupied by the phase 2 flagellin encoding genes in STm. These resistant genes were flanked by a number of insertion sequences (IS), and co-localized with mercury tolerance genes. Our findings identify AMR genes, possibly associated with multiple IS26 copies, in the genetic region between STM2759 and iroB genes replacing phase 2 flagellin encoding fljAB genes in Thai S. 4,5,12:i:- isolates.