Overview of Salmonella Genomic Island 1-Related Elements Among Gamma-Proteobacteria Reveals Their Wide Distribution Among Environmental Species.

The aim of this study was to perform an in silico analysis of the available whole-genome sequencing data to detect syntenic genomic islands (GIs) having homology to Salmonella genomic island 1 (SGI1), analyze the genetic variations of their backbone, and determine their relatedness. Eighty-nine non-redundant SGI1-related elements (SGI1-REs) were identified among gamma-proteobacteria. With the inclusion of the thirty-seven backbones characterized to date, seven clusters were identified based on integrase homology: SGI1, PGI1, PGI2, AGI1 clusters, and clusters 5, 6, and 7 composed of GIs mainly harbored by waterborne or marine bacteria, such as Vibrio, Shewanella, Halomonas, Idiomarina, Marinobacter, and Pseudohongiella. The integrase genes and the backbones of SGI1-REs from clusters 6 and 7, and from PGI1, PGI2, and AGI1 clusters differed significantly from those of the SGI1 cluster, suggesting a different ancestor. All backbones consisted of two parts: the part from attL to the origin of transfer (oriT) harbored the DNA recombination, transfer, and mobilization genes, and the part from oriT to attR differed among the clusters. The diversity of SGI1-REs resulted from the recombination events between GIs of the same or other families. The oriT appeared to be a high recombination site. The multi-drug resistant (MDR) region was located upstream of the resolvase gene. However, most SGI1-REs in Vibrio, Shewanella, and marine bacteria did not harbor any MDR region. These strains could constitute a reservoir of SGI1-REs that could be potential ancestors of SGI1-REs encountered in pathogenic bacteria. Furthermore, four SGI1-REs did not harbor a resolvase gene and therefore could not acquire an integron. The presence of mobilization genes and AcaCD binding sites indicated that their conjugative transfer could occur with helper plasmids. The plasticity of SGI1-REs contributes to bacterial adaptation and evolution. We propose a more relevant classification to categorize SGI1-REs into different clusters based on their integrase gene similarity.

Mobilisation of plasmid-mediated blaVEB-1 gene cassette into distinct genomic islands of Proteus mirabilis after ceftazidime exposure.

OBJECTIVES: We sought to integrate a VEB-1-encoding gene cassette into the integron of the MDR region of genomic islands (GIs) harboured by Proteus mirabilis strains after antibiotic exposure. METHODS: An IncP1 plasmid from Achromobacter xylosoxidans carrying the cassette array dfrA14-blaVEB-1-aadB was introduced by conjugation into five strains of P. mirabilis: PmBRI, PmABB, PmSCO and Pm2CHAMA harbouring Salmonella GI 1 and PmESC harbouring Proteus GI 1. Circular intermediates of the cassettes were amplified by PCR. blaVEB-harbouring P. mirabilis were exposed to increasing concentrations of ceftazidime each day. Presence of blaVEB-1 in the GI was assessed by PCR. The complete MDR regions were mapped and sequenced in positive clones. RESULTS: Circular intermediates were detected for dfrA14 and blaVEB-1-aadB and dfrA14-blaVEB-1-aadB cassettes arrays in A. xylosoxidans, and for aadA2 in P. mirabilis. Insertion of blaVEB-1 into the GIs occurred under ceftazidime pressure. In all cases, the three cassettes from IncP1 were integrated. They replaced the cassette array of PmBRI, PmABB and PmSCO in which floRc, tet(A)G and blaPSE-1 were conserved, whereas they replaced an integron and the IS26-flanked region in Pm2CHAMA. In PmESC, they only replaced aadB, with aadA2 being conserved. blaVEB-1 integration occurred just after conjugation for Pm2CHAMA but required ceftazidime exposure for the other strains. CONCLUSION: Homologous recombination of gene cassettes conferring resistance to clinically important antibiotics may occur under antibiotic pressure between an integron located on a plasmid and a co-resident GI. This feature participates in the acquisition, maintenance and spread of antibiotic resistance genes.

Genomic islands related to Salmonella genomic island 1; integrative mobilisable elements in trmE mobilised in trans by A/C plasmids.

