Typhimurium Phage Type DT104 Research Articles

Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature

Salmonella enterica serovar Typhimurium (S. Typhimurium) remains an important cause of food-borne infection in the developed world. In order to establish infection within a host, Salmonella must survive and recover from a range of environmental stresses. S. Typhimurium strain SL1344 is among the most extensively studied pathogenic Salmonella strains, while S. Typhimurium phage type DT104 is an important type that has been associated with pandemic spread and a high number of food-borne disease outbreaks over the last two decades. In this study, we have compared the abilities of these two S. Typhimurium types to recover from stress exposures commonly encountered in food production, including 685 mM NaCl, pH 3.8, low temperature (6 °C) and combinations thereof. Following removal from prolonged (8 days) stress, DT104 cultures that had been exposed to low temperature, with or without additional stress, resumed exponential growth more rapidly than SL1344 cultures exposed to the same conditions. SL1344 showed higher levels of filamentation than DT104 in response to NaCl exposure at low temperature. Further, SL1344 incurred higher levels of membrane damage in response to elevated NaCl and pH 3.8 at both temperatures compared with DT104. However, both strains recovered normal cell division and membrane integrity within 6 h when all stresses were removed. Expression of the Salmonella pathogenicity island 1 gene prgH, the first gene in the prg/org operon, was monitored using a chromosomal reporter in which gfp(+) expression was driven by the prgH promoter. Recovery of prgH expression was comparable for SL1344 and DT104 exposed to stress at 22 °C. However, DT104 cultures exposed to pH 3.8 or combined NaCl and low-pH stress at low temperature resumed prgH expression more rapidly than SL1344. Both strains recovered maximal levels of prgH expression after 6 h recovery from all stresses and, interestingly, maximal levels of prgH expression were significantly higher in SL1344, consistent with prgH expression in late-exponential, non-stressed SL1344 and DT104 cultures. Together, these data show that S. Typhimurium is capable of rapid recovery from environmental and food-related stresses, and give insight into the enhanced ability of DT104 compared with SL1344 to adapt to such stresses, which may contribute to the success of this globally disseminated pathogenic phage type.

Involvement of aSalmonellaGenomic Island 1 Gene in the Rumen Protozoan-Mediated Enhancement of Invasion for Multiple-Antibiotic-ResistantSalmonella entericaSerovar Typhimurium

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that may be more virulent than related strains lacking the multiresistance phenotype. Salmonella enterica serotype Typhimurium phage type DT104 is the most prevalent of these multiresistant/hypervirulent strains. Multiresistance in DT104 is conferred by an integron structure, designated Salmonella genomic island 1 (SGI1), while we recently demonstrated DT104 hyperinvasion mediated by rumen protozoa (RPz) that are normal flora of cattle. Hyperinvasion was also observed in other Salmonella strains, i.e., other S. enterica serovar Typhimurium phage types and other S. enterica serovars, like S. enterica serovar Infantis, possessing SGI1, while DT104 strains lacking SGI1 were not hyperinvasive. Herein we attempted to identify SGI1 genes involved in the RPz-mediated hyperinvasion of Salmonella strains bearing SGI1. Transposon mutagenesis, coupled with a novel reporter system, revealed the involvement of an SGI1 gene previously designated SO13. Disruption of SO13 expression led to an abrogation of hyperinvasion as assessed by tissue culture invasion assays and by bovine challenge experiments. However, hyperinvasion was not observed in non-SGI1-bearing strains of Salmonella engineered to express SO13. That is, SO13 and another SGI1 gene(s) may coordinately upregulate invasion in DT104 exposed to RPz.

Variant Salmonella genomic island 1 antibiotic resistance gene clusters in Salmonella enterica serovar Derby isolates from humans in Taiwan

