Salmonella Genomic Island 1 Research Articles

A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella entericaserotype Typhimurium

BackgroundTyphimurium is the main serotype of Salmonella enterica subsp. enterica implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid pSLT and Salmonella genomic island 1 (SGI1)) as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104). To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (sul1) and beta-lactam (blaTEM) resistance. Finally, the intI1 determinant encoding integrase from class 1 integron was also investigated.ResultsA total of 538 unrelated S. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, intI1, sul1 and blaTEM determinants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.ConclusionThe GeneDisc® assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

The Salmonella Genomic Island 1 Is Specifically Mobilized In Trans by the IncA/C Multidrug Resistance Plasmid Family

BackgroundThe Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55.Methodology/Principal FindingsHere, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency <10−9). In our collection, ESBL gene-carrying plasmids were mainly from the IncHI2 and I1 groups and thus were unable to mobilize SGI1. However, the horizontal transfer of SGI1 was shown to be specifically mediated by conjugative helper plasmids of the broad-host-range IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10−3 to 10−6 transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase bla CMY-2 gene were shown to mobilize in trans SGI1.Conclusions/SignificanceThe ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic resistance genomic island SGI1 and its close derivatives.

Occurrence and Distribution of Various Genetic Structures of Class 1 and Class 2 Integrons inSalmonella entericaIsolates from Foodborne Disease Patients in Korea for 16 Years

We have investigated the distribution of integrons among 752 multidrug-resistant Salmonella isolates from human febrile and/or diarrheal patients during 1992-2007 and analyzed their genetic characteristics. Here, we report extensive integron analysis results within human isolates during the last 16 years. The gene or gene cassette(s) in the class 1 integrons found in the isolates were dfrA7, dfrA12-orfF-aadA2, aadA2, bla(PSE1), dfrA1-aadA1, dfrA17-aadA5, bla(OXA1)-aadA1, aadB-aadA1, aadA22, aadA1, and aac6'Ib-bla(OXA1)-aadA2. Class 2 integrons harboring dfrA1-sat2-aadA1 gene cassette were also found in four isolates. Twenty-nine isolates including one Salmonella Schleissheim isolate had two integrons harboring aadA2 and bla(PSE1) in their variable regions of 1.0 and 1.2 kb amplicons, respectively, which have been also found in Salmonella genomic island 1 (SGI1) of multidrug-resistant Salmonella Typhimurium DT104. The presence of SGI1 in Salmonella Schleissheim isolate was proved by SGI1-specific polymerase chain reaction. We first report a Salmonella Schleissheim having SGI1, Salmonella Typhimurium and Salmonella Heidelberg having the class 2 integron with dfrA1-sat2-aadA1 cassettes, Salmonella London with the aac6'Ib-bla(OXA1)-aadA2 gene cassette, Salmonella Chailey with the gene cassette of aadA22, and coexistence of two class 1 integrons carrying aadA22 and dfrA12-orfF-aadA2 in Salmonella Typhimurium.

Salmonella Genomic Islands and Antibiotic Resistance in Salmonella Enterica

Antibiotic resistance in several Salmonella enterica serovars that cause gastrointestinal disease in humans is due to a set of related genomic islands carrying a class 1 integron, which carries the resistance genes. Salmonella genomic island 1 (SGI1), the first island of this type, was found in S. enterica serovar Typhimurium DT104 isolates, which are resistant to ampicillin, chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamides and tetracycline. Several Salmonella serovars and Proteus mirablis have since been shown to harbor SGI1 or related islands carrying various sets of resistance genes and some distinct groups have emerged. SGI1 is an integrative mobilizable element and can be transferred experimentally into Escherichia coli. However, within serovars, isolates recovered from different parts of the world appear to be clonal, indicating that SGI1 movement may be rare. Potential reservoirs in food-producing animals or in ornamental fish have been identified for some serovars.

Evaluation of the pathogenicity and virulence of three strains of Salmonella organisms in calves and pigs

