Application of machine learning algorithm and modified high resolution DNA melting curve analysis for molecular subtyping of Salmonella isolates from various epidemiological backgrounds in northern Thailand.

Food poisoning from consumption of food contaminated with non-typhoidal Salmonella spp. is a global problem. A modified high resolution DNA melting curve analysis (m-HRMa) was introduced to provide effective discrimination among closely related HRM curves of amplicons generated from selected Salmonella genome sequences enabled Salmonella spp. to be classified into discrete clusters. Combination of m-HRMa with serogroup identification (ms-HRMa) helped improve assignment of Salmonella spp. into clusters. In addition, a machine learning (dynamic time warping) algorithm (DTW) was employed to provide a simple and rapid protocol for clustering analysis as well as to create phylogeny tree of Salmonella strains (n = 40) collected from home, farms and slaughter houses in northern Thailand. Applications of DTW and ms-HRMa clustering analyses were capable of generating molecular signatures of the Salmonella isolates, resulting in 25 ms-HRM and 28 DTW clusters compared to 14 clusters from a standard HRM analysis, and the combination of both analyses permitted molecular subtyping of each Salmonella isolate. Results from DTW and ms-HRMa cluster analyses were in good agreement with that obtained from enterobacterial repetitive intergenic consensus sequence PCR clustering. While conventional serotyping of Clusters 1 and 2 revealed six different Salmonella serotypes, the majority being S. Weltevraden, the new Salmonella subtyping protocol identified five S. Weltevraden subtypes with S.Weltevreden subtype DTW4-M1 being predominant. Based on knowledge of the sources of Salmonella subtypes, transmission of S. Weltevraden in northern Thailand was likely to be farm-to-farm through contaminated chicken stool. In conclusion, the rapid, robust and specific Salmonella subtyping developed in the study can be performed in a local setting, enabling swift control and preventive measures to be initiated against potential epidemics of salmonellosis.

CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica.

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 ß-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 ß-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.

Effectiveness of BOX-PCR in Differentiating Genetic Relatedness among Salmonella enterica Serotype 4,[5],12:i:- Isolates from Hospitalized Patients and Minced Pork Samples in Northern Thailand.

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.

The interaction dynamics of a negative feedback loop regulates flagellar number in Salmonella enterica serovar Typhimurium.

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD(4) C(2) activity. FlhD(4) C(2) 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 σ(28) activity. We have found that efficient growth-dependent regulation of FlhD(4) C(2) 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 FlhD(4) C(2) activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD(4) C(2) circuit. When FlhD(4) C(2) was not bound to DNA, FliT disrupted the FlhD(4) C(2) complex. Interestingly, when FlhD(4) C(2) was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD(4) C(2) activity by preventing the formation of the FlhD(4) C(2) :DNA complex. Our data would suggest that this level of endogenous regulation of FlhD(4) C(2) activity allows the flagellar system to efficiently respond to external signals.

Interactions between antiplasmodial 3,6-diamino-1'-dimethyl-9-anilinoacridine and hematin and concanamycin A.

Antiplasmodial 9-anilinoacridine derivatives exert their effects either by inhibiting DNA topoisomerase (topo) II or by interfering with heme crystallization within the parasite acidic food vacuole. Previous studies have shown that analogs of 9-anilinoacridine containing 3,6-diamino substitutions (in the acridine ring) inhibit Plasmodium falciparum DNA topo II in situ, whereas those with a 3,6-diCl substitution act by inhibiting beta-hematin formation, a property also seen with 3,6-diamino-1'-dimethyl-9-anilinoacridine (DDAA). To understand this seemingly anomalous property of DDAA, studies of its interaction with hematin and localization within the parasite food vacuole were undertaken. A weak interaction with hematin was demonstrated spectroscopically. Antagonism of DDAA inhibition of Plasmodium falciparum growth in culture by concanamycin A, a macrolide antibiotic inhibitor of vacuolar H(+)-ATPase derived from Streptomyces sp, was equivocal.