Sequence Analyses and Phenotypic Characterization Revealed Multidrug Resistant Gene Insertions in the Genomic Region Encompassing Phase 2 Flagellin Encoding fljAB Genes in Monophasic Variant Salmonella enterica Serovar 4,5,12:i:- Isolates From Various Sources in Thailand

Abstract

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.

Salmonella Shedding in Slaughter Pigs and the Use of Esterified Formic Acid in the Drinking Water as a Potential Abattoir-Based Mitigation Measure

“…This rise in the prevalence of the monophasic variant of S. Typhimurium appears to be related with some evolutionary advantage, in part likely associated with the accumulation of mobile resistance elements, which in turn would have contributed to its monophasic phenotype [38,39]. In fact, the monophasic variant of S. Typhimurium has been usually characterized by displaying AMR to ampicillin, streptomycin, sulphonamides, and tetracycline (ASSuT) [7,[40][41][42].…”

Highly prevalent MDR, frequently carrying virulence genes and antimicrobial resistance genes in Salmonella enterica serovar 4,[5],12:i:- isolates from Guizhou Province, China

“…Significantly, extended-Spectrum β-lactamase (ESBLs) Salmonella is considered a severe global public health problem [ 10 ]. In this study, bla TEM-1 was the most frequent ESBLs gene consistent with Eastern China [ 37 ] and Thailand [ 46 ]. It is known that bla CTX-M is a plasmid-mediated ESBLs enzyme that preferentially hydrolyzes ceftriaxone or cefotaxime, becoming an effective mechanism of Salmonella resistance to broad-spectrum cephalosporin [ 47 ].…”

Evidence of international transmission of mobile colistin resistant monophasic Salmonella Typhimurium ST34

“…H1-120 was isolated from frozen food in 2010 and demonstrated resistance to Amp, C, S, T, and sulfamethoxazole/trimethoprim (Sxt) 33 . Genetic studies of the three isolates were previously reported 30 , 33 . Their PCR patterns indicated the absence of fljAB - hin region.…”

“…Twenty-two human isolates and 31 non-human isolates of Thai S. 4,[5],12,i:- collected during 2009–2012 were previously sequenced 30 . The presence of mcr genes were identified using ResFinder 31 .…”

“…The short read sequencing was previously done 30 . The accession number and the numbers of reads of all selected isolates were shown in Supplementary Table 8.…”