Typhimurium DT104 Research Articles

Use of Saccharomyces cerevisiae fermentation product on growth performance and microbiota of weaned pigs during Salmonella infection1

Anaerobically fermented yeast products are a rich source of nutritional metabolites, mannanoligosaccharides, and β-glucans that may optimize gut health and immunity, which can translate into better growth performance and a reduced risk of foodborne pathogens. The objective of this study was to quantify the effects of Saccharomyces cerevisiae fermentation product (Diamond V Original XPC) inclusion in nursery diets on pig performance and gastrointestinal microbial ecology before, during, and after an oral challenge with Salmonella. Pigs (n = 40) were weaned at 21 d of age, blocked by BW, and assigned in a 2 × 2 factorial arrangement consisting of diet (control or 0.2% XPC) and inoculation (sterile broth or Salmonella). Pigs were fed a 3-phase nursery diet (0 to 7 d, 7 to 21 d, and 21 to 35 d) with ad libitum access to water and feed. On d 14, pigs were orally inoculated with 10(9) cfu of Salmonella enterica serovar Typhimurium DT104 or sterile broth. During d 17 to 20, all pigs were treated with a 5 mg/kg of BW intramuscular injection of ceftiofur-HCl. Growth performance and alterations in the gastrointestinal microbial ecology were measured during preinoculation (PRE; 0 to 14 d), sick (SCK; 14 to 21 d), and postinoculation (POST; 21 to 35 d). Body weight and ADG were measured weekly. Rectal temperature (RT) was measured weekly during PRE and POST, and every 12 h during SCK. Diet had no effect on BW, ADG, or RT during any period (P = 0.12 to 0.95). Inclusion of XPC tended (P < 0.10) to increase Salmonella shedding in feces during SCK. Consumption of XPC altered the composition of the gastrointestinal microbial community, resulting in increased (P < 0.05) populations of Bacteroidetes and Lactobacillus after Salmonella infection. Pigs inoculated with Salmonella had decreased ADG and BW, and increased RT during SCK (P < 0.001). Furthermore, fecal Salmonella cfu (log(10)) was modestly correlated (P = 0.002) with BW (r = -0.22), ADFI (r = -0.27), ADG (r = -0.36), G:F (r = -0.18), and RT (r = 0.52) during SCK. After antibiotic administration, all Salmonella-infected pigs stopped shedding. During POST, an interaction between diet and inoculation (P = 0.009) on ADG indicated that pigs infected with Salmonella grew better when eating XPC than the control diet. The addition of XPC to the diets of weanling pigs resulted in greater compensatory BW gains after infection with Salmonella than in pigs fed conventional nursery diets. This increase in BW gain is likely associated with an increase in beneficial bacteria within the gastrointestinal tract.

An imported enteric fever caused by a quinolone-resistantSalmonella typhi

Recent reports indicate that nalidixic acid susceptibility correlates well with the clinical outcome of patients with Salmonella Typhi infection treated with quinolones. We report a case of enteric fever caused by S Typhi in which the isolate was resistant to nalidixic acid, but showed in vitro susceptibility to ciprofloxacin. Following treatment with ciprofloxacin, the clinical outcome was not satisfactory and the patient had a relapse. However, after using a higher dose of ciprofloxacin, the patient was cured. We recommend that all Salmonella systemic infections resistant to nalidixic acid with in vitro but decreased susceptibility to fluoroquinolones be treated with other antibiotics like third-generation cephalosporins or azithromycin. These patients should be closely followed up and observed for further relapse.

Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain?

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1

Characterization of Resistance Genes in Multidrug-Resistant Salmonella enterica Serotype Typhimurium Isolated from Diseased Cattle in France (2002 to 2007)

We report here the results of the survey of antimicrobial resistance in 148 serotype Typhimurium strains isolated from cattle in France from 2002 to 2007 and displaying more than two antimicrobial resistances. Salmonella enterica serotype Typhimurium of definitive phage type 104 strains that are commonly resistant to ampicillin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfonamides, and tetracycline (ACSSuT phenotype) harbored resistance genes clustered on a complex class 1 integron In104 of the Salmonella genomic island 1 (SGI1). In our isolates, the most common antimicrobial resistance pattern was ACSSuT (77.7%) or ACSSuT combined to additional resistances. SGI1 was detected in 143 strains and constituted thus the main structure involved in resistance to antimicrobials in these strains. In spite of the high recombination potential of In104, SGI1 variability was quite limited among these strains since only two SGI1 variants, SGI1-B and SGI1-C, were identified. One hundred and thirty-eight out of the 143 SGI1-positive isolates belonged to the DT104 complex. Pulsed-field gel electrophoresis profile A was the most prevalent in 135 SGI1-positive isolates, confirming the diffusion of the DT104 clone. However, changes in phages susceptibility have occurred in three serotype Typhimurium strains of phage type DT12, as they displayed the same pulsed-field gel electrophoresis profile as the SGI1-positive serotype Typhimurium DT104. No variant harboring an additional resistance gene was identified, but the risk of recombination between SGI1 and any other mobile structure carrying other antimicrobial resistance genes is still an issue in serotype Typhimurium.

