DT104 Strains Research Articles

Intra-macrophage expression of ArtAB toxin gene in Salmonella.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. Worthington, and S. bongori produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on a prophage in Salmonella, and prophage induction by SOS-inducing agents is associated with increases in ArtAB production in vitro. However, little is known about the expression of artAB in vivo. Here, we showed a significant increase in artAB transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of artAB in DT104 and S. bongori was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in S. Worthington. Upregulation of oxyR, a major regulator of oxidative stress, and cI, a repressor of prophage induction, was observed in S. Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of oxyR was increased, artAB was upregulated in S. bongori, which lacks the cI gene in the incomplete artAB-encoded prophage. Taken together, oxidative stress plays a role in the production of artAB toxins in macrophages, and high expression levels of oxyR and cI are responsible for the low expression of artAB. Therefore, strain variation in the level of artAB expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.

Genomic Characterization of Salmonella typhimurium DT104 Strains Associated with Cattle and Beef Products

Salmonella enterica subsp. enterica serovar Typhimurium DT104, a multidrug-resistant phage type, has emerged globally as a major cause of foodborne outbreaks particularly associated with contaminated beef products. In this study, we sequenced three S. Typhimurium DT104 strains associated with a 2009 outbreak caused by ground beef, including the outbreak source strain and two clinical strains. The goal of the study was to gain a stronger understanding of the genomics and genomic epidemiology of highly clonal S. typhimurium DT104 strains associated with bovine sources. Our study found no single nucleotide polymorphisms (SNPs) between the ground beef source strain and the clinical isolates from the 2009 outbreak. SNP analysis including twelve other S. typhimurium strains from bovine and clinical sources, including both DT104 and non-DT104, determined DT104 strains averaged 55.0 SNPs between strains compared to 474.5 SNPs among non-DT104 strains. Phylogenetic analysis separated the DT104 strains from the non-DT104 strains, but strains did not cluster together based on source of isolation even within the DT104 phage type. Pangenome analysis of the strains confirmed previous studies showing that DT104 strains are missing the genes for the allantoin utilization pathway, but this study confirmed that the genes were part of a deletion event and not substituted or disrupted by the insertion of another genomic element. Additionally, cgMLST analysis revealed that DT104 strains with cattle as the source of isolation were quite diverse as a group and did not cluster together, even among strains from the same country. Expansion of the analysis to 775 S. typhimurium ST19 strains associated with cattle from North America revealed diversity between strains, not limited to just among DT104 strains, which suggests that the cattle environment is favorable for a diverse group of S. typhimurium strains and not just DT104 strains.

Characterisation of Phage Susceptibility Variation in Salmonellaenterica Serovar Typhimurium DT104 and DT104b.

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.

Isolation, Molecular Detection and Antibiogram of Multi-drug Resistant Salmonella Typhimurium DT104 from Selected Dairy Farms in Mymensingh, Bangladesh

The Salmonella typhimurium DT104, an emerging cause of human illness has received an increasing attention due to its multidrug resistant properties. Since it has been isolated from human and other sources including food-producing animals around the world; it has become a worldwide public health concern. Therefore, the present study was designed to isolate, identify and study the antibiogram profile of multidrug resistant S. typhimurium DT104 from several dairy farms in Mymensingh district, Bangladesh. A total of 135 fecal samples from diarrhoeic cattle were collected aseptically and subjected for bacterial isolation, molecular detection using PCR followed by antibiogram study. Salmonella spp. could be isolated from a total of 39 (28.88%) samples based on cultural and staining methods which were further confirmed by PCR using invA gene specific primers. However, out of 39 Salmonella spp., 6 isolates were confirmed as S. typhimurium DT104 strain. Results of the antibiotic resistance patterns demonstrated that 100% (39/39) isolates were resistant to erythromycin followed by tetracycline (73.68%), colistin (89.47%), ampicillin (47.36%), gentamicin (21.05%), ciprofloxacin (31.57%), streptomycin (42.10%), enrofloxacin (10.52%) and chlormphenicol (31.57%). Moreover, about 23.07% isolates were resistant to more than 5 antibiotics. However, all the isolates were found to be sensitive to amikacin. These results suggest that antibiotic resistant S. typhimurium DT104 strain has been circulating in dairy cattle in Bangladesh which is alarming and may impose threat to livestock and public health due to lack of proper hygienic management. This study will be helpful for the selection of proper antibiotics against salmonellosis in cattle.