To assess in pigs the pathogenicity and virulence of 3 strains of Salmonella spp capable of causing atypical salmonellosis in cattle. 36 Holstein calves and 72 pigs experimentally infected with Salmonella spp. Representative Salmonella strains associated with 3 new disease phenotypes (protozoa-mediated hypervirulence, multisystemic cytopathicity, and encephalopathy) that have been characterized in cattle during the past 10 years were orally inoculated into pigs. Clinical manifestations were compared with those observed in cattle. Samples were collected from various tissues, and the presence of Salmonella organisms was assessed qualitatively and quantitatively by use of Salmonella-selective media. Of the 3 unique Salmonella disease phenotypes observed in cattle, only protozoa-mediated hypervirulence was observed in pigs. Hypervirulence was related to a more rapid onset of disease and higher pathogen burden in pigs than in cattle. This phenotype was observed in pigs inoculated with multiresistant Salmonella enterica serotypes Typhimurium or Choleraesuis bearing the Salmonella genomic island 1 (SGI1) integron. Salmonella hypervirulence was identified in pigs noculated with SGI1-bearing strains exposed to free-living protozoa. Additionally, an SGI1-bearing strain of Salmonella Choleraesuis was detected that resulted in augmented virulence in pigs. Therefore, it appeared that protozoa-associated salmonellosis was analogous in pigs and cattle. Salmonella-mediated encephalopathy and multisystemic cytopathicity did not appear to be relevant diseases in pigs.

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.

Genetic analysis of multidrug-resistant Salmonella enterica serovars Stanley and Typhimurium from cattle

During 2005–2008, a longitudinal study was conducted in southern Japan to detect and characterize multidrug-resistant Salmonella enterica serovars recovered from cattle diagnostic specimens. Determination of antimicrobial resistance phenotypes and genotypes, identification of Salmonella genomic island 1 (SGI1), detection of virulence genes, plasmid analysis, conjugal transfer experiments, and sequencing of class 1 integrons were conducted. Multidrug-resistant Salmonella detected were serovars Stanley, Typhimurium, and O4:d. Salmonella Stanley isolates exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, trimethoprim, and kanamycin (ACSSuT+) encoded by bla TEM, catA, aadA2, tetA, sul1, dfrA12, and aphA1 genes, respectively. Sequencing analysis revealed that aadA2 and dfrA12 were integrated as gene cassettes within the class 1 integrons of 1.5 kb size. Importantly, the isolates harboured easily transferable plasmids of ca. 210 kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. Moreover, Salmonella Typhimurium DT104 isolates with typical SGI1 were detected and presented ACSSuT+ resistance phenotype encoded by bla PSE-1 and bla TEM; floR; aadA1; sul1; and tetA and tetG, respectively. Salmonella Typhimurium isolates carried plasmids of variable sizes ranging from 3.5 to 100 kb with DT104 isolates harbouring plasmids of ca. 90 kb. Salmonella serovar O4:d had ACSSuT+ resistance phenotype mediated by bla TEM, catA, aadA1, sul1, tetA, and aphA1 genes. A virulence gene invA was found in all multidrug-resistant Salmonella Typhimurium, Stanley and O4:d clinical isolates. In conclusion, this is the first report describing the occurrence of multidrug-resistant Salmonella Stanley from bovine species. The emergence of Salmonella Stanley isolates exhibiting plasmid-encoded high-level multidrug resistance is an important health concern because this new pathogenecity was associated with mortality in cattle.

An Additional Novel Antimicrobial Resistance Gene Cluster inSalmonellaGenomic Island 1 of aSalmonella entericaSerovar Typhimurium DT104 Human Isolate

The multi-antimicrobial resistance gene cluster and its derivatives have been detected in Salmonella genomic island 1 (SGI1), which has been identified in the Salmonella enterica serovar Typhimurium, phage types DT104, DT12, DT120, and U302, as well as other Salmonella serovars, including Agona, Paratyphi B, Albany, Meleagridis, Newport, Cerro, Derby, Dusseldorf, Infantis, Kiambu, and Emek. We acquired 53 Salmonella Typhimurium DT104 isolates from diarrheal patients in Korea. From these isolates, we identified a novel antimicrobial resistance gene cluster as an additional gene cassette in SGI1 from a multi-antimicrobial resistant isolate. The minimum inhibitory concentration for this isolate against ampicillin and chloramphenicol was two to four times higher than those for other multi-antimicrobial-resistant Salmonella Typhimurium DT104 isolates. The new antimicrobial resistance gene cluster detected in this isolate consisted of bla(PSE-1), sul1 Delta, floR, and tetR, in that order. The order of this gene cluster was shuffled as compared to that of the known In104 in SGI1. This report is, to the best of our knowledge, the first to identify and describe an additional shuffled antimicrobial resistance gene cluster in SGI1.

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.