Comparison between pre-enrichment in single- or double-strength buffered peptone water for recovery of Salmonella enterica serovar Typhimurium DT104 from acidic marinade sauces containing spices

The ability of the standard pre-enrichment procedure in buffered peptone water (BPW) to recover Salmonella Typhimurium from acidic marinade sauces containing spices was tested by inoculating marinade sauces with known numbers of an antibiotic-resistant marker strain of Salmonella Typhimurium DT104 prior to pre-enrichment. Viable numbers of salmonellae present in BPW after 24 h incubation depended on the inoculum level. If initial cell numbers were low (below 10 3 cfu per 250 ml BPW) final cell concentrations were also low and, in some cases, no growth occurred. The problem was overcome by use of double-strength BPW that neutralised the acidity and allowed good recovery from otherwise inhibitory marinade sauces.

Epidemiology of plasmid-mediated quinolone resistance in Salmonella enterica serovar Typhimurium Isolates from Food-Producing Animals in Japan

A total of 225 isolates of Salmonella enterica serovar Typhimurium from food-producing animals collected between 2003 and 2007 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, namely qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')Ib-cr, in Japan. Two isolates (0.8%) of S. Typhimurium DT104 from different dairy cows on a single farm in 2006 and 2007 were found to have qnrS1 on a plasmid of approximately 9.6-kbp. None of the S. Typhimurium isolates had qnrA, qnrB, qnrC, qnrD, qepA and acc(6')-Ib-cr. Currently in Japan, the prevalence of the PMQR genes among S. Typhimurium isolates from food animals may remain low or restricted. The PFGE profile of two S. Typhimurium DT104 isolates without qnrS1 on the farm in 2005 had an identical PFGE profile to those of two S. Typhimurium DT104 isolates with qnrS1. The PFGE analysis suggested that the already existing S. Typhimurium DT104 on the farm fortuitously acquired the qnrS1 plasmid.

Salmonella serovars and their antimicrobial resistance in British turkey flocks in 1995 to 2006

Serovar and antimicrobial resistance data from the scanning surveillance of British turkey flocks for Salmonella between 1995 and 2006 were analysed and compared with prevalence data from other livestock and animal feed. A total of 2753 incidents of 57 different serovars were reported. The five most prevalent serovars were Salmonella Typhimurium (20.8%), Salmonella Newport (14.7%), Salmonella Derby (10.6%), Salmonella Indiana (8.3%) and Salmonella Agona (6.4%). S. Typhimurium reports peaked in the mid- to late 1990s; this occurred in parallel with the S. Typhimurium DT104 epidemic in other livestock species. S. Enteritidis reports peaked in mid- to late 1990s, followed by a considerable decrease after 2000, which was also noted in flocks of domestic fowl. S. Newport, Salmonella Montevideo, Salmonella Senftenberg and Salmonella Binza occurred in marked clusters, indicating that they were introduced into one or more flocks at a certain time (i.e. via contaminated feed or infected 1-day-old chicks). A proportion of 43.1% of the reported Salmonella isolates were resistant to at least one antimicrobial, while 17.7% were multi-resistant. No isolates were resistant to ciprofloxacin or to the third-generation cephalosporins ceftazidime and cefotaxime. Resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide compounds and tetracycline was common, and it was mainly a characteristic of S. Typhimurium DT104 compared with S. Typhimurium non-DT104 and non-S. Typhimurium isolates (P<0.001). Resistance to nalidixic acid decreased from 16.9% in 1995 to 11.8% in 2006. Nalidixic acid resistance was most frequently found in Salonella Hadar (71.4%), S. Typhimurium DT104 (30.0%), S. Newport (17.9%) and S. Typhimurium non-DT104 (11.1%).