Reduction of Salmonella contamination on the surface of chicken skin using bacteriophage

BackgroundEnteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively.MethodsSalmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h.ResultsThe median levels of Salmonella reduction following phage treatment were 1.38 log10 MPN (Enteritidis) and 1.83 log10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls (p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface.ConclusionsThe results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Distinct evolutionary origins of common multi-drug resistance phenotypes in Salmonella typhimurium DT104: a convergent process for adaptation under stress.

Antimicrobial resistance makes pathogenic bacteria hard to control, but little is known about the general processes of resistance gain or loss. Here, we compared distinct S. typhimurium DT104 strains resistant to zero, two, five, or more of the tested antimicrobials. We found that common resistance phenotypes could be encoded by distinct genes, on SGI-1 or plasmid. We also demonstrated close clonality among all the tested non-resistant and differently resistant DT104 strains, demonstrating dynamic acquisition or loss (by total deletion or gradual decaying of multi-drug resistance gene clusters) of the genetic traits. These findings reflect convergent processes to make the bacteria resistant to multiple antimicrobials by acquiring the needed traits from stochastically available origins. When the antimicrobial stress is absent, the resistance genes may be dropped off quickly, so the bacteria can save the cost for maintaining unneeded genes. Therefore, this work reiterates the importance of strictly controlled use of antimicrobials.

Multidrug Resistance Salmonella Genomic Island 1 in a Morganella morganii subsp. morganii Human Clinical Isolate from France.

Salmonella genomic island 1 (SGI1) is a multidrug resistance integrative mobilizable element that harbors a great diversity of antimicrobial resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. A serious threat to public health was revealed in the recent description in P.mirabilis of a SGI1-derivative multidrug resistance island named PGI1 (Proteus genomic island 1) carrying extended-spectrum-β-lactamase (ESBL) and metallo-β-lactamase resistance genes, blaVEB-6 and blaNDM-1, respectively. Here, we report the first description of Salmonella genomic island 1 (SGI1) in a multidrug-resistant clinical Morganella morganii subsp. morganii strain isolated from a patient in France in 2013. Complete-genome sequencing of the strain revealed SGI1 variant SGI1-L carrying resistance genes dfrA15, floR, tetA(G), blaPSE-1 (now referred to as blaCARB-2), and sul1, conferring resistance to trimethoprim, phenicols, tetracyclines, amoxicillin, and sulfonamides, respectively. The SGI1-L variant was integrated into the usual chromosome-specific integration site at the 3' end of the trmE gene. Beyond Salmonella enterica and Proteus mirabilis, the SGI1 integrative mobilizable element may thus also disseminate its multidrug resistance phenotype in another genus belonging to the Proteae tribe of the family Enterobacteriaceae. IMPORTANCE Since its initial identification in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium DT104 strains, several SGI1 variants, SGI1 lineages, and SGI1-related elements (SGI2, PGI1, and AGI1) have been described in many bacterial genera (Salmonella, Proteus, Morganella, Vibrio, Shewanella, etc.). They constitute a family of multidrug resistance site-specific integrative elements acquired by horizontal gene transfer, SGI1 being the best-characterized element. The horizontal transfer of SGI1/PGI1 elements into other genera is of public health concern, notably with regard to the spread of critically important resistance genes such as ESBL and carbapenemase genes. The identification of SGI1 in Morganella morganii raises the issue of (i) the potential for SGI1 to emerge in other human pathogens and (ii) its bacterial host range. Further surveillance and research are needed to understand the epidemiology, the spread, and the importance of the members of this SGI1 family of integrative elements in contributing to antibiotic resistance development.

Rapid detection of Salmonella enterica serovar Typhimurium DT104 strains by the polymerase chain reaction.