Sir, A chromosomal genomic island called Salmonella genomic island 1 (SGI1) has been initially described in epidemic multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium phage type DT104 strains (Salmonella Typhimurium DT104). SGI1 contains an antibiotic resistance gene cluster and most often confers resistance to ampicillin, chloramphenicol/ florfenicol, streptomycin/spectinomycin, sulphonamides and tetracyclines. The 43 kb SGI1 is located between the thdF and int2 genes of the chromosome of Salmonella Typhimurium. The int2 gene is part of a retron sequence that has been reported to date only in Salmonella Typhimurium. In other S. enterica serovars, SGI1 is located between the thdF and yidY genes. The antibiotic resistance gene cluster is located near the 30 end of SGI1 and constitutes a complex class 1 integron (Figure 1). At the first attI1 site of this complex integron, the cassette carries the aadA2 gene, which confers resistance to streptomycin and spectinomycin, and a 30-conserved segment (30-CS) with a truncated sul1 (sul1delta) gene is present. At the second attI1 site, the cassette contains the b-lactamase gene blaPSE-1 conferring resistance to ampicillin and the 30-CS comprises a complete sul1 gene conferring resistance to sulphonamides. Flanked by the two cassettes are the floR gene, which confers cross-resistance to chloramphenicol and florfenicol, and the tetracycline-resistance genes tetR and tet(G). SGI1 has been recently demonstrated to be an integrative mobilizable element. Variant SGI1 antibiotic resistance gene clusters have been described in a wide variety of S. enterica serovars. SGI1 variant antibiotic resistance gene clusters were accordingly classified from SGI1-A to SGI1-L. These gene clusters were probably generated after chromosomal recombinational events or by antibiotic resistance gene cassette replacement at one of the attI1 sites. We reasoned that MDR of Salmonella Derby isolated in Taiwan might be due to the spread of SGI1 and the presence of SGI1 antibiotic resistance gene clusters was thus investigated. The 22 MDR isolates of Salmonella Derby isolated in 2003 and 2004 from humans examined in this study were provided by the Center of Disease Control, Taiwan. Detection of SGI1, its location, and mapping of antibiotic resistance genes were performed by PCR on extracted genomic DNA using primers and conditions described previously (Figure 1). Among the 22 Salmonella Derby isolates examined, 13 isolates showed three different MDR resistance phenotypes indicative of the presence of SGI1 antibiotic resistance gene clusters. SGI1 was detected in these 13 isolates based on PCRs using primer pairs corresponding to the left junction and right junction of SGI1 in the Salmonella chromosome. PCR results were positive for the left junction of SGI1 with the chromosomal thdF gene and for the right junction with the chromosomal yidY gene, but negative for the junction with the int2 gene. Thus, the 13 Salmonella Derby strains were shown to harbour SGI1 at the same chromosomal location, i.e. between the thdF and yidY genes, as in non-Typhimurium serovars. In seven Salmonella Derby strains showing the typical Salmonella Typhimurium DT104 MDR phenotype, PCR mapping of the antibiotic resistance gene cluster showed the presence of fragments A, B, C, D, E, F and floR of the sizes expected (Figure 1). These isolates thus carried the SGI1 antibiotic resistance gene cluster classically found in MDR Salmonella Typhimurium DT104. For the five Salmonella Derby strains showing the chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides, tetracycline and trimethoprim MDR phenotype, PCR mapping yielded fragments A–D and floR. However, fragment E specific for the blaPSE-1 gene cassette was negative whereas a PCR using the forward primer of PCR E and the reverse primer of PCR H was positive, with a size slightly different from that expected from an SGI1-carrying Salmonella Typhimurium DT104 control strain, suggesting the presence of another gene cassette(s) at the second attI1 site of the SGI1 antibiotic resistance gene cluster. To confirm this result and to identify the gene cassette(s), PCR amplification of class 1 integrons using primers 50-conserved segment and 30-CS was performed as described previously. This PCR yielded two fragments, one of 1 kb corresponding to the aadA2 gene cassette and one of 1.3 kb, which was sequenced. Nucleotide sequencing identified the dfrA1 (conferring resistance to trimethoprim) and orfC (unknown function) gene cassettes. This array of gene cassettes was previously identified in the SGI1 antibiotic resistance gene cluster called SGI1-I of a Salmonella Derby human isolate from Malaysia. Thus, in these five Salmonella Derby strains the blaPSE-1 gene was replaced by the dfrA1 and orfC gene cassettes and corresponded to the SGI1-I antibiotic resistance gene cluster.

Detection of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Phage Types DT102, DT104, and U302 by Multiplex PCR

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.

Multi-Drug Resistance in Salmonella enterica: Efflux Mechanisms and Their Relationships with the Development of Chromosomal Resistance Gene Clusters

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

The genetics of Salmonella genomic island 1

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104, resistant to ampicillin, chloramphenicol/florfenicol, streptomycin, sulfonamides, and tetracycline, has disseminated worldwide. The resistance genes reside on the 43-kb Salmonella genomic island 1 (SGI1), which is transferable. Drug-resistant variants of SGI1 have been identified in numerous serotypes. Strains harboring SGI1 may be more virulent and have a tendency to rapidly disseminate.