Aquaculture and Florfenicol Resistance inSalmonella entericaSerovar Typhimurium DT104

In his letter (1), Cabello makes 2 observations regarding the debate concerning the origin of the floR gene in Salmonella enterica serovar Typhimurium DT104. The first observation is that the plasmid PASPPFLO contained cloned segments of an indigenous Vibrio damsela plasmid. However, PASPPFLO is not the name of a plasmid but is the GenBank locus identifier associated with the sequence (GenBank accession no. D37826) of a 3,745-bp region of the V. damsela plasmid pSP92088 that contained pp-flo (2,3). The second observation is that sequences flanking the floR gene in S. enterica serovar Typhimurium DT104 (GenBank accession no. AF071555) are homologous to those flanking the pp-flo gene sequenced from the V. damsela plasmid pSP92088 (4). On the basis of this homology, he seems to assume that these flanking sequences must have originated in V. damsela and, therefore, that they constitute a molecular signature that firmly establishes the aquaculture origin of this florfenicol resistance. What Cabello does not mention is that sequences flanking a wide range of floR genes, including those in plasmid R55 (GenBank accession no. AF332662), are also homologous to those found in S. enterica serovar Typhimurium DT104 (5,6). These data suggest that during horizontal transfer between species and genera, the association of floR with its flanking regions has been conserved (5,6). However, the data provide no evidence for postulating a unique association of these flanking sequences with V. damsela, and, therefore, do not provide evidence for an aquaculture origin of floR. If Cabello believes that sequences flanking floR in S. enterica serovar Typhimurium DT04 constitute a molecular signature that firmly establishes the aquaculture origin of floR in S. enterica serovar Typhimurium DT104, he should provide some explanation as to how this signature was also present in the R55 plasmid detected in a Klebsiella pneumoniae strain isolated in 1969 (5,7).

Influence of a Probiotic Strain ofEnterococcus faeciumonSalmonella entericaSerovar Typhimurium DT104 Infection in a Porcine Animal Infection Model

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.

Effects of reduction in beef surface water activity on the survival ofSalmonellaTyphimurium DT104 during heating

To investigate the influence of reducing beef surface water activity (a(w)) on the survival of Salmonella Typhimurium DT104 during heating. Beef discs were surface inoculated with S. Typhimurium DT104 and either untreated or dried to achieve surface a(w) values of 0.95, 0.85 and 0.70. The samples were vacuum packed, heat-treated at 60 degrees C and removed at predetermined times. The inactivation curves were influenced by a(w) and treatment time. Biphasic inactivation curves were observed for S. Typhimurium DT104 heat-treated on beef samples with altered a(w) values, which were characterized by an initial decline in cell numbers at commencement of heating followed by a much slower rate of inactivation during the remaining treatment period. Point estimates of the heating time required to achieve a 1 log reduction on beef surfaces with a(w) of 0.99, 0.95, 0.85 and 0.70 were 0.5, 1.55, 11.25 and 17.79 min, respectively. A decrease in beef surface a(w) can substantially enhance the survival of S. Typhimurium DT104 after heating. Caution needs to be taken using dry air as a decontamination method as this may rapidly decrease product surface and pathogen a(w) values resulting in enhanced survival.

Prevalence and antibiotic-resistance of Salmonella isolated from food in Morocco

Salmonellosis remains one of the most frequent food-borne diseases worldwide, especially in developing countries. The emergence of antimicrobial resistance in Salmonella isolates from food can potentially compromise the treatment of these infections. This investigation was conducted for the first time in Morocco both to detect the occurrence of Salmonella in foods as well as to determine the antibiotic resistance profile of the Salmonella isolates. In total, 11,516 food samples collected from 2002 to 2005 were investigated. Isolated Salmonella were characterized by serotyping and susceptibilities were determined for 15 antimicrobial drugs using the disc diffusion assay. The overall percentage of Salmonella prevalence (n=105) was 0.91% with rates of 71% for slaughterhouses and 9% for seafood. Sixteen different serotypes were identified among 104 Salmonella enterica isolates including serotypes Infantis (n=25), Bredeney (n=13), Blokley (n=11), Typhimurium (n=9), Mbandaka (n=8), Branderup II (n=7), and Kiambu (n=6); 1 isolate of Salmonella enterica belonged to subspecies II salamae. Twenty-nine percent of isolates (n=30/105) were resistant to at least one antimicrobial. Resistance to tetracycline was the most common finding (21%), followed by resistance to ampicillin (13%), amoxicillin+clavulanic acid (9%), streptomycin (7%), chloramphenicol (4%) and nalidixic acid (3,8%). None of the isolates was resistant to 3rd-cephalosporin and fluoroquinolones (i.e. ciprofloxacin). Multidrug resistance (MDR) was seen in 9.5% of the isolates, mainly in S.. Typhimurium DT104 with R-type ACSSuT and S. Hadar. Despite a low frequency of Salmonella isolation, S. Typhimurium DT104 was identified in the first step of the food chain. The study points out the need control antibiotic resistance in Salmonella isolated from food in Morocco to avoid the spread of MDR.

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.