Salmonella genomic island 1 (SGI1), an integrative mobilisable element (IME), was first reported 20 years ago, in the multidrug resistant Salmonella Typhimurium DT104 clone. Since this first report, many variants and relatives have been found in Salmonella enterica and Proteus mirabilis. Thanks to whole genome sequencing, more and more complete sequences of SGI1-related elements (SGI1-REs) have been reported in these last few years among Gammaproteobacteria. Here, the genetic organisation and main features common to SGI1-REs are summarised to help to classify them. Their integrases belong to the tyrosine-recombinase family and target the 3'-end of the trmE gene. They share the same genetic organisation (integrase and excisionase genes, replicase module, SgaCD-like transcriptional activator genes, traN, traG, mpsB/mpsA genes) and they harbour AcaCD binding sites promoting their excision, replication and mobilisation in presence of A/C plasmid. SGI1-REs are mosaic structures suggesting that recombination events occurred between them. Most of them harbour a multiple antibiotic resistance (MAR) region and the plasticity of their MAR region show that SGI1-REs play a key role in antibiotic resistance and might help multiple antibiotic resistant bacteria to adapt to their environment. This might explain the emergence of clones with SGI1-REs.

New insights regarding Acinetobacter genomic island-related elements.

The objective of this study was to mobilise the Acinetobacter genomic island 1-A (AGI1-A) from Enterobacter hormaechei EclCSP2185 (E. cloacae complex) and to search for the distribution and structure of AGI1-related elements in the NCBI database. AGI1-A was transferred to Escherichia coli. Analysis of the attachment (att) sites could locate the possible recombination crossover in the att sequences at position 10-11 (GG) in the last 18 bp of trmE. In silico detection of AGI backbones in the WGS database identified AGI variants in Salmonella enterica (83 strains), Vibrio cholerae (33), E. hormaechei (12), Acinetobacter baumannii (2), most belonging to prevalent clones (ST40, ST69, ST114 and ST25, respectively), but also in E. coli (1) and Klebsiella pneumoniae (1). Two groups of backbone were identified: one similar to AGI1, the other with a short segment from a Shewanella element upstream of ORF A022. The MDR regions were inserted by transposition at the res site in four different positions ATAGG (A. baumannii), CATAG (S. enterica and V. cholerae), TAGGT (S. enterica and K. pneumoniae) and TGCAC (S. enterica) representing four different lineages. In some V. cholerae, E. hormaechei and E. coli, deletion events occurred that eliminated part of the backbone at the left junction. Analysis of the right junction identified a fifth lineage in V. cholerae and E. hormaechei (CCATA). In conclusion, based on the position of the MDR region, AGI-related elements belonged to five groups of closely related genomic islands (AGI1-AGI5), with differences in backbones that evolved independently over time.

Two New SGI1-LK Variants Found in Proteus mirabilis and Evolution of the SGI1-HKL Group of Salmonella Genomic Islands.

Integrative mobilizable elements belonging to the SGI1-H, -K, and -L Salmonella genomic island 1 (SGI1) variant groups are distinguished by the presence of an alteration in the backbone (IS1359 replaces 2.8 kb of the backbone extending from within traN [S005] to within S009). Members of this SGI1-HKL group have been found in Salmonella enterica serovars and in Proteus mirabilis Two novel variants from this group, designated SGI1-LK1 and SGI1-LK2, were found in the draft genomes of antibiotic-resistant P. mirabilis isolates from two French hospitals. Both variants can be derived from SGI1-PmGUE, a configuration found previously in another P. mirabilis isolate from France. SGI1-LK1 could arise via an IS26-mediated inversion in the complex class 1 integron that duplicated the IS26 element and the target site in IS6100 SGI1-LK1 also has a larger 8.59-kb backbone deletion extending from traN to within S013 and removing traG and traH. However, SGI1-LK1 was mobilized by an IncC plasmid. SGI1-LK2 can be derived from a hypothetical progenitor, SGI1-LK0, that is related to SGI1-PmGUE but lacks the aphA1 gene and one copy of IS26. The integron of SGI1-LK2 could arise via deletion of DNA adjacent to an IS26 and a deletion occurring via homologous recombination between duplicated copies of part of the integron 3'-conserved segment. SGI1-K can also be derived from SGI1-LK0. This would involve an IS26-mediated deletion and an inversion via homologous recombination of a segment between inversely oriented IS26s. Similar events can explain the configuration of the integrons in other SGI1-LK variants.IMPORTANCE Members of the SGI1-HKL subgroup of SGI1-type integrative mobilizable elements have a characteristic alteration in their backbone. They are widely distributed among multiply antibiotic-resistant Salmonella enterica serovars and Proteus mirabilis isolates. The SGI1-K type, found in the globally disseminated multiply antibiotic-resistant Salmonella enterica serovar Kentucky clone ST198 (sequence type 198), and various configurations in the original SGI1-LK group, found in other multiresistant S. enterica serovars and Proteus mirabilis isolates, have complex and highly plastic resistance regions due to the presence of IS26 However, how these complex forms arose and the relationships between them had not been analyzed. Here, a hypothetical progenitor, SGI1-LK0, that can be formed from the simpler SGI1-H is proposed, and the pathways to the formation of new variants, SGI1-LK1 and SGI1-LK2, found in P. mirabilis and other reported configurations via homologous recombination and IS26-mediated events are proposed. This led to a better understanding of the evolution of the SGI1-HKL group.

SGI0, a relative of Salmonella genomic islands SGI1 and SGI2, lacking a class 1 integron, found in Proteus mirabilis.

Several groups of integrative mobilizable elements (IMEs) that harbour a class 1 integron carrying antibiotic resistance genes have been found at the 3'-end of the chromosomal trmE gene. Here, a new IME, designated SGI0, was found in trmE in the sequenced and assembled genome of a French clinical, multiply antibiotic resistant Proteus mirabilis strain, Pm1LENAR. SGI0 shares the same gene content as the backbones of SGI1 and SGI2 (overall 97.6% and 97.7% nucleotide identity, respectively) but it lacks a class 1 integron. However, SGI0 is a mosaic made up of segments with >98.5% identity to SGI1 and SGI2 interspersed with segments sharing 74-95% identity indicating that further diverged backbone types exist and that recombination between them is occurring. The structure of SGI1-V, here re-named SGI-V, which lacks two SGI1 (S023 and S024) backbone genes and includes a group of additional genes in the backbone, was re-examined. In regions shared with SGI1, the backbones shared 97.3% overall identity with the differences distributed in patches with various levels of identity. The class 1 integron is also in a slightly different position with the target site duplication AAATT instead of ACTTG for SGI1 and variants, indicating that it was acquired independently. The Pm1LENAR resistance genes are in the chromosome, in Tn7 and an ISEcp1-mobilised segment.

Identification of AGI1-A, a variant of Acinetobacter genomic island 1 (AGI1), in a French clinical isolate belonging to the Enterobacter cloacae complex.

Objectives: To characterize the structure of a variant of Acinetobacter genomic island 1 (AGI1) in the Enterobacter cloacae clinical isolate EclC2185 responsible for an 8 year outbreak in Dijon University Hospital. Methods: The genome was sequenced (Miseq) and de novo assembled (Velvet). PCR and Sanger sequencing were performed to determine the sequence of the genomic island. Results: The new variant of AGI1 named AGI1-A (41.7 kb), located at the 3' end of the chromosomal trmE, was detected in an E. cloacae complex isolate identified as Enterobacter hormaechei subsp. oharae. The backbone of AGI1-A lacked A008, part of A025, A026 and the resolvase gene. Its MDR region (19.7 kb) contained two integrons followed by a hybrid transposon Tn502/Tn5053. The former integron was a typical In4-type class 1 integron carrying aadA1 and the latter integron carrying dfrA1 did not belong to a class described to date. The nucleotide sequence intI-dfrA1 was surrounded by a 78 bp imperfect repeat sequence in inverse orientation. An external circular form of AGI1-A was detected, suggesting potential mobility. AGI1-A was also detected in 15 isolates from the outbreak selected at random. They belonged to the ST114 high-risk clone. Conclusions: AGI1-A, a variant of AGI1 described in Acinetobacter baumannii, is the first genomic resistance island belonging to the Salmonella genomic island/Proteus genomic island/Acinetobacter genomic island family detected in E. cloacae. One might, therefore, fear for interspecies dissemination of genomic islands from this family.

Genomic context of resistance genes within a French clinical MDR Proteus mirabilis: identification of the novel genomic resistance island GIPmi1.

Objectives: To determine the location of the antibiotic resistance genes in the MDR Proteus mirabilis PmPHI clinical isolate. Methods: WGS and de novo assembly were performed. BLAST searches were used to identify relevant contigs. PCR and Sanger sequencing were used to link the fragments of interest and fill the gaps. Results: P. mirabilis PmPHI was resistant to six classes of antibiotics: penicillins, aminoglycosides, phenicols, tetracyclines, folate inhibitors and fluoroquinolones. A novel genomic resistance island (GIPmi1) of 55.8 kb was located at the 3' end of trmE. The backbone shared 93% identity with a genomic sequence of Enterobacter cloacae DSM 16690. The MDR region was composed of two class 1 integrons [one Tn402-type (estX-qacE) and one In5-type (aadB-aadA2)], separated by a region containing many parts of transposons. An external circular form of GIPmi1 was detected; however, mobilization by an A/C plasmid failed. In addition, an SXT/R391 integrative and conjugative element (ICEPmiFra1) was inserted into prfC. It carried floR, the sul2-strA-strB cluster and a composite transposon flanked by two copies of a tISPpu12 element that contains a class 1 integron (dfrA32-ereA1-aadA2), Tn4352 (aphA1a) and tetA(C). A class 2 integron (dfrA1-sat2-aadA1) was also identified on Tn7 as well as point mutations in gyrA and parC accounting for quinolone resistance. Conclusions: The finding of the new genomic island GIPmi1 belonging to the same superfamily of genomic islands as SGI1/SGI2/PGI1/AGI1 and of the integrative conjugative element ICEPmiFra1 (SXT/R391 family) suggested that these genetic elements might be key mediators of resistance gene acquisition in P. mirabilis.

Two new Salmonella genomic islands 1 from Proteus mirabilis and description of blaCTX-M-15 on a variant (SGI1-K7).

Objectives: To characterize the structure of Salmonella genomic islands 1 (SGI1s) from two clinical Proteus mirabilis isolates: one producing an ESBL and the other a penicillinase. Methods: WGS completed by PCR and Sanger sequencing was performed to determine sequences of SGI1s from Pm2CHAMA and Pm37THOMI strains. Results: Two new variants of SGI1 named SGI1-Pm2CHAMA (53.6 kb) and SGI1-K7 (55.1 kb) were identified. The backbone of SGI1-Pm2CHAMA shared 99.9% identity with that of SGI1. Its MDR region (26.3 kb) harboured two class 1 integrons (an In2-type integron and an In4-type integron) containing in particular a qacH cassette (encoding a quaternary ammonium compound efflux pump). These two integrons framed a complex region (harbouring among others blaCARB-4) resulting from transposon insertions mediated by IS26 and successive transposition events of ISs (ISAba14 isoform and the new ISPmi2). The second variant (SGI1-K7) had the same backbone as SGI1-K. Its MDR region (29.7 kb) was derived from that of SGI1-K and was generated by three events. The two main events were mediated by IS26: inversion of a large portion of the MDR region of SGI1-K and insertion of a structure previously reported on plasmids carried by prevalent and successful MDR clones of Enterobacteriaceae. This last event led to the insertion of the blaCTX-M-15 gene into SGI1-K7. Conclusions: This study confirmed the great plasticity of the MDR region of SGI1 and its potential key role for the dissemination of clinically significant antibiotic resistance among Enterobacteriaceae.

"Does the Salmonella Genomic Island 1 (SGI1) confer invasiveness properties to human isolates?"

BACKGROUND: In the eighties, a multidrug resistant clone of Salmonella Typhimurium DT104 emerged in UK and disseminated worldwide. This clone harbored a Salmonella genomic island 1 (SGI1) that consists of a backbone and a multidrug resistant region encoding for penta-resistance (ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides and tetracycline (ACSSuT)). Several authors suggested that SGI1 might have a potential role in enhancement of virulence properties of Salmonella enterica. The aim of this study was to investigate whether nontyphoidal S. enterica isolates carrying SGI1 cause more severe illness than SGI1 free ones in humans. METHODS: From 2011 to 2016, all patients infected with nontyphoidal S. enterica in our hospital were retrospectively included. All nontyphoidal S. enterica isolates preserved in our University Hospital (Dijon, France) were screened for the presence of SGI1. Clinical and biological data of patients were retrospectively collected to evaluate illness severity. Statistical analysis of data was performed by Kruskal-Wallis test or Fisher's exact test for univariate analysis, and by logistic regression for multivariate analysis. RESULTS: A total of 100 isolates of S. enterica (22 serovars) were collected. Twelve isolates (12%) belonging to 4 serovars harbored SGI1: S. Typhimurium, S. Infantis, S. Kentucky, S. St Paul. The severity of the disease was age-related (for invasive infection, sepsis and inflammatory response) and was associated with immunosuppression (for invasive infection, sepsis and bacteremia) but not with the presence of SGI1 or with antimicrobial resistance. CONCLUSION: A rather high proportion (12%) of human clinical isolates belonging to various serovars (for the first time serovar St Paul) and harboring various antimicrobial resistance profile carried SGI1. Diseases due to SGI1-positive S. enterica or to antimicrobial resistant isolates were not more severe than the others. This first clinical observation should be confirmed by a multicenter and prospective study.

Nosocomial Infections with IMP-19-Producing Pseudomonas aeruginosa Linked to Contaminated Sinks, France.

We isolated IMP-19-producing Pseudomonas aeruginosa from 7 patients with nosocomial infections linked to contaminated sinks in France. We showed that blaIMP-19 was located on various class 1 integrons among 8 species of gram-negative bacilli detected in sinks: P. aeruginosa, Achromobacter xylosoxidans, A. aegrifaciens, P. putida, Stenotrophomonas maltophilia, P. mendocina, Comamonas testosteroni, and Sphingomonas sp.

Occurence of ArmA and RmtB Aminoglycoside Resistance 16S rRNA Methylases in Extended-Spectrum ß-Lactamases Producing Escherichia coli in Algerian Hospitals.

The aim of this study, was to characterize the extended-spectrum-ß-lactamases (ESBLs) producing clinical strains of Escherichia coli isolated between January 2009 and June 2012 from Algerian hospitals and to determine the prevalence of 16S rRNA methylase among them. Sixty-seven ESBL-producers were detected among the 239 isolates included: 52 CTX-M-15-producers, 5 CTX-M-3-producers, 5 CTX-M-1-producers, 2 CTX-M-14-producers, 2 SHV-12-producers and one TEM-167-producer. Among the ESBL-producing strains twelve harbored 16S rRNA methylase genes: 8 rmtB and 4 armA. rmtB was located on a IncFIA plasmid and armA was located either on a IncL/M or a IncFIA plasmid. RmtB-producing isolates were genotypically related and belonged to the sequence type ST 405 whereas ArmA-producing isolates belonged to ST10, ST 167, and ST 117. This first description of 16S rRNA methylases among E. coli in Algerian hospitals pointed out the necessity to establish control measures to avoid their dissemination.

Outbreak of Extended-Spectrum Beta-Lactamase Producing Enterobacter cloacae with High MICs of Quaternary Ammonium Compounds in a Hematology Ward Associated with Contaminated Sinks.

OBJECTIVE: To investigate an outbreak of extended-spectrum beta-lactamase (ESBL) producing Enterobacter cloacae that occurred in the Hematology ward (24-bed unit) of the François Mitterrand University Hospital (Dijon, France) between January 2011 and December 2013. The outbreak involved 43 patients (10 infected and 33 colonized). DESIGN: We performed environmental analysis to detect multiresistant E. cloacae for comparison with clinical isolates (genotyping by pulsed-field gel electrophoresis and MLST as well as ESBL-typing) and determined the MICs of the quaternary ammonium compounds (QACs) alkyldimethylbenzylammonium chloride (ADBAC) and didecyldimethylammonium chloride (DDAC). A bleach-based cleaning-disinfection program was implemented in December 2012 after mechanical removal of the biofilm in all sinks. RESULTS: We have detected 17 ESBL-producing E. cloacae in patients sink drains, shower drains and medical sink drains. Sequencing of the bla genes performed on 60 strains recovered from patients and environment (n = 43 clinical and n = 17 environmental) revealed that bla CTX-M15 was predominant (37 isolates) followed by bla CTX-M9 plus bla SHV-12 (20 isolates). We observed a great diversity among the isolates: 14 pulsotypes (11 STs) in clinical isolates and 9 pulsotypes (7 STs) in environmental isolates. Six pulsotypes were identical between clinical and environmental isolates. MICs of the quaternary ammonium compounds widely used for disinfection were very high in clinical and environmental isolates. Immediately after the implementation of the disinfection program we noticed a substantial fall in cases number. Our findings demonstrate the role of drains as important reservoir of ESBL-producing E. cloacae and highlight the necessity to settle drains accessible to achieve correct cleaning as well as to use disinfectant with proved activity against nosocomial pathogens.

Mobilization of the Salmonella genomic island SGI1 and the Proteus genomic island PGI1 by the A/C2 plasmid carrying blaTEM-24 harboured by various clinical species of Enterobacteriaceae.

OBJECTIVES: The objective of this study was to transfer the Salmonella genomic islands (GIs) SGI1 and SGI1-V and the Proteus GI PGI1-PmESC to clinical isolates of Enterobacteriaceae harbouring an A/C2 plasmid. METHODS: The entire genetic structures of SGI1 and PGI1-PmESC from Salmonella Typhimurium and Proteus mirabilis, respectively, were characterized by PCR and DNA sequencing. Ten enterobacterial isolates from different species carrying blaTEM-24 on an A/C2 plasmid were used for the mobilization of SGI1: Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter aerogenes, Citrobacter freundii, Klebsiella oxytoca, Proteus vulgaris, Providencia stuartii and Serratia marcescens. SGI1-V and PGI1-PmESC were transferred to E. aerogenes. Conjugation attempts were also performed using the wild strain E. aerogenes BOL and E. coli K-12 with or without pA/C2. Detection and location of the GI in the transconjugants were assessed by PCR targeting their junctions. RESULTS: The multidrug resistance region of PGI1-PmESC contained a class 1 integron (aadB and aadA2) and regions deriving from transposon Tn501 and a hybrid Tn502/Tn5053 transposon, whereas SGI1 harboured the known determinants responsible for the pentaresistance. The transfer of SGI1 occurred from Salmonella Typhimurium to the 10 enterobacterial isolates, and transfer of SGI1-V and PGI1-PmESC occurred from P. mirabilis to E. aerogenes. In all transconjugants the GI was located at the 3'-end of trmE. SGI1 was also transferred to E. aerogenes BOL (pA/C2) and E. coli K-12 (pA/C2), but not to E. aerogenes BOL and E. coli K-12. CONCLUSIONS: This is the first known description of SGI1 mobilization into a broad range of enterobacterial species harbouring an A/C2 plasmid and the first demonstration of PGI1 movement. The A/C2 plasmid is responsible for the GI mobilization.

Survey of multidrug resistance integrative mobilizable elements SGI1 and PGI1 in Proteus mirabilis in humans and dogs in France, 2010-13.

OBJECTIVES: To characterize MDR genomic islands related to Salmonella genomic island 1 (SGI1) and Proteus genomic island 1 (PGI1) in Proteus mirabilis from human and animal sources in France in light of the previously reported cases. METHODS: A total of 52 and 46 P. mirabilis clinical strains from human and animal sources, respectively, were studied for the period 2010-13. MDR was assessed by antimicrobial susceptibility testing, PCR detection of SGI1 and PGI1 and PCR mapping of the MDR regions. The diversity of the SGI1/PGI1-positive P. mirabilis strains was assessed by PFGE. RESULTS: Twelve P. mirabilis strains (5 humans and 7 dogs) were found to harbour an MDR island related to SGI1 or PGI1. Among them, several SGI1 variants were identified in diverse P. mirabilis genetic backgrounds. The variant SGI1-V, which harbours the ESBL bla VEB-6 gene, was found in closely genetically related human and dog P. mirabilis strains. The recently described PGI1 element was also identified in human and dog strains. Finally, one strain harboured a novel SGI genomic island closely related to SGI1 and SGI2 without an insertion of the MDR region. CONCLUSION: This study reports for the first time, to our knowledge, SGI1-positive and PGI1-positive P. mirabilis strains from dogs in France. The genetic diversity of the strains suggests several independent horizontal acquisitions of these MDR elements. The potential transmission of SGI1/PGI1-positive P. mirabilis strains between animals and humans is of public health concern, notably with regard to the spread of ESBL and carbapenemase genes, i.e. bla VEB-6 and bla NDM-1.

Proteus genomic island 1 (PGI1), a new resistance genomic island from two Proteus mirabilis French clinical isolates.

OBJECTIVES: To analyse the genetic environment of the antibiotic resistance genes in two clinical Proteus mirabilis isolates resistant to multiple antibiotics. METHODS: PCR, gene walking and whole-genome sequencing were used to determine the sequence of the resistance regions, the surrounding genetic structure and the flanking chromosomal regions. RESULTS: A genomic island of 81.1 kb named Proteus genomic island 1 (PGI1) located at the 3'-end of trmE (formerly known as thdF) was characterized. The large MDR region of PGI1 (55.4 kb) included a class 1 integron (aadB and aadA2) and regions deriving from several transposons: Tn2 (blaTEM-135), Tn21, Tn6020-like transposon (aphA1b), a hybrid Tn502/Tn5053 transposon, Tn501, a hybrid Tn1696/Tn1721 transposon [tetA(A)] carrying a class 1 integron (aadA1) and Tn5393 (strA and strB). Several ISs were also present (IS4321, IS1R and IS26). The PGI1 backbone (25.7 kb) was identical to that identified in Salmonella Heidelberg SL476 and shared some identity with the Salmonella genomic island 1 (SGI1) backbone. An IS26-mediated recombination event caused the division of the MDR region into two parts separated by a large chromosomal DNA fragment of 197 kb, the right end of PGI1 and this chromosomal sequence being in inverse orientation. CONCLUSIONS: PGI1 is a new resistance genomic island from P. mirabilis belonging to the same island family as SGI1. The role of PGI1 in the spread of antimicrobial resistance genes among Enterobacteriaceae of medical importance needs to be evaluated.

Emergence of Salmonella genomic island 1 (SGI1) among Proteus mirabilis clinical isolates in Dijon, France.

OBJECTIVES: Salmonella genomic island 1 (SGI1) is often encountered in antibiotic-resistant Salmonella enterica and exceptionally in Proteus mirabilis. We investigated the prevalence of SGI1-producing clinical isolates of P. mirabilis in our hospital (Dijon, France). METHODS: A total of 57 strains of P. mirabilis resistant to amoxicillin and/or gentamicin and/or trimethoprim/sulfamethoxazole isolated from August 2011 to February 2012 as well as 9 extended-spectrum ß-lactamase (ESBL)-producing P. mirabilis from our collection were tested for the presence of SGI1 by PCR. The complete SGI1 structure from positive isolates [backbone and multidrug resistance (MDR) region] was sequenced. RESULTS: SGI1 was detected in 7 isolates; 5 out of the 57 isolates collected during the study period (9%) and 2 out of the 9 ESBL-producing strains of our collection. The structures of the seven SGI1s were distinct. Three different backbones were identified: one identical to the SGI1 backbone from the epidemic Salmonella Typhimurium DT104, one with variations already described in SGI1-K from Salmonella Kentucky (deletion and insertion of IS1359 in the region spanning from S005 to S009) and one with a variation never detected before (deletion from S005 to S009). Six different MDR regions were identified: four simple variants containing resistance genes already described and two variants harbouring a very complex structure including regions derived from several transposons and IS26 elements with aphA1a never reported to date in SGI1. CONCLUSIONS: SGI1 variants are widely distributed among P. mirabilis clinical strains and might spread to other commensal Enterobacteriaceae. This would become a serious public health problem.

Epidemiology and resistance of Achromobacter xylosoxidans from cystic fibrosis patients in Dijon, Burgundy: first French data.

BACKGROUND: Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis (CF) patients recognised as causal agent of inflammation. The prevalence of infection or colonisation is variable among CF centres. We report here the first epidemiological data about A. xylosoxidans in a French CF centre: Dijon, Burgundy. METHODS: All isolates recovered from the patients affiliated with our centre in 2010 since their first visit were included. Antimicrobial susceptibility was determined by disk diffusion method and E-test. Molecular epidemiology was performed by Pulsed Field Gel Electrophoresis (PFGE) and compared with repetitive sequence-based PCR (rep-PCR, DiversiLab®). We also sequenced the constitutive bla-oxa-114 gene. RESULTS: Out of 120 patients, 21 (17.5%) had at least one positive culture with A. xylosoxidans since they started to receive routine care in our CF centre (447 isolates). Median age at first positive culture was 16 years (range 3-34 years). Most patients were colonised by their own strain, cross-contamination was very rare. We observed two cases of intra-family spread. DiversiLab® is a useful tool as efficient as PFGE to compare isolates recovered simultaneously from different patients when an outbreak is suspected. However, PFGE remains the reference method for long-term survey of chronically colonised patients. We detected new OXA-114 variants and the new oxacillinase OXA-243 (88% amino acid identity with OXA-114). Acquired resistance to ciprofloxacin, ceftazidime and carbapenems was frequent. In 2010, 7 patients harboured strains resistant to ceftazidime, 6 patients strains with decreased susceptibility to carbapenems (especially meropenem) and 12 patients strains resistant to ciprofloxacin. CONCLUSIONS: In our centre, the high prevalence of colonisation is not due to cross-contamination. Our main concern is the high rate of antimicrobial resistance.

The new variant of Salmonella genomic island 1 (SGI1-V) from a Proteus mirabilis French clinical isolate harbours blaVEB-6 and qnrA1 in the multiple antibiotic resistance region.

OBJECTIVES: The clinical strain of Proteus mirabilis VB1248 isolated from a blood culture in August 2009 was multiresistant (i.e. resistant to ß-lactams, fluoroquinolones, aminoglycosides and sulphonamides). We searched for the presence of a Salmonella genomic island 1 (SGI1). METHODS: The whole genetic structure surrounding the genes involved in antibiotic resistance was characterized by PCR or gene walking followed by DNA sequencing. RESULTS: The new variant SGI1-V (42.9 kb) was located downstream of the thdF chromosomal gene. Genes sharing homology with phage-related genes were detected on a structure of 8.3 kb located between the right junction of the SGI1-V and the hipB/hipA genes. Some genetic rearrangements occurred in the SGI1-V backbone: an insertion of 2349 bp within the open reading frame (ORF) S014, and a deletion of 3766 bp in the region spanning from ORFs S021 to S025 leading to the lack of ORFs S023 and S024. The multidrug resistance (MDR) region of 17.1 kb was located on a complex class 1 integron extremely different from those described so far. The cassette array included aacA4, aadB and dhfrA1. Adjacent to this classical structure, bla(VEB-6) was found flanked by 135 bp elements and bracketed by two 3'-conserved segments (3'-CS). Downstream of the second copy of 3'-CS, the qnrA1 gene was associated with common region 1. CONCLUSIONS: We have identified in P. mirabilis the new variant SGI1-V containing the bla(VEB-6) and qnrA1 genes in the MDR region. This is the first report of an extended-spectrum ß-lactamase-encoding gene and a qnr determinant conferring resistance to quinolones on an SGI1-like structure. It might constitute a source of spread of resistance to other bacterial species.