BackgroundInfection 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.ResultsWe 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.ConclusionsThe ST104 primers exhibit specificity to DT104 strains and are suitable for epidemiological applications.

Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections

Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less understood in comparison with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). To study interactions of weaned piglets with Salmonella Derby, we compared the course of infections with Salmonella Derby De1 and Salmonella Typhimurium DT104 strains, both isolated from pig herds with a long history of asymptomatic infection.Salmonella Derby strain used was shed during the 28-day experiment period, while Salmonella Typhimurium strain was not found in faeces after day 17 post-infection. When the piglets were co-infected with both strains, Salmonella Derby was present in faeces until the end of the experiment, whilst Salmonella Typhimurium disappeared after day 21 post-infection. At the end of the experiment, Salmonella Derby was present in more tissues when compared with Salmonella Typhimurium. Piglets infected with Salmonella Typhimurium responded earlier with synthesis of anti-lipopolysaccharide IgM and IgG antibodies and with higher antibody levels compared to piglets infected with Salmonella Derby. Cellular immune response to both strains was very low and was detected later than was the onset of IgG antibody production.

Identification of Virulence Properties in Salmonella Typhimurium DT104 Using Caenorhabditis elegans

Salmonella enterica serover Typhimurium definitive phage type DT104, resistant to multiple antibiotics, is one of the most widespread Salmonella species in human infection worldwide. Although several cohort studies indicate that DT104 carrying the multidrug resistance (MDR) locus on salmonella genomic island 1 is a possible hyper-virulent strain compared to DT104 strains without MDR, or other Salmonella enterica serotypes, existing experimental evidence regarding virulence properties associated with the MDR region is controversial. To address this question, we constructed an isogenic MDR deletion (∆MDR) mutant strain of DT104, SNS12, by allelic exchange and used Caenorhabditis elegans as a host model to assess differences in virulence between these two strains. SNS12 exhibited decreased virulence in C. elegans, and we observed increased colonization and proliferation of the intestine of C. elegans by DT104. The immune response against MDR-carrying DT104 appears to function through a non-canonical Unfolded Protein Response (UPR) pathway, namely prion-like-(QN-rich)-domain-bearing protein pathway (PQN), in a ced-1 dependent manner in C. elegans. Further, we also demonstrate that genes of the PQN pathway and antimicrobial peptide gene abf-2, are expressed at higher transcriptional levels in worms immediately following exposure to DT104, in comparison with worms exposed to SNS12. Altogether, our results suggest that the MDR region of Salmonella Typhimurium DT104 has a direct role in virulence against Caenorhabditis elegans.

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.

Genomic Analysis ofSalmonella entericaSerovar Typhimurium Definitive Phage Type 104

Genomic Analysis of<i>Salmonella enterica</i>Serovar Typhimurium Definitive Phage Type 104

Pathogenic potential of Salmonella Typhimurium DT104 following sequential passage through soil, packaged fresh-cut lettuce and a model gastrointestinal tract.

From a quantitative microbial risk assessment perspective it is important to know whether certain food environments influence the pathogenic potential of pathogens and to what extent. The purpose of the present study was to examine the pathogenic potential of S. Typhimurium DT104, measured as the capability to survive a simulated gastrointestinal tract system and the capability of adhering to and invading differentiated Caco-2 cells, after sequential incubation (without intermediate culturing) into soil, lettuce and cut lettuce stored under modified atmosphere (MAP) conditions. Two S. Typhimurium DT104 strains were used, one isolated from a pig carcass and one isolated from lettuce. The most important result of the present study is that the sequential incubation of S. Typhimurium in soil and lettuce slightly increased the capability of surviving the simulated gastric fluid, increased the capability to grow in the simulated intestinal fluid but decreased the capability of epithelial attachment and invasion and decreased the overall survival probability of the gastrointestinal tract system. Some variation in responses between the strains was observed, with the lettuce strain maintaining higher epithelial attachment capability and the carcass strains maintaining higher epithelial invasion capability. This study provided quantitative data on the effect of environmental and food matrices on the pathogenic potential of S. Typhimurium DT104 using a realistic system of sequential incubations in environmental and food matrices, followed by simulated gastrointestinal tract passage without intermediate culturing. These results could aid the development of more realistic quantitative microbial risk assessments.