Association of IS26-composite transposons and complex In4-type integrons generates novel multidrug resistance loci in Salmonella genomic island 1

Clinical isolates of Salmonella enterica serovar Haifa and Newport, which displayed extended multidrug resistance phenotypes, were investigated for the presence of Salmonella genomic island 1 (SGI1) and the genetic organization of its antibiotic resistance gene clusters. The S. enterica strains were isolated from humans in France in 2003 and 2004. Antibiotic susceptibility tests and various molecular techniques were used for detection and characterization of SGI1. We identified SGI1 integrated in the 3' end of the chromosomal thdF gene in six multidrug-resistant serovar Haifa and Newport strains. Two strains, of serovar Haifa and Newport, harboured the previously described SGI1-H variant. A new variant of the novel SGI1-Ks group, named SGI1-K6, revealed IS26-mediated rearrangements of the antibiotic resistance gene cluster in two serovar Newport strains. Two other serovar Newport strains harboured the SGI1-L complex class 1 integron containing the dfrA15 and bla(PSE-1) resistance gene cassettes. In addition, these variants of SGI1 also contained large IS26-composite transposons inserted by a transposition event in the SGI1 backbone. These IS26-composite transposons showed a similar genetic structure to the SGI1-K variants containing an In4-type integron, a mercury resistance operon and parts of Tn1721 and Tn5393. These extended resistance gene clusters containing up to 10 antibiotic resistance genes were named SGI1-L1 and -L2. The serovar Haifa represents the 16th S. enterica serovar in which SGI1 has been identified. The genomic island SGI1 appears to be a hotspot of acquisition of antibiotic resistance genes by the transposition of In4-type integrons and large IS26-composite transposons.

Characterisation of antimicrobial resistance-associated integrons and mismatch repair gene mutations in Salmonella serotypes

In this study, we examined the presence of integrons and Salmonella genomic island 1 (SGI1) and assessed their contribution to antimicrobial resistance as well as determining the extent of the mutator phenotype in Salmonella isolates. A total of 81 Salmonella enterica serotype Typhimurium isolates were examined for the presence of integrons and SGI1 and for hypermutators using polymerase chain reaction (PCR) and the mutator assay, respectively. An additional 336 Salmonella isolates were also used to screen for hypermutators. Fourteen S. Typhimurium isolates carried class 1 integrons, of which six were shown to possess SGI1. Five putative mutators, S. Typhimurium ST20751, S. enterica serotype Heidelberg 22396 and S. enterica serotype Enteritidis 17929, 17929N and 17929R, were identified among the 417 Salmonella isolates. Complementation analysis with the wild-type mutH, mutL, mutS and uvrD genes indicated that none of the five mutators contained defective mismatch repair (MMR) system alleles. DNA sequence analysis revealed that single point mutations resulting in aspartic acid (codon 87) substitution in the gyrA gene conferred resistance to nalidixic acid and/or other fluoroquinolone drugs (ciprofloxacin and enrofloxacin) among four isolates. Our findings indicated that integrons and SGI1 play an important role in multidrug resistance in Salmonella. The incidence of hypermutators owing to defective MMR in Salmonella appears to be rare.

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.

Real-Time PCRs and Fingerprinting Assays for the Detection and Characterization ofSalmonellaGenomic Island-1 Encoding Multidrug Resistance: Application to 445 European Isolates ofSalmonella, Escherichia coli, Shigella, andProteus

Salmonella Genomic Island-1 (SGI-1) harbors a cluster of genes encoding multidrug resistance (MDR). SGI-1 is horizontally transmissible and is therefore of significant public health concern. This study presents two novel realtime PCRs detecting three SGI-1 protein-coding genes and a SGI-1 fingerprinting assay. These assays were applied to 445 European enterobacterial isolates. Results from real-time PCRs were comparable to those obtained from gelbased PCRs used for the detection of SGI-1, but were rapid to perform and suitable for large-scale screening. Furthermore, real-time PCRs also detected SGI-1 even when only part of the island was present in bacterial isolates. No trace of SGI-1 was detected in isolates other than Salmonella enterica. The fingerprints showed that regions of SGI-1 outside the MDR region exhibited genomic variations between isolates. In conclusion, the realtime PCRs described here are suitable for the detection of SGI-1 in bacterial isolates. Further studies are necessary to elucidate divergence in its non-MDR region.

Salmonella Genomic Island 1 (SGI1) and genetic characteristics of animal and food isolates of Salmonella Typhimurium DT104 in Hungary

To study the genetic characteristics of DT104 strains of Salmonella Typhimurium and the prevalence of Salmonella Genomic Island (SGI1) in Hungary, 140 recent Salmonella strains of food and animal origin were examined. For the first time in Hungary, the SGI1 was found in 17 out of 59 S. Typhimurium isolates (all proven to be DT104 phage type). These 17 strains were then subtyped by pulsed-field gel electrophoresis (PFGE) into 6 pulsotypes which were less correlated with the geographic origin than with the animal species of origin.

Biofilm formation in field strains of Salmonella enterica serovar Typhimurium: Identification of a new colony morphology type and the role of SGI1 in biofilm formation

In this study we examined the extent of biofilm formation in field strains of Salmonella enterica serovar Typhimurium ( S. Typhimurium), an important foodborne pathogen. Ninety-four field strains of S. Typhimurium were tested for their ability to form biofilm and components contributing to its formation. Most S. Typhimurium strains were highly capable of biofilm formation except for strains of phage type DT2 originating from pigeons. The most efficient biofilm forming strains were those of phage type DT104 positive for Salmonella genomic island 1 (SGI1). A comparison of SGI1 positive and negative strains indicated that the increased biofilm formation of SGI1 positive strains was associated with the presence of this genomic island. Finally, in five strains we found an alternative strategy of biofilm formation independent of curli fimbriae and cellulose production but solely dependent on an overproduction of capsular polysaccharide. Due to a mucoid and brown appearance on Congo Red agar we designated these strains as belonging to the SBAM (smooth brown and mucoid) morphotype.

Salmonella genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in proteus mirabilis clinical and food isolates from China.

Salmonella genomic island 1 (SGI1) and variants (SGI1-I and the new variant SGI1-O) were mapped in five strains of Proteus mirabilis isolated from humans and food in China. Sequencing showed that SGI1 and variants were integrated at the 3' end of the chromosomal thdF gene as previously described for Salmonella strains.

Less frequent Salmonella serovars as a reservoir of antimicrobial resistance

Sir, Salmonella enterica serovars Enteritidis and Typhimurium are the most frequent Salmonella serovars isolated from humans. However, a significant proportion of human Salmonella isolates belong to other serovars, for which antimicrobial susceptibility patterns are rarely reported. In Denmark, the frequency of isolation of these less frequent Salmonella serovars from human clinical samples has increased in recent years. They represented 30% of all human Salmonella isolates, which corresponded to an incidence of 10.5 per 100 000 inhabitants in 2005. From 2003 to 2005, 530 isolates belonging to these Salmonella serovars were tested for their antimicrobial susceptibility by broth microdilution (Sensititre; Trek Diagnostic Systems Ltd, East Grinstead, UK) at the Statens Serum Institut. These included 106 Salmonella Virchow, 95 Salmonella Newport, 91 Salmonella Stanley, 80 Salmonella Dublin, 53 Salmonella Hadar, 38 Salmonella Saintpaul, 32 Salmonella Derby, 20 Salmonella Uganda and 15 Salmonella Anatum obtained from sporadic clinical infections, isolated at the 16 clinical microbiology laboratories in Denmark. The MICs for these 530 human clinical Salmonella isolates were determined for ampicillin (1–32 mg/L), cefalotin (4–32 mg/L), chloramphenicol (2–64 mg/L), ciprofloxacin (0.03–4 mg/L), gentamicin (1–32 mg/L), nalidixic acid (8–64 mg/L), sulfamethoxazole (64–1024 mg/L), streptomycin (4–64 mg/L), tetracycline (2–32 mg/L) and trimethoprim (4–32 mg/L). The CLSI (formerly NCCLS) breakpoints were used. For ciprofloxacin, a breakpoint of 0.125 mg/L was also used, and for streptomycin, a breakpoint of 32 mg/L was used. Escherichia coli ATCC 25922 was used for quality control. The number of tested isolates and the percentages of resistance are reported in Table 1. Isolates belonging to some serovars, e.g. Salmonella Dublin, Salmonella Saintpaul and Salmonella Derby, were susceptible to most antimicrobials tested, whereas isolates belonging to other serovars showed much higher frequencies of resistance, e.g. Salmonella Virchow, Salmonella Newport and Salmonella Stanley. A high prevalence of multidrug resistance (resistance to four or more antimicrobial agents) was observed among isolates belonging to the following serovars: Salmonella Hadar (30%), Salmonella Virchow (25%), Salmonella Stanley (22%) and Salmonella Newport (19%). Resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline was present in Salmonella Newport (15%), Salmonella Stanley (8%) and Salmonella Virchow (1%). Resistance to these five antimicrobials could indicate the presence of the multidrug-resistant region Salmonella genomic island 1 (SGI1). This region is well described and common in Salmonella Typhimurium phage type DT104. It has also been found in other Salmonella serovars, e.g. Salmonella Newport. Of the tested Salmonella Hadar and Salmonella Virchow isolates, 87% and 80%, respectively, were resistant to nalidixic acid. However, these isolates were not resistant to ciprofloxacin when the CLSI breakpoint (4 mg/L) was used. When using the EUCAST breakpoint for ciprofloxacin (0.125 mg/L), a good consistency was observed between nalidixic acid and ciprofloxacin resistance frequencies, even though minor differences were

