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

Genomic context of resistance genes within a French clinical MDR Proteus mirabilis:... Abstract 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. Introduction Proteus mirabilis is an opportunistic human pathogen that is generally susceptible to all classes of antibiotic agents active against Gram-negative bacilli, with the exception of tetracyclines and polymyxins. Antibiotic resistance is often due to the acquisition of resistance genes associated with integrons, transposons or ISs.1 Class 1 and class 2 integrons are those most frequently encountered in P. mirabilis.2,3 The former are generally plasmid-borne and the latter are found on transposon Tn7 mainly inserted into the chromosomal glmS transcriptional terminator.4,5 Sometimes the resistance genes are clustered into structures named genomic islands (GIs). Several types of GIs have been described to date. The most frequently encountered in P. mirabilis are Salmonella genomic island 1 (SGI1)6–8 and Proteus genomic island 1 (PGI1)9,10, belonging to the same superfamily of GIs that also includes SGI2 and AGI1, identified in Acinetobacter baumannii.11 SGI1, initially found in Salmonella enterica serovars, and PGI1 are large integrative mobilizable elements.12,13 They target the last 18 bp of the chromosomal trmE gene, encode related integrases and consist of a backbone and an MDR region located adjacent to the res gene. Different variants with backbone alterations and/or MDR region variations have been described.14 Besides these GIs, SXT/R391-related integrative conjugative elements (ICEs) have been described in P. mirabilis.15,16 These large elements, originally discovered in Vibrio cholerae (SXT) or Providencia rettgeri (R391), are integrated into the peptide chain release factor 3 gene (prfC).17 Various genes have been identified in hot spots (HS1–HS5) and variable regions (VRI–VRIV) of ICEs. Genetic diversity of these ICEs has been reported in MDR P. mirabilis in China.18 During our continuous survey of SGI1 and PGI1 among MDR P. mirabilis, we used PCRs with primers targeting the ORF S026 and the left and right junctions of these GIs.8 The isolate PmPHI, lacking the ORF S026 and with an undetectable intact trmE, was retained for characterization of its resistance genes content and their genetic context using WGS. Materials and methods Bacterial strain P. mirabilis PmPHI was isolated in August 2012 from a patient hospitalized in the haematology ward of Dijon hospital (France). Antibiotic susceptibility testing performed by the disc diffusion method according to the Antibiogram Committee of the French Society for Microbiology (CASFM)/EUCAST guidelines included: (i) β-lactams (amoxicillin, amoxicillin/clavulanic acid, ticarcillin, imipenem, cefalotin, cefoxitin, cefotaxime and ceftazidime); (ii) aminoglycosides (streptomycin, spectinomycin, kanamycin, gentamicin, tobramycin and amikacin); (iii) phenicols (chloramphenicol); (iv) tetracyclines (doxycycline); (v) folate inhibitors (sulfamethoxazole and trimethoprim); (vi) quinolones (nalidixic acid and ciprofloxacin); and (vii) rifampicin. DNA sequencing Genomic DNA was extracted using the DNeasy blood and tissue kit (QIAGEN). Class 1 integrons (sul1-type with primers L1-R1 and Tn402-type with L1-RH506) and class 2 integrons were searched and sequenced by the Sanger method.19 The whole genome was sequenced with an Illumina MiSeq sequencer using the Nextera XT DNA sample preparation kit, according to the manufacturer’s instructions. De novo assembly was performed with Velvet from the galaxy.prabi.fr web service (http://galaxy.prabi.fr/). All contigs were searched for resistance genes and potential foreign genetic structures using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). PCR linkage between genes belonging to non-repeated genetic elements and Sanger sequencing were performed to fill the gap between contigs of interest. The Illumina reads were mapped on the assembled sequences using the BWA (Burrows–Wheeler Aligner) read aligner (Galaxy) and visualized with the Integrative Genomics Viewer (IGV) (Broad Institute, http://software.broadinstitute.org/software/igv/) to identify potential base mismatches. Conjugation experiments and mobilization assays Conjugation experiments were carried out by a broth mating method using the rifampicin-resistant Escherichia coli K-12 C600 recipient strain. Transconjugants were selected on sorbitol MacConkey agar supplemented with rifampicin (50 mg/L) and gentamicin (4 mg/L) (for the transfer of GIPmi1) or with rifampicin (50 mg/L) and chloramphenicol (50 mg/L) (for the transfer of ICEPmiFra1). When transfer failed, mobilization assays were carried out using Enterobacter aerogenes 409 harbouring an A/C2 plasmid (pEA409TEM24 GenBank accession number MG764534) or its E. coli K-12 C600 transconjugant, as previously described, to mobilize SGI1 and PGI1.13 Nucleotide sequence accession number The nucleotide sequences of GIPmi1, ICEPmiFra1 and Tn7 were assigned GenBank accession numbers MF490433, MF490434 and MG701057, respectively. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PUXR00000000. The version described in this article is version PUXR01000000. Results and discussion Antibiotic susceptibility, integron content and genome analysis P. mirabilis PmPHI was resistant to: amoxicillin and ticarcillin; streptomycin, spectinomycin, kanamycin, gentamicin and tobramycin; chloramphenicol; doxycycline; sulfamethoxazole and trimethoprim; nalidixic acid and ciprofloxacin. Various cassette arrays were detected on two sul1-type class 1 integrons (aadB-aadA2 and dfrA32-ereA1-aadA2), one Tn402-type class 1 integron (estX-qacE), and one class 2 integron (dfrA1-sat2-aadA1). Genome analysis revealed that these integrons were embedded in three acquired genetic structures. The first one was a GI named GIPmi1 inserted at the 3′ end of trmE. The second one was an ICE named ICEPmiFra1 inserted within prfC. The last one was a Tn7 located downstream of glmS. Resistance to quinolones was due to the well-known mutations in the quinolone resistance-determining regions of gyrA (S83I) and parC (S84I). No plasmids were found. Characterization of GIPmi1 The 55.8 kb GI, GIPmi1, consisted of a backbone and an MDR region. Its backbone shared 93% identity with a structure detected in Enterobacter cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 (accession number CP017184). Both structures were inserted by site-specific recombination into the last 18 bp of trmE, as for SGI1 and PGI1. Unlike the GI from E. cloacae, GIPmi1 carried an MDR region close to a resolvase gene. The backbone of the GI (18.7 kb) contained 14 ORFs with a GC content equal to 43.5% (Figure 1a). The integrase gene was similar to those of SGI1, SGI2, PGI1 and AGI1 (64%–67% DNA identity). A higher similarity was found with other integrase genes: 95% identity with that of E. cloacae DSM 16690, 93% and 92% with an integrase gene found at the end of trmE in Shewanella putrefaciens 200 (CP002457) and S. putrefaciens CN-32 (CP000681), respectively; 98% with a partial integrase gene in S. enterica subsp. enterica serovar Senftenberg strain ATCC 43845 (CP019194). The resolvase gene and the nucleotide sequence of the three ORFs of unknown function at the right end of GIPmi1 shared 92%–99% identity with those found in these four strains. Other ORFs encoded products of putative function: a transcriptional regulator of the excisionase family, a helicase, a hydroxy acid dehydrogenase (virulence protein of the RhuM family), a DNA methyltransferase, an ATP-binding protein and a DEAD/DEAH-box helicase (ORFs RI02, RI03, RI05, RI08, RI09 and RI10, respectively). No reading frames encoding proteins with possible conjugative functions were found. Figure 1. View largeDownload slide Structure of GIPmi1. (a) Comparison of the backbone of GIPmi1 with the GI of E. cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 and (b) genetic organization of the MDR region of GIPmi1. Arrows indicate ORFs and genes, and their transcriptional orientation. (a) Black arrows represent chromosomal genes and white arrows the GI backbone: for E. cloacae, ORFs are indicated by the last three figures of the corresponding locus_tag (e.g. trmE: locus_tag = BFV67_22270). attL and attR sites are represented. (b) The 5′-CS and 3′-CS of the integrons are shown as filled arrows and the IRs (IRi and IRt) are indicated. Target site duplications are framed. Transposons and IS elements are shown as grey and hatched arrows, respectively, IRs are indicated by vertical bars. Antibiotic resistance genes are shown as dotted arrows. Figure 1. View largeDownload slide Structure of GIPmi1. (a) Comparison of the backbone of GIPmi1 with the GI of E. cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 and (b) genetic organization of the MDR region of