SummaryEach Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD4C2 activity. FlhD4C2 activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of s 28 activity. We have found that efficient growth-dependent regulation of FlhD4C2 requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhD4C2 activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD4C2 circuit. When FlhD4C2 was not bound to DNA, FliT disrupted the FlhD4C2 complex. Interestingly, when FlhD 4C2 was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD4C2 activity by preventing the formation of the FlhD4C2:DNA complex. Our data would suggest that this level of endogenous regulation of FlhD4C2 activity allows the flagellar system to efficiently respond to external signals.

Salmonella enterica Serotype 4,[5],12:i:-, a monophasic variant of S. Typhimurium, with high virulence and multidrug resistance is distributed globally causing pathogenicity to both humans and domesticated animals. BOX-A1R-based repetitive extragenic palindromic-PCR (BOX)-PCR proved to be superior to three other repetitive element-based PCR typing methods, namely, enterobacterial repetitive intergenic consensus (ERIC)-, poly-trinucleotide (GTG)5-, and repetitive extragenic palindromic (REP)-PCR (carried out under a single optimized amplification condition), in differentiating genetic relatedness among S. 4,[5],12:i:- isolates from feces of hospitalized patients (n=12) and isolates from minced pork samples of S. 4,[5],12:i:- (n=6), S. Typhimurium (n=6), and Salmonella Serogroup B (n=4) collected from different regions of northern Thailand. Construction of phylogenetic trees from amplicon size patterns allowed allocation of Salmonella isolates into clusters of similar genetic relatedness, with BOX-PCR generating more unique clusters for each serotype than the other three typing methods. BOX-, (GTG)5-, and REP-PCR indicated significant genetic relatedness between S. 4,[5],12:i:- isolates 1 and 9 from hospitalized patients and S. 4,[5],12:i:- isolate en 29 from minced pork, suggesting a possible route of transmission. Thus, BOX-PCR provides a suitable molecular typing method for discriminating genetic relatedness among Salmonella spp. of the same and different serotypes and should be suitable for application in typing and tracking route of transmission in Salmonella outbreaks.

Background Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans. Method A small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis. Results CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles. Conclusion CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

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Background: Nontyphoidal Salmonella spp. transmitted through various routes are a major concern of food poisoning due to the consumption of contaminated food. Objective: To establish a molecular-based protocol for simple and rapid subtyping of Salmonella isolates from various sources. Materials and methods: Sensitive High-Resolution Melting-curve analysis (S-HRMa) and Dynamic Time Warping assessment (DTW) were applied for serotyping forty Salmonella spp. isolates from various origins and locations in seven provinces in the north of Thailand; the results were compared to those from conventional serotyping and ERIC- PCR. Results: HRM serotyping of forty Salmonella spp. initially produced fourteen melting-curves with two predominant clusters: C1 (n=18) and C2 (n=9). Applying S-HRMa and serogroups generated twenty-five sensitive clusters. Conventional serotyping revealed that cluster C1 and C2 comprised of six different Salmonella serotypes with S. Weltevradent (n=14) as the predominant one. The S-HRMa also suggested the possible subtyping in some serotypes. In addition, DTW was performed to cluster those forty Salmonella spp. into twenty-eight clusters, assigned into different four clades corresponding to S-HRMa. The two clustering methods indicated that the S. Weltevreden was the predominant subtype (DTW4-S1, n=6). Three ERIC clusters at 92% similarity index also corresponded to the results of those two clustering methods. With important and related epidemiological data, S. Derby and S. Monophasic were suggested to be related to the slaughterhouse and swine. In this study, the ERIC cluster 10 comprising two Salmonella isolates of S. Weltevraden suggested the transmission route was likely to be farm-to-farm in the same province. Conclusions: The DTW assessment and S-HRMa effectively increased the discrimatory power of clustering to the same level as that of ERIC - PCR and were a simple and rapid protocol to perform Salmonella subtyping for the epidemiological research.