Bactericidal effect of saffron ( Crocus sativus L.) on Salmonella enterica during storage

The presence of pathogenic bacteria in spices represents a public health risk as a possible cause of food contamination. Salmonella has been found in several spices and it has been involved in food-borne outbreaks, but this bacterium has not been reported as a contaminant of saffron ( Crocus sativus L.). We examined a possible antibacterial effect of saffron using samples from Iran, Greece and Spain which were artificially contaminated with clinical isolates belonging to five different serovars of Salmonella. We detected a loss of viability during the room-temperature storage of the saffron samples, with bacteria being undetectable at day 16 except in the case of the DT104 strain of the Typhimurium serovar, in all of the samples, and of the Hadar serovar in the Iranian sample, both of which gave negative culture at day 32. The laboratory strain LT2 of the Typhimurium serovar was undetectable at day 4. To gain an insight into the basis for this bactericidal effect, we measured the inhibitory and bactericidal concentrations of safranal and crocin, the main compounds responsible for the flavouring and colouring capabilities of saffron. They were in the order of 8–16 mg/mL and 64–128 mg/mL for safranal and crocin, respectively. These data suggest that these compounds, and probably their chemical relatives, are involved in the antibacterial activity of saffron, and that this effect can significantly reduce the risk of food contamination with Salmonella by this spice.

Epidemiological Characteristics of Salmonella enterica Serovar Typhimurium from Healthy Pigs in Japan

We characterized 53 Salmonella enterica ser. Typhimurium strains recovered from healthy pigs during 1998-1999 (n=12) and 2004-2005 (n=41) as to their carriage of DT104 spacer region, class 1 and 2 integrons, virulence genes (spvC, rck, and pefA), and XbaI- and BlnI-Pulsed-Field Gel Electrophoresis (PFGE) profiles. No DT104 strain was detected in 1998-1999, whereas 65.9% (27/41) of the strains in 2004-2005 were DT104 showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and cephaloridine (R-type ACSSuT+). Class 1 intergron-associated genes, aadA2 (1.0-kbp amplicon) and pse1 (1.2-kbp amplicon), were found in all the DT104 strains (27/27). One strain showing resistance to streptomycin, sulfonamides, tetracyclin, and trimethoprim (R-type SsuTTm) harbored another class 1 integron-associated gene (dhfrXII-orfF- aadA2) on 1.9-kbp amplicon. Virulence gene spvC was found in 92.5% (49/53) and rck and pefA were found in 88.7% (47/53) of the strains, whereas spvC, rck, and pefA were found in all the DT104 strains. Ser. Typhimurium strains were categorized into four clusters (X1, X2, X3, and X4a/X4b) by XbaI-PFGE, or into nine clusters (B1, B2, B3a/B3b, B4, B5, B6, B7, B8, B9a/B9b) by BlnI-PFGE analyses. DT104 strains were restricted into X2, or into B2, B3a/B3b, and B6 clusters, indicating that our multidrug-resistant DT104 strains from healthy pigs might have derived from at least three independent clones, with the most widespread clone being the cluster B6 strains isolated in Kanto, Tokai, Chugoku, and Kyushu regions.

Variation in Antimicrobial Resistance in Sporadic and Outbreak-relatedSalmonella entericaSerovar Typhimurium

The prevalence of different antimicrobial resistance profiles and variants of the Salmonella genomic island 1 (SGI1) was reported for Salmonella enterica serovar Typhimurium DT104 strains isolated from patients in Denmark. Variation in antimicrobial resistance and corresponding changes of SGI1 were shown among isolates from a foodborne outbreak.

Characterization of the Genomes of a Diverse Collection of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.

An Approach for Mapping the Number and Distribution of Salmonella Contamination on the Poultry Carcass

Mapping the number and distribution of Salmonella on poultry carcasses will help guide better design of processing procedures to reduce or eliminate this human pathogen from poultry. A selective plating media with multiple antibiotics (xylose-lysine agar medium [XL] containing N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) and the antibiotics chloramphenicol, ampicillin, tetracycline, and streptomycin [XLH-CATS]) and a multiple-antibiotic-resistant strain (ATCC 700408) of Salmonella Typhimurium definitive phage type 104 (DT104) were used to develop an enumeration method for mapping the number and distribution of Salmonella Typhimurium DT104 on the carcasses of young chickens in the Cornish game hen class. The enumeration method was based on the concept that the time to detection by drop plating on XLH-CATS during incubation of whole chicken parts in buffered peptone water would be inversely related to the initial log number (N0)of Salmonella Typhimurium DT104 on the chicken part. The sampling plan for mapping involved dividing the chicken into 12 parts, which ranged in average size from 36 to 80 g. To develop the enumeration method, whole parts were spot inoculated with 0 to 6 log Salmonella Typhimurium DT104, incubated in 300 ml of buffered peptone water, and detected on XLH-CATS by drop plating. An inverse relationship between detection time on XLH-CATS and N0 was found (r =−0.984). The standard curve was similar for the individual chicken parts and therefore, a single standard curve for all 12 chicken parts was developed. The final standard curve, which contained a 95% prediction interval for providing stochastic results for N0, had high goodness of fit (r2 = 0.968) and was N0 (log) = 7.78±0.61 − (0.995 × detention time). Ninety-five percent of N0 were within ± 0.61 log of the standard curve. The enumeration method and sampling plan will be used in future studies to map changes in the number and distribution of Salmonella on carcasses of young chickens fed the DT104 strain used in standard curve development and subjected to different processing procedures.

Novel Insertion Sequence- and Transposon-Mediated Genetic Rearrangements in Genomic Island SGI1 ofSalmonella entericaSerovar Kentucky

Salmonella genomic island 1 (SGI1) is an integrative mobilizable element that harbors a multidrug resistance (MDR) gene cluster. Since its identification in epidemic Salmonella enterica serovar Typhimurium DT104 strains, variant SGI1 MDR gene clusters conferring different MDR phenotypes have been identified in several S. enterica serovars and classified as SGI1-A to -O. A study was undertaken to characterize SGI1 from serovar Kentucky strains isolated from travelers returning from Africa. Several strains tested were found to contain the partially characterized variant SGI1-K, recently described in a serovar Kentucky strain isolated in Australia. This variant contained only one cassette array, aac(3)-Id-aadA7, and an adjacent mercury resistance module. Here, the uncharacterized part of SGI1-K was sequenced. Downstream of the mer module similar to that found in Tn21, a mosaic genetic structure was found, comprising (i) part of Tn1721 containing the tetracycline resistance genes tetR and tet(A); (ii) part of Tn5393 containing the streptomycin resistance genes strAB, IS1133, and a truncated tnpR gene; and (iii) a Tn3-like region containing the tnpR gene and the beta-lactamase bla(TEM-1) gene flanked by two IS26 elements in opposite orientations. The rightmost IS26 element was shown to be inserted into the S044 open reading frame of the SGI1 backbone. This variant MDR region was named SGI1-K1 according to the previously described variant SGI1-K. Other SGI1-K MDR regions due to different IS26 locations, inversion, and partial deletions were characterized and named SGI1-K2 to -K5. Two new SGI1 variants named SGI1-P1 and -P2 contained only the Tn3-like region comprising the beta-lactamase bla(TEM-1) gene flanked by the two IS26 elements inserted into the SGI1 backbone. Three other new variants harbored only one IS26 element inserted in place of the MDR region of SGI1 and were named SGI1-Q1 to -Q3. Thus, in serovar Kentucky, the SGI1 MDR region undergoes recombinational and insertional events of transposon and insertion sequences, resulting in a higher diversity of MDR gene clusters than previously reported and consequently a higher diversity of MDR phenotypes.

Aquaculture and Florfenicol Resistance inSalmonella entericaTyphimurium DT104

To the Editor: In June 2006, the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the World Organisation for Animal Health (OIE) convened an Expert Consultation to consider the risks to human health represented by the use of antimicrobial drugs in aquaculture. This would, therefore, appear to be an opportune time to reexamine some of the arguments that have been presented with respect to the assessment of these risks. In their contributions to the debate regarding the risks associated with the use of antimicrobial agents in aquaculture, Angulo (1), Angulo and Griffin (2), Ribot et al. (3), and, more recently, Cabello (4) have argued that the available molecular evidence suggests that the flo gene that encodes chloramphenicol and florfenicol resistance in Salmonella enterica serovar Typhimurium DT104 (DT104) originally emerged in Japanese aquaculture and may have transferred horizontally from this host to DT104. This argument also appears in the report of the WHO/FAO/OIE consultation (ftp://ftp.fao.org/ag/agn/food/aquaculture_rep_13_16june2006.pdf). These authors (1–4) have based their argument on the assertions that florfenicol was first used in Japan and that flo gene–mediated resistance to this agent was first identified in bacteria isolated from Japanese fish farms. In attempting to identify the date of the emergence of florfenicol resistance in Japanese aquaculture, Angulo and Griffin (2) state that florfenicol had been used in this country since the early 1980s. However, Schering Plough, the manufacturer of florfenicol, reports first marketing this agent for aquacultural use in Japan in 1990 (D. Schofield, pers. comm.), and this date for the introduction of florfenicol is also provided by Kim et al. (5). It should, however, be noted that the flo gene encodes resistance to both florfenicol and chloramphenicol and, therefore, flo-containing bacteria could be selected for by the use of either agent. As a consequence, arguments about the chronology of the first use of florfenicol may have limited relevance. Florfenicol resistance in Japanese aquaculture (5) was first reported in strains of Pasteurella piscicida (now renamed Vibrio damsela). The sequence of the gene that encoded florfenicol resistance in these strains (6) was demonstrated to have a 97% nucleotide sequence similarity to that found in strains of DT104 resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline (ACSSuT DT104) (7). The available data (5,8) allow a reasonably accurate estimate of the date when these florfenicol-resistant strains first emerged in Japanese aquaculture. The strains of florfenicol-resistant P. piscicida were first isolated in Japan in 1992 (5). However, a previous study (8) had demonstrated 100% susceptibility to florfenicol of P. piscicida strains isolated from 1989 through 1991. Because this study examined 175 P. piscicida strains isolated from fish farms distributed over a wide geographic area in Japan, the data it generated provide strong support for the conclusions that flo-mediated florfenicol resistance in P. piscicida first emerged in Japanese aquaculture in 1992 (5). However, the presence of the floR gene has been demonstrated in a strain of ACSSuT DT104 isolated in the United States in 1985 (3). Thus, the floR gene was present in DT104 strains isolated in the United States at least 7 years before the first bacteria containing this gene (6) were isolated from an aquaculture setting in Japan (5). There are, however, data indicating that the flo gene was present in terrestrial bacteria associated with humans long before it was detected in multidrug -resistant DT104. A gene with a 95%–97% nucleotide identity with the flo gene of DT104 was detected in the Inc C plasmid R55 (9). This plasmid was originally identified in a chloramphenicol-resistant strain of Klebsiella pneumoniae isolated from a person in Paris in 1969 (10). The earliest report of the isolation of a bacterium whose florfenicol resistance was encoded by a flo gene (6) and the earliest accession date for a flo gene sequence in GenBank (www.ncbi.nlm.nih.gov/Genbank) both related to P. damsela isolated from Japanese aquaculture. Publication and accession dates do not, however, constitute evidence of the date of the first isolation of a bacterium containing this gene. Analysis of the available chronological and molecular data presented here indicates that a variant of the floR gene in DT104 (9) was present in a terrestrial bacterium isolated in 1969 (10), 23 years before the first isolation, in 1992, of a bacterium associated with aquaculture that contained this gene (5). It further demonstrates that this gene was present in strains of DT104 isolated in 1985. Thus, these data provide no support for the arguments (1–4) that implicate aquacultural use of florfenicol or the subsequent occurrence of florfenicol-resistant P. damsela in the emergence of the flo gene in DT104.