Exposure to Rumen Protozoa Leads to Enhancement of Pathogenicity of and Invasion by Multiple-Antibiotic-ResistantSalmonella entericaBearing SGI1

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that has been purported to be more virulent than antibiotic-sensitive counterparts. The paradigm for this multiresistant/hyperpathogenic phenotype is Salmonella enterica serotype Typhimurium phage type DT104 (DT104). The basis for the multiresistance in DT104 is related to an integron structure designated SGI1, but factors underlying hyperpathogenicity have not been completely identified. Since protozoa have been implicated in the alteration of virulence in Legionella and Mycobacterium spp., we attempted to assess the possibility that protozoa may contribute to the putative hypervirulence of DT104. Our study reveals that DT104 can be more invasive, as determined by a tissue culture invasion assay, after surviving within protozoa originating from the bovine rumen. The enhancement of invasion was correlated with hypervirulence in a bovine infection model in which we observed a more rapid progression of disease and a greater recovery rate for the pathogen. Fewer DT104 cells were recovered from tissues of infected animals when protozoa were lysed by preinfection chemical defaunation of the bovine or ovine rumen. The protozoan-mediated hypervirulence phenotype was observed only in DT104 and other Salmonella strains, including serovars Agona and Infantis, possessing SGI1.

Characterization ofSalmonella entericaSerovar Typhimurium and MonophasicSalmonellaSerovar 1,4,[5],12:i:- Isolates in Thailand

Duplex PCR was developed to screen Salmonella enterica serovar Typhimurium phage type DT104 and related strains in Thailand because a phage typing laboratory of serovar Typhimurium is not available. Of 46 isolates of serovar Typhimurium and 32 isolates of S. enterica serovar 1,4,[5],12:i:-, 15 (33%) and 30 (94%) were duplex PCR positive, respectively. All isolates were submitted for phage typing to analyze the specificity of the PCR assay. Among serovar Typhimurium isolates that yielded positive duplex PCRs, only seven isolates were phage types DT104 or U302, and eight isolates were undefined types, whereas the negative PCR isolates were either other phage types, including DT7, DT12, DT66, DT79, DT166, DT170, DT193, and DT208 or an undefined type. The serovar Typhimurium and serovar 1,4,[5],12:i:- isolates that were duplex PCR positive were further subtyped by using XbaI PFGE to reveal their genetic relatedness. All serovar Typhimurium phage type DT104 strains had indistinguishable chromosomal patterns. The isolates of phage type U302 and most of the serovar 1,4,[5],12:i:- isolates that were duplex PCR positive yielded similar pulsed-field gel electrophoresis patterns. The patterns of PCR-negative isolates distinctly differed from the patterns of PCR-positive isolates. A total of 26% of all isolates had a dominant R-type ACSSuTG that was not found in the isolates of phage type DT104.

International dissemination of antibiotic resistant strains of bacterial pathogens

The increasing incidence of methicillin and multiple resistant Staphylococcus aureus (MRSA) in nosocomial infections is mainly associated with a wide, international dissemination of well defined clonal lineages (epidemic MRSA) which are clearly different from community acquired MRSA by molecular typing patterns and structure of the staphylococcal cassette chromosome containing the mecA gene. Although belonging to a definite subpopulation within the species Enterococcus faecium, hospital associated vancomycin resistant isolates also containing the esp gene have very likely evolved by acquisition of glycopeptide resistance gene clusters at different occasions and at different times by a susceptible already disseminated clonal lineage. There is obviously a continuous selection of new types of extended spectrum β-lactamases in enterobacteriaceae and also horizontal spread of bla-genes. Intrahospital dissemination of particular strains has often been observed, however, an international dissemination until now has been described only for Salmonella enterica serovar Typhimurium producing the CTX-M-3 enzyme. Multiresistant isolates of Salmonella enterica serovar Typhimurium phage type DT104 harbour a multiresistant gene cluster with resistance genes from taxonomically more unrelated species (tetG, floR, blaPSE1). Although in vitro transduction has been demonstrated, this gene cluster has only rarely been reported from isolates exhibiting other phage patterns of the same serovar or from other serovars of S. enterica.

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6 kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment’s nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415 bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.

In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.

Molecular characterization, spread and evolution of multidrug resistance in Salmonella enterica typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has emerged during the last decade as a global health problem because of its involvement in diseases in animals and humans. Multidrug-resistant DT104 strains are mostly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines (ACSSuT resistance type). The genes coding for such resistances are clustered on the chromosome. This paper reviews new developments in the characterization of S. enterica Typhimurium DT104, its chromosomal antibiotic resistance genes and their spread among other S. enterica Typhimurium phage types and other S. enterica serovars, the development of specific detection methods, virulence characteristics, and the evolution of multidrug-resistance with regard to the emergence of quinolone resistance.

Multiple clones within multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104. The Greek Nontyphoidal Salmonella Study Group.

Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3'')-Ia, pse-1, and tem-1.