Potential integration sites of the Salmonella genomic island 1 in Proteus mirabilis and other bacteria

Sir, We read with great interest the article of Ahmed et al. which described the occurrence of a variant Salmonella genomic island 1 (SGI1) in a clinical isolate of Proteus mirabilis. SGI1 was described initially in epidemic multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) phage type DT104 strains. The 43 kb SGI1 is located between the thdF and int2 genes of the chromosome of S. Typhimurium and contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides and tetracyclines. The int2 gene is part of a retron sequence which has been reported only in S. 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 called In104. Variant SGI1 antibiotic resistance gene clusters have been described in a wide variety of S. enterica serovars and have been named SGI1-A to SGI1-L. – 5 These gene clusters were likely generated after chromosomal recombinational events or by antibiotic resistance gene cassette replacement at one of the attI1 sites. Recently, SGI1 has been shown to be mobilized using the R55 conjugative plasmid and thus transferred by conjugation to Salmonella or Escherichia coli recipient strains. The site specific integration in Salmonella or E. coli occurs at the last 18 bp of the 30 end of the thdF gene named attB. The attP site, which is almost identical to attB, is present on the mobile circular form of SGI1. Thus integration or excision of SGI1 into the chromosome occurs through homologous recombination between the attB and attP sites mediated by the integrase int gene which is the first gene found at the 50 end of SGI1. SGI1 is thus considered to be an integrative mobilizable element. Interestingly, for the first time, Ahmed et al. identified SGI1 in a bacterium other than Salmonella. The antibiotic resistance gene cluster identified corresponded to that of the SGI1-L complex integron reported previously in S. Newport where the aadA2 gene cassette is replaced by a dfrA15 trimethoprim resistance gene cassette at the first attI1 site of this complex integron. However, although a circular extrachromosomal form of SGI1 was detected in P. mirabilis, the authors failed to identify the integration site in the P. mirabilis chromosome. The authors used the primers U7-L12 (targeting the Salmonella thdF gene) and LJ-R1 (targeting the SGI1 int gene) to confirm by PCR the location of SGI1 downstream of thdF in the Salmonella chromosome. Since this PCR failed to produce an amplicon in P. mirabilis 18306, the authors concluded that the integration site of SGI1 into the chromosome of P. mirabilis 18306 may be different from that of S. enterica. Using the Blastn algorithm (http://www.ncbi.nlm.nih.gov/) we searched for a thdF gene homologue in the P. mirabilis strain HI4320 sequenced genome (http://www.sanger.ac.uk/ cgi-bin/blast/submitblast/p_mirabilis) and identified one gene that is 70% identical in nucleotide sequence to that of the S. Typhimurium thdF gene. The last 18 bp of the putative P. mirabilis thdF gene had a nucleotide sequence that was 100% identical to that of the SGI1 attP site and thus likely constitutes the attB site in P. mirabilis. In S. enterica serovars or E. coli, the attB sites usually differ in 1–2 bp from the SGI1 attP site. Analysis of the potential U7-L12 (20-mer primer) binding site in the thdF gene of the P. mirabilis strain HI4320 sequenced genome revealed seven nucleotide differences which may explain why the PCR failed to generate an amplicon. The identification of SGI1 in a P. mirabilis clinical isolate is of great interest as the spread of the multidrug resistance phenotype in conjunction with the potential for enhanced virulence has significant clinical implications. Since the SGI1 may also spread to other bacterial species, we searched for potential attB target sites of SGI1 in bacteria other than Salmonella and E. coli, using several databases and Blastn. The bacteria found with a potential attB SGI1 target site differing in no more than three nucleotides from the SGI1 attP site are listed in Table 1. The fact that potential attB sites were identified in diverse bacteria such as Shigella spp., Vibrio spp., Pseudomonas spp., Brucella spp., Legionella pneumophila and Klebsiella pneumoniae raises the potential for SGI1 to emerge in other human pathogens. Of particular interest in the evolution of SGI1 is that in some Shewanella genome sequencing projects, parts of SGI1 are found downstream of the respective thdF gene, also named trmE in the following database (http://genome.jgi-psf.org/ mic_home.html). In conclusion, the horizontal transfer of SGI1 may play an important role in the dissemination of multidrug resistance and potential virulence not only among S. enterica serovars but also among other bacterial genera of clinical importance.