The survival ofSalmonella entericaserovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5 degrees C or 10 degrees C). The influence of low water activity (a(w)) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10 degrees C) in an environmental cabinet. Salmonella counts declined during storage at low a(w) (75% RH) conditions at 5 degrees C or 10 degrees C. Salmonella counts increased during storage at high a(w) (96% RH) at 10 degrees C only. At 5 degrees C, TVCs increased during storage at high a(w), but not at low a(w). TVCs increased on all samples from carcasses stored at high or low a(w) at 10 degrees C, except those samples taken from areas of surface fat. This suggests that substrate composition dictates growth rates under low a(w) conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth. The data obtained in this study provides useful insights on the influence of a(w) and temperature on pathogen survival on meat surfaces at chill temperature.

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.

Antimicrobial resistance of Salmonella enterica serovar Typhimurium isolated from cattle in Japan

The antimicrobial susceptibility of 144 Salmonella enterica serovar Typhimurium isolates collected from all over Japan between 1973 and 1998 were investigated. All the isolates exhibited resistance to four or more antimicrobials and 22 resistance patterns were observed. Isolates showing resistance patterns to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su) and tetracycline (T), which are typical resistance patterns for S. Typhimurium DT104 (DT104), were predominant. Thirty-six of the 68 isolates that exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 by phage typing. Another 103 S. Typhimurium strains gathered from cattle between 1977 and 1999 in a limited area of Japan were analyzed for molecular epidemiological studies. Results using fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis suggest that clonal exchange of S. Typhimurium among cattle in Japan has occurred since 1992, and that contemporary strains show a remarkable degree of homogeneity with DT104 at a molecular level. The clonal replacement by DT104 affected the antimicrobial resistance pattern of S. Typhimurium from cattle in Japan.

A PORTABLE RAMAN SYSTEM FOR THE IDENTIFICATION OF FOODBORNE PATHOGENIC BACTERIA

ABSTRACT Raman spectroscopy is emerging as an important nondestructive, noninvasive, analytical tool for the analysis of biologic materials. This study presents a procedure to make use of commercial off‐the‐shelf components to construct a portable dispersive Raman system and evaluates it for discrimination of bacteria by surface‐enhanced Raman scattering (SERS). The system consists of a semiconductor laser (784.8 nm), a fiber optic probe (∼135 µm focal spot), a mini spectrometer and a computer. UV‐visible spectroscopy and transmission electron microscopy analysis of four silver colloid preparations produced in this study, together with the SERS spectra of Listeria innocua adsorbed on colloidal particles, indicated that silver colloids with the extinction maximum at &gt;415 nm (particle size &gt;75 nm) and a larger long wavelength tail are capable of promoting SERS of bacteria. The SERS spectra of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica were acquired with the system, leading to an unambiguous identification of these bacterial foodborne pathogens on the basis of their unique spectral bands. This study demonstrated the feasibility of constructing a low‐cost compact Raman system using commercially available components to perform the SERS analysis of bacteria.PRACTICAL APPLICATIONSCommercial off‐the‐shelf components such as a semiconductor laser, a fiber optic probe and a mini spectrometer can be used to construct a portable, low‐cost dispersive Raman system. Such system allows for the acquisition of SERS spectra of bacteria adsorbed on silver colloidal nanoparticles, as exemplified by three important bacterial foodborne pathogens L. monocytogenes (serotype 4b), E. coli O157:H7 and S. enterica (serotype Typhimurium DT 104). An unambiguous identification of these pathogens was achieved based on their unique spectral bands, indicating that an inexpensive dispersive Raman system such as the one described here may be built for the rapid characterization of bacteria isolates from food, clinical and environment samples using SERS spectral fingerprints.

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.

Secondary Chromosomal Attachment Site and Tandem Integration of the Mobilizable Salmonella Genomic Island 1

BackgroundThe Salmonella genomic island 1 is an integrative mobilizable element (IME) originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104. SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. Previous studies have shown that SGI1 integrates site-specifically into the S. enterica, Escherichia coli, or Proteus mirabilis chromosome at the 3′ end of thdF gene (attB site).Methodology/Principal FindingsHere, we report the transfer of SGI1 to a ΔthdF mutant of S. Typhimurium LT2. In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude. Through DNA sequencing SGI1 was shown to integrate specifically into a secondary attachment site (2nd attB), which is located in the intergenic region between the chromosomal sodB and purR genes. At this 2nd attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of ΔthdF mutant) contained tandem SGI1 arrays. Moreover, in wild type S. Typhimurium LT2 transconjugants, SGI1 integrated into both attachment sites, i.e., thdF and sodB-purR. The formation of SGI1 tandem arrays occurred in both specific attB sites. There was heterogeneity in the size of the SGI1 tandem arrays detected in single transconjugant colonies. Some arrays consisted as far as six SGI1s arranged in tandem. These tandem arrays were shown to persist during serial passages with or without antibiotic selection pressure.Conclusions/SignificanceThe ability of integration into two distinct chromosomal sites and tandem array formation of SGI1 could contribute to its spread and persistence. The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1st or 2nd specific SGI1 attB sites of Salmonella.