GIPmi1. Arrows indicate ORFs and genes, and their transcriptional orientation. (a) Black arrows represent chromosomal genes and white arrows the GI backbone: for E. cloacae, ORFs are indicated by the last three figures of the corresponding locus_tag (e.g. trmE: locus_tag = BFV67_22270). attL and attR sites are represented. (b) The 5′-CS and 3′-CS of the integrons are shown as filled arrows and the IRs (IRi and IRt) are indicated. Target site duplications are framed. Transposons and IS elements are shown as grey and hatched arrows, respectively, IRs are indicated by vertical bars. Antibiotic resistance genes are shown as dotted arrows. The complex MDR region (37.1 kb) was flanked by a 5 bp duplication of the target sequence (CTGGT), as generated by insertion via transposition (Figure 1b). It was composed of two class 1 integrons separated by a structure of 22.9 kb. The Tn402-type class 1 integron at the left-hand end, with the tni module located adjacent to the resolvase gene, carried the estX-qacE cassette array. The four genes of the transposition module [87% identity with Tn402 (X72585)] were a structural chimera [tniR, tniQ, tniB and tniA sharing, respectively, 100%, 99%, 100% and 95% identities with their homologues in Tn402, Tn5053 (L40585), Tn502 (EU306743) and Tn502, respectively]. The sul1-type class 1 integron (In5-type) at the right-hand end contained the aadB-aadA2 cassette array. Both integrons were in inverse orientation compared with their classical position in other genetic structures such as SGI1 or PGI1. The mosaic structure between these two integrons was made of several parts of transposons and IS elements. It contained: (i) parts of transposons Tn1721, Tn5393 (strA-strB), Tn21 and the complete transposon Tn2 (blaTEM-1b); and (ii) the following IS elements: IS1133 harboured by Tn5393, IS26, IS4321, which targeted the IRtnp end of Tn21 and IS1R inserted 58 bp from the IRmer of Tn21 as in Tn2670. It is noteworthy that all components of this central region including the plasmid genes (topB and parB) have already been found in the MDR region of PGI1-PmCHA.9 However, they are not in the same arrangement. An extrachromosomal circular form of GIPmi1 was detected by a two-step PCR protocol, suggesting its potential mobility (primers used for PCRs are listed in Table S1, available as Supplementary data at JAC Online, and the attG site in the circular form of GIPmi1 is represented in Figure S1). However, the conjugative transfer of GIPmi1 and its mobilization by an A/C plasmid failed despite three independent attempts to select gentamicin-resistant transconjugants. This result is not surprising because GIPmi1 does not carry tra genes. Characterization of ICEPmiFra1 The ICEPmiFra1 of 104.2 kb belonging to the SXT/R391 family was inserted into the 5′ end of prfC. Its backbone (72.7 kb) shared high sequence identity (99%) with that of ICEPmiChn1 recently described in China.20 ICEPmiFra1 harboured floR and the sul2-strA-strB cluster in the variable region VRIII, as already described by Li et al.18 (Figure 2). A composite transposon flanked by two copies of a transporter ISPpu12 element (tISPpu12) (AY128707) composed of four ORFs (a transposase and three passenger genes) was inserted in the hot spot HS4. The right-hand copy shared 99% identity with AY128707, but the left-hand one had a 2 nt deletion in tnpA leading to a shortened transposase (tISPpu12 isoform). An 8 bp target site duplication (TAAAGAAA) was detected, characteristic of a transposition event. This composite transposon carried a partial tetA(C)-tetR(C) mobile unit flanked by two copies of IS26, one of them being truncated by the tISPpu12 isoform, the transposon Tn4352 (aphA1a) and a sul1-type class 1 integron (dfrA32-ereA1-aadA2) with truncated 5′-CS (conserved sequence) and 3′-CS. Tn4352 might have been generated by incorporation at an existing IS26 of a translocatable unit made up of aphA1a and an IS26 as recently described.21 Figure 2. View largeDownload slide Genetic organization of ICEPmiFra1. The white rectangles represent the backbone of ICEPmiFra1. The variable region VRIII and the hot spot HS4 are detailed. Antibiotic resistance genes and IS elements are shown as dotted arrows and hatched arrows, respectively. The 5′-CS of the integron is shown as filled arrows. Target site duplications are framed. Figure 2. View largeDownload slide Genetic organization of ICEPmiFra1. The white rectangles represent the backbone of ICEPmiFra1. The variable region VRIII and the hot spot HS4 are detailed. Antibiotic resistance genes and IS elements are shown as dotted arrows and hatched arrows, respectively. The 5′-CS of the integron is shown as filled arrows. Target site duplications are framed. The conjugative transfer of ICEPmiFra1 to E. coli K-12 C600 occurred at a frequency of 10−8 chloramphenicol-resistant transconjugants per donor. Conclusions The MDR P. mirabilis PmPHI harboured two large GIs including a new ICE of the SXT/R391 family (ICEPmiFra1) and a new genomic resistance island (GIPmi1). GIPmi1 is the first member of a new lineage of genomic resistance island belonging to the superfamily of GIs proposed by Hamidian et al.11 including SGI1, SGI2, PGI1 and AGI1. Indeed, it meets the criteria defining this superfamily: insertion at the 3′ end of trmE and similar integrases. Nevertheless, the gene synteny was not conserved between GIPmi1 and SGI1/SGI2/PGI1/AGI1 backbones. The presence of a resolvase gene within GIPmi1 allowed the integration of a class 1 integron. Multiple rearrangements involving fragments of known transposons and a IS26 element led to a very complex MDR region, which might easily incorporate genes conferring resistance to antibiotics of medical importance, as already described for SGI1 and PGI1. Funding This work was supported by the Association Dijonnaise des Bactériologistes (ADIBAC) and by the University Hospital Research Division (for genome sequencing). Transparency declarations None to declare. Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online. References 1 Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria . FEMS Microbiol Rev 2011 ; 35 : 820 – 55 . Google Scholar CrossRef Search ADS PubMed 2 Leverstein-van Hall MA , Blok HEM , Donders ART et al. Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin . J Infect Dis 2003 ; 187 : 251 – 9 . Google Scholar CrossRef Search ADS PubMed 3 Wei Q , Hu Q , Li S et al. A novel functional class 2 integron in clinical Proteus mirabilis isolates . J Antimicrob Chemother 2014 ; 69 : 973 – 6 . Google Scholar CrossRef Search ADS PubMed 4 Hansson K , Sundström L , Pelletier A et al. IntI2 integron integrase in Tn7 . J Bacteriol 2002 ; 184 : 1712 – 21 . Google Scholar CrossRef Search ADS PubMed 5 DeBoy RT , Craig NL. Target site selection by Tn7: attTn7 transcription and target activity . J Bacteriol 2000 ; 182 : 3310 – 3 . Google Scholar CrossRef Search ADS PubMed 6 Ahmed AM , Hussein AI , Shimamoto T. Proteus mirabilis clinical isolate harbouring a new variant of Salmonella genomic island 1 containing the multiple antibiotic resistance region . J Antimicrob Chemother 2007 ; 59 : 184 – 90 . Google Scholar CrossRef Search ADS PubMed 7 Doublet B , Poirel L , Praud K et al. European clinical isolate of Proteus mirabilis harbouring the Salmonella genomic island 1 variant SGI1-O . J Antimicrob Chemother 2010 ; 65 : 2260 – 2 . Google Scholar CrossRef Search ADS PubMed 8 Siebor E , Neuwirth C. Emergence of Salmonella genomic island 1 (SGI1) among Proteus mirabilis clinical isolates in Dijon, France . J Antimicrob Chemother 2013 ; 68 : 1750 – 6 . Google Scholar CrossRef Search ADS PubMed 9 Siebor E , Neuwirth C. Proteus genomic island 1 (PGI1), a new resistance genomic island from two Proteus mirabilis French clinical isolates . J Antimicrob Chemother 2014 ; 69 : 3216 – 20 . Google Scholar CrossRef Search ADS PubMed 10 Girlich D , Dortet L , Poirel L et al. Integration of the blaNDM-1 carbapenemase gene into Proteus genomic island 1 (PGI1-PmPEL) in a Proteus mirabilis clinical isolate . J Antimicrob Chemother 2015 ; 70 : 98 – 102 . Google Scholar CrossRef Search ADS PubMed 11 Hamidian M , Holt KE , Hall RM. Genomic resistance island AGI1 carrying a complex class 1 integron in a multiply antibiotic-resistant ST25 Acinetobacter baumannii isolate . J Antimicrob Chemother 2015 ; 70 : 2519 – 23 . Google Scholar CrossRef Search ADS PubMed 12 Doublet B , Boyd D , Mulvey MR et al. The Salmonella genomic island 1 is an integrative mobilizable element . Mol Microbiol 2005 ; 55 : 1911 – 24 . Google Scholar CrossRef Search ADS PubMed 13 Siebor E , de Curraize C , Amoureux L et al. 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 . J Antimicrob Chemother 2016 ; 71 : 2167 – 70 . Google Scholar CrossRef Search ADS PubMed 14 Hall RM. Salmonella genomic islands and antibiotic resistance in Salmonella enterica . Future Microbiol 2010 ; 5 : 1525 – 38 . Google Scholar CrossRef Search ADS PubMed 15 Harada S , Ishii Y , Saga T et al. Chromosomally encoded blaCMY-2 located on a novel SXT/R391-related integrating conjugative element in a Proteus mirabilis clinical isolate . Antimicrob Agents Chemother 2010 ; 54 : 3545 – 50 . Google Scholar CrossRef Search ADS PubMed 16 Mata C , Navarro F , Miró E et al. Prevalence of SXT/R391-like integrative and conjugative elements carrying blaCMY-2 in Proteus mirabilis . J Antimicrob Chemother 2011 ; 66 : 2266 – 70 . Google Scholar CrossRef Search ADS PubMed 17 Wozniak RA , Fouts DE , Spagnoletti M et al. Comparative ICE genomics: insights into the evolution of the SXT/R391 family of ICEs . PLoS Genet 2009 ; 5 : e1000786 . Google Scholar CrossRef Search ADS PubMed 18 Li X , Du Y , Du P et al. SXT/R391 integrative and conjugative elements in Proteus species reveal abundant genetic diversity and multidrug resistance . Sci Rep 2016 ; 6 : 37372 . Google Scholar CrossRef Search ADS PubMed 19 Post V , Recchia GD , Hall RM. Detection of gene cassettes in Tn402-like class 1 integrons . Antimicrob Agents Chemother 2007 ; 51 : 3467 – 8 . Google Scholar CrossRef Search ADS PubMed 20 Lei CW , Zhang AY , Wang HN et al. Characterization of SXT/R391 integrative and conjugative elements in Proteus mirabilis isolates from food-producing animals in China . Antimicrob Agents Chemother 2016 ; 60 : 1935 – 8 . Google Scholar CrossRef Search ADS PubMed 21 Harmer CJ , Hall RM. IS26-mediated precise excision of the IS26-aphA1a translocatable unit . MBio 2015 ; 6 : e01866 – 15 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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

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Abstract

Abstract 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. Introduction Proteus mirabilis is an opportunistic human pathogen that is generally susceptible to all classes of antibiotic agents active against Gram-negative bacilli, with the exception of tetracyclines and polymyxins. Antibiotic resistance is often due to the acquisition of resistance genes associated with integrons, transposons or ISs.1 Class 1 and class 2 integrons are those most frequently encountered in P. mirabilis.2,3 The former are generally plasmid-borne and the latter are found on transposon Tn7 mainly inserted into the chromosomal glmS transcriptional terminator.4,5 Sometimes the resistance genes are clustered into structures named genomic islands (GIs). Several types of GIs have been described to date. The most frequently encountered in P. mirabilis are Salmonella genomic island 1 (SGI1)6–8 and Proteus genomic island 1 (PGI1)9,10, belonging to the same superfamily of GIs that also includes SGI2 and AGI1, identified in Acinetobacter baumannii.11 SGI1, initially found in Salmonella enterica serovars, and PGI1 are large integrative mobilizable elements.12,13 They target the last 18 bp of the chromosomal trmE gene, encode related integrases and consist of a backbone and an MDR region located adjacent to the res gene. Different variants with backbone alterations and/or MDR region variations have been described.14 Besides these GIs, SXT/R391-related integrative conjugative elements (ICEs) have been described in P. mirabilis.15,16 These large elements, originally discovered in Vibrio cholerae (SXT) or Providencia rettgeri (R391), are integrated into the peptide chain release factor 3 gene (prfC).17 Various genes have been identified in hot spots (HS1–HS5) and variable regions (VRI–VRIV) of ICEs. Genetic diversity of these ICEs has been reported in MDR P. mirabilis in China.18 During our continuous survey of SGI1 and PGI1 among MDR P. mirabilis, we used PCRs with primers targeting the ORF S026 and the left and right junctions of these GIs.8 The isolate PmPHI, lacking the ORF S026 and with an undetectable intact trmE, was retained for characterization of its resistance genes content and their genetic context using WGS. Materials and methods Bacterial strain P. mirabilis PmPHI was isolated in August 2012 from a patient hospitalized in the haematology ward of Dijon hospital (France). Antibiotic susceptibility testing performed by the disc diffusion method according to the Antibiogram Committee of the French Society for Microbiology (CASFM)/EUCAST guidelines included: (i) β-lactams (amoxicillin, amoxicillin/clavulanic acid, ticarcillin, imipenem, cefalotin, cefoxitin, cefotaxