Karine Praud

Emergence of Extended-Spectrum-β-Lactamase (CTX-M-9)-Producing Multiresistant Strains of Salmonella enterica Serotype Virchow in Poultry and Humans in France

ABSTRACT During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum β-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The blaCTX-M-9 gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum β-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.

Clonal Emergence of Extended-Spectrum β-Lactamase (CTX-M-2)-Producing Salmonella enterica Serovar Virchow Isolates with Reduced Susceptibilities to Ciprofloxacin among Poultry and Humans in Belgium and France (2000 to 2003)

ABSTRACT Antibiotic treatment is not required in cases of Salmonella enterica gastroenteritis but is essential in cases of enteric fever or invasive salmonellosis or in immunocompromised patients. Although fluoroquinolones and extended-spectrum cephalosporins are the drugs of choice to treat invasive Salmonella, resistance to these antibiotics is increasing worldwide. During the period 2000 to 2003, 90 Salmonella enterica serovar Virchow poultry and poultry product isolates and 11 serovar Virchow human isolates were found to produce an extended-spectrum β-lactamase, CTX-M-2, concomitantly with a TEM-1 β-lactamase. The blaCTX-M-2 gene was located on a large conjugative plasmid (>100 kb). Pulsed-field gel electrophoresis indicated a clonal relationship between the poultry and human isolates. All these isolates displayed additional resistance to trimethoprim-sulfamethoxazole and tetracycline as well as a reduced susceptibility to ciprofloxacin (MICs of between 0.5 and 1 μg/ml). CTX-M-2-producing Salmonella with a reduced susceptibility to fluoroquinolones constitutes a major concern, since such strains could disseminate on a large scale and jeopardize classical antibiotic therapy in immunocompromised patients.

Dissemination of an Extended-Spectrum-β-Lactamase blaTEM-52 Gene-Carrying IncI1 Plasmid in Various Salmonella enterica Serovars Isolated from Poultry and Humans in Belgium and France between 2001 and 2005

ABSTRACT We report here the dissemination of a conjugative IncI1 plasmid carrying blaTEM-52 on a Tn3 transposon conferring resistance to extended-spectrum cephalosporins in Salmonella enterica serovar Agona, Derby, Infantis, Paratyphi B dT+, and Typhimurium isolates from poultry and humans in Belgium and France from 2001 to 2005. The most prevalent serovar spreading this resistance was serovar Infantis.

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

Background The 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 Findings Here, 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/Significance The 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.

IncI1 Plasmid Carrying Extended-Spectrum-β-Lactamase Gene blaCTX-M-1 in Salmonella enterica Isolates from Poultry and Humans in France, 2003 to 2008

ABSTRACT We report the dissemination of a conjugative IncI1 plasmid carrying blaCTX-M-1, conferring resistance to extended-spectrum cephalosporins, in Salmonella enterica isolates from poultry and humans in France from 2003 to 2008. By IncI1 plasmid subtyping, this plasmid was shown to be genetically related to that found in Escherichia coli isolates from healthy poultry in France.

Novel insertion sequence- and transposon-mediated genetic rearrangements in genomic island SGI1 of Salmonella enterica serovar Kentucky.

ABSTRACT 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 β-lactamase blaTEM-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 β-lactamase blaTEM-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.

Comparative Analysis of IncHI2 Plasmids Carrying blaCTX-M-2 or blaCTX-M-9 from Escherichia coli and Salmonella enterica Strains Isolated from Poultry and Humans

ABSTRACT Salmonella enterica bla CTX-M-2 and blaCTX-M-9 plasmid backbones from isolates from Belgium and France were analyzed. The blaCTX-M-2-plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the blaCTX-M-9 plasmids were closely related to the IncHI2 R478 plasmid.

Variant Salmonella Genomic Island 1-L Antibiotic Resistance Gene Cluster in Salmonella enterica Serovar Newport

ABSTRACT We report a new Salmonella genomic island 1 variant antibiotic resistance gene cluster called SGI1-L in a Salmonella enterica serovar Newport isolate containing a dfrA15 gene cassette conferring resistance to trimethoprim. The isolate carried another class 1 integron containing the aacC5 and aadA7 gene cassettes conferring resistance to gentamicin and streptomycin/spectinomycin, respectively.

European clinical isolate of Proteus mirabilis harbouring the Salmonella genomic island 1 variant SGI1-O

Sir, The 43 kb Salmonella genomic island 1 (SGI1) is a Salmonelladerived site-specific integrative mobilizable element that was initially characterized in multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium DT104 (where DT stands for definitive phage type) strains. SGI1 is integrated into the last 18 bp of the chromosomal trmE gene (also called thdF). SGI1 has been shown to be mobilized in trans using a conjugative helper plasmid that can be transferred to Salmonella and Escherichia coli recipient strains. SGI1 contains a complex class 1 integron, named In104, responsible for the MDR phenotype and located in the 3′ part of the island. The complex integron In104 possesses two attachment sites (attI1) for resistance gene cassettes, and numerous variants named SGI1-A to SGI1-T have been described in several S. enterica serovars. – 5 In 2007–08, two studies reported the identification of SGI1-like variants in Proteus mirabilis clinical and food isolates from China and Palestine. Chromosomal insertion of the SGI1-like element has been shown in those P. mirabilis strains. It was subsequently speculated that the target site for SGI1 integration in P. mirabilis could be also, as observed in S. enterica, the trmE gene, since an homologous gene was identified in the chromosome of P. mirabilis. The presence of SGI1 in clinical isolates of P. mirabilis is of interest, since it may further contribute to multidrug resistance in P. mirabilis. Here, we have analysed an MDR P. mirabilis clinical strain, NKU, recovered from rectal and nasal swabs of a patient hospitalized at the Bicêtre hospital in 2007. This patient did not report any recent history of travel abroad. This strain displayed a broad-spectrum b-lactam resistance profile, and in addition was resistant to chloramphenicol, streptomycin, tetracycline, sulphonamides, trimethoprim and fluoroquinolones. Phenotypic detection of extended-spectrum b-lactamase (ESBL) production performed as described previously was positive. PCRs to detect ESBL genes (blaTEM, blaSHV and blaCTX-M) performed using previously described primers followed by sequencing identified the blaTEM-52 ESBL gene. 6 Conjugation experiments to transfer the ESBL resistance phenotype were unsuccessful. However, electroporation of the P. mirabilis plasmid DNA to E. coli strain XL1-Blue permitted transfer of the blaTEM-52 resistance gene without any additional resistance marker. Thus, the blaTEM-52 gene was located as a single resistance gene on a non-conjugative plasmid. The lack of identification of other plasmid-borne resistance determinants prompted us to investigate the possible presence of an SGI1-like element in P. mirabilis NKU. Detection of SGI1 was performed by PCR using primers listed in Table S1 (available as Supplementary data at JAC Online; http://jac.oxfordjournals.org/), as previously described. The PCR result was positive for the left junction, indicating that SGI1 was inserted in the chromosome at the 3′ end of the chromosomally located trmE gene (Figure 1). However, fragment 2, corresponding to the right junction between SGI1 and the downstream chromosomal hipB gene, was not obtained. This suggested a genetic variation, possibly involving deletions at the SGI1 3′ end and/or in the adjacent chromosomal region. PCR mapping (PCRs 4–6) and Southern blot hybridization of XbaI-digested genomic DNA, performed as described previously (see also Table S1, Figure 1), confirmed the presence of the entire SGI1. To identify the genetic content of the SGI1 complex integron, PCR mapping was performed as described previously, but gave negative results, indicating that P. mirabilis NKU probably harboured a variant of SGI1. The integron cassette array was detected by PCR using primers CS1 and CS2, yielding a single 1.3 kb fragment, and sequencing identified the dfrA1-orfC gene cassettes previously described in the SGI1-O variant from P. mirabilis. Further PCR mapping confirmed the genetic organization of SGI1-O (Table S1, Figure 1). The SGI1-O variant harboured the dfrA1 and sul1 genes, conferring resistance to trimethoprim and sulphonamides, respectively, and has been previously identified in P. mirabilis in China. The 3′ end of SGI1-O was conserved in P. mirabilis NKU, but the absence of detection of the right junction suggested a likely genetic rearrangement in the chromosome sequence of P. mirabilis strain NKU. To better analyse this rearrangement in the downstream chromosomal region of SGI1, another reverse primer was designed 2 kb downstream of the direct repeat right of SGI1 in the chromosome sequence of P. mirabilis strain HI4320, according to GenBank accession number NC_010554 (Table S1, Figure 1). PCR results revealed a 1714 bp deletion in the chromosome of P. mirabilis strain NKU compared with the genome sequence of P. mirabilis strain HI4320 (Figure 1). The nucleotide sequence obtained (deposited in GenBank under accession number HM560958) indicated that this deletion was likely to be the result of a precise recombinational event between 49 bp perfect direct repeats bracketing the hipBA gene. In P. mirabilis strain NKU, the variant SGI1-O was thus inserted in its specific attachment site (the last 18 bp of the trmE gene) and followed by a single 49 bp perfect direct repeat, which resulted from the loss of the hipBA module (Figure 1). Moreover, this Research letters

Early strains of multidrug-resistant Salmonella enterica serovar Kentucky sequence type 198 from Southeast Asia harbor Salmonella genomic island 1-J variants with a novel insertion sequence.

ABSTRACT Salmonella genomic island 1 (SGI1) is a 43-kb integrative mobilizable element that harbors a great diversity of multidrug resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. The majority of SGI1 variants contain an In104-derivative complex class 1 integron inserted between resolvase gene res and open reading frame (ORF) S044 in SGI1. Recently, the international spread of ciprofloxacin-resistant S. enterica serovar Kentucky sequence type 198 (ST198) containing SGI1-K variants has been reported. A retrospective study was undertaken to characterize ST198 S. Kentucky strains isolated before the spread of the epidemic ST198-SGI1-K population in Africa and the Middle East. Here, we characterized 12 ST198 S. Kentucky strains isolated between 1969 and 1999, mainly from humans returning from Southeast Asia (n = 10 strains) or Israel (n = 1 strain) or from meat in Egypt (n = 1 strain). All these ST198 S. Kentucky strains did not belong to the XbaI pulsotype X1 associated with the African epidemic clone but to pulsotype X2. SGI1-J subgroup variants containing different complex integrons with a partial transposition module and inserted within ORF S023 of SGI1 were detected in six strains. The SGI1-J4 variant containing a partially deleted class 1 integron and thus showing a narrow resistance phenotype to sulfonamides was identified in two epidemiologically unrelated strains from Indonesia. The four remaining strains harbored a novel SGI1-J variant, named SGI1-J6, which contained aadA2, floR2, tetR(G)-tetA(G), and sul1 resistance genes within its complex integron. Moreover, in all these S. Kentucky isolates, a novel insertion sequence related to the IS630 family and named ISSen5 was found inserted upstream of the SGI1 complex integron in ORF S023. Thus, two subpopulations of S. Kentucky ST198 independently and exclusively acquired the SGI1 during the 1980s and 1990s. Unlike the ST198-X1 African epidemic subpopulation, the ST198-X2 subpopulation mainly from Asia harbors variants of the SGI1-J subgroup that are encountered mainly in the Far East, as previously described for S. enterica serovars Emek and Virchow.