Project description:It has been 30 years since the initial emergence and subsequent rapid global spread of multidrug-resistant Salmonella entericaserovar Typhimurium DT104 (MDR DT104). Nonetheless, its origin and transmission route have never been revealed. We used whole-genome sequencing (WGS) and temporally structured sequence analysis within a Bayesian framework to reconstruct temporal and spatial phylogenetic trees and estimate the rates of mutation and divergence times of 315S Typhimurium DT104 isolates sampled from 1969 to 2012 from 21 countries on six continents. DT104 was estimated to have emerged initially as antimicrobial susceptible in ∼1948 (95% credible interval [CI], 1934 to 1962) and later became MDR DT104 in ∼1972 (95% CI, 1972 to 1988) through horizontal transfer of the 13-kb Salmonella genomic island 1 (SGI1) MDR region into susceptible strains already containing SGI1. This was followed by multiple transmission events, initially from central Europe and later between several European countries. An independent transmission to the United States and another to Japan occurred, and from there MDR DT104 was probably transmitted to Taiwan and Canada. An independent acquisition of resistance genes took place in Thailand in ∼1975 (95% CI, 1975 to 1990). In Denmark, WGS analysis provided evidence for transmission of the organism between herds of animals. Interestingly, the demographic history of Danish MDR DT104 provided evidence for the success of the program to eradicate Salmonellafrom pig herds in Denmark from 1996 to 2000. The results from this study refute several hypotheses on the evolution of DT104 and suggest that WGS may be useful in monitoring emerging clones and devising strategies for prevention of Salmonella infections.
Project description:Recently a chromosomal locus possibly specific for Salmonella enterica serovar Typhimurium DT104 has been reported that contains a multiple antibiotic resistance gene cluster. Evidence is provided that Salmonella enterica serovar Agona strains isolated from poultry harbor a similar gene cluster including the newly described floR gene, conferring cross-resistance to chloramphenicol and florfenicol.
Project description:Infection with Salmonella enterica is a major public health concern in developed countries, and multidrug-resistant strains have become increasingly prevalent. S. enterica serovar Typhimurium DT104 (DT104) strains are prevalent in livestock in Japan and include numerous strains of multidrug-resistant S. enterica. Epidemiological analysis of these strains is critical for both agriculture and public health; however, diagnostic tests for these strains have yielded inconsistent results.We developed a rapid, simple, and inexpensive polymerase chain reaction test to detect multi-drug resistant DT104 strains. We designed primers specific to the prophage ST104 sequence encoded by DT104 strains and assessed the specificity of these primers by assaying a panel of 50 S. enterica isolates. Amplification products of the expected size were generated from the genomes of each of the DT104 strains; however, the ST104 primers failed to amplify products from non-DT104 strains of S. enterica serovar Typhimurium or other S. enterica serovars. Furthermore, a probe generated using the ST104 primers detected a restriction fragment encoding the ST104 region of DT104 by Southern hybridization.The ST104 primers exhibit specificity to DT104 strains and are suitable for epidemiological applications.
Project description:Salmonella enterica serotype typhimurium (S. typhimurium) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes floS. typhimurium (floSt), int, invA, and spvC were produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene floSt, reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the floSt-positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene floSt, described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the floSt gene or its resulting phenotype.
Project description:Salmonella enterica serotype Typhimurium definitive phage type 104 was isolated several times from the same patient over a period of 2 years. The strain developed reduced sensitivity to fluoroquinolones, and a mutation in the gyrA gene that is associated with reduced sensitivity to quinolones was identified.
Project description:Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.
Project description:PCR was used to identify antibiotic resistance determinants in 31 Canadian Salmonella serovar Typhimurium DT104 isolates. Genes encoding resistance to ampicillin (pse1 or blaP1), chloramphenicol (pasppflo-like), streptomycin-spectinomycin (aadA2), sulfonamide (sulI), and tetracycline [tet(G)] were mapped to a 13-kb region of DNA of one isolate. Two copies of sulI were identified and mapped to the 3' end of either pse1 or aadA2 integrons. The two integrons were separated by the pasppflo-like gene and the tet(G) gene. The kanamycin resistance determinant (aphA-1) was present on a 2.0-MDa plasmid (five isolates) or on the chromosome (three isolates).
Project description:Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.
Project description:Salmonella enterica serovar Typhimurium is a common cause of nontyphoidal salmonellosis in humans and animals. Multidrug-resistant serovar Typhimurium phage type DT104, which emerged in the 1990s, has become widely distributed in many countries. A total of 104 clinical isolates of Salmonella serogroup B were collected from three major hospitals in Taiwan during 1997 to 2003 and were examined by a multiplex PCR targeting the resistance genes and the spv gene of the virulence plasmid. A total of 51 isolates (49%) were resistant to all drugs (ACSSuT [resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline]), and all contained a 1.25-kb PCR fragment of integron that is part of the 43-kb Salmonella genomic island 1 (SGI1). The second group was resistant to SSu (28%), and the third was susceptible to all five drugs (13%). Fifty-nine isolates were serotyped to be serovar Typhimurium by the tube agglutination method using H antisera. The virulence plasmid was found in 54 (91.5%) of the 59 serovar Typhimurium isolates. A majority (94.1%) of the Salmonella serogroup B isolates with the ACSSuT resistance pattern harbored a virulence plasmid. Phage typing identified three major phage types: DT104, DT120, and U302. Analysis of the isolates by pulsed-field gel electrophoresis showed six genotypes. We found two genotypes in DT104 strains, two in DT120, and the other two in U302. The presence of a monophasic serovar (4,5,12:i:-) has added difficulty in the determination of the serovars of multidrug-resistant Salmonella serogroup B isolates. Nevertheless, the multiplex PCR devised in the present study appears to be efficient and useful in the rapid identification of ACSSuT-type serovar Typhimurium with SGI1, irrespective of their phage types.
Project description:The surge in mortality and morbidity rates caused by multidrug-resistant (MDR) bacteria prompted a renewal of interest in bacteriophages (phages) as clinical therapeutics and natural biocontrol agents. Nevertheless, bacteria and phages are continually under the pressure of the evolutionary phage-host arms race for survival, which is mediated by co-evolving resistance mechanisms. In Anderson phage typing scheme of Salmonella Typhimurium, the epidemiologically related definitive phage types, DT104 and DT104b, display significantly different phage susceptibility profiles. This study aimed to characterise phage resistance mechanisms and genomic differences that may be responsible for the divergent phage reaction patterns in S. Typhimurium DT104 and DT104b using whole genome sequencing (WGS). The analysis of intact prophages, restriction-modification systems (RMS), plasmids and clustered regularly interspaced short palindromic repeats (CRISPRs), as well as CRISPR-associated proteins, revealed no unique genetic determinants that might explain the variation in phage susceptibility among the two phage types. Moreover, analysis of genes coding for potential phage receptors revealed no differences among DT104 and DT104b strains. However, the findings propose the need for experimental assessment of phage-specific receptors on the bacterial cell surface and analysis of bacterial transcriptome using RNA sequencing which will explain the differences in bacterial susceptibility to phages. Using Anderson phage typing scheme of Salmonella Typhimurium for the study of bacteria-phage interaction will help improving our understanding of host-phage interactions which will ultimately lead to the development of phage-based technologies, enabling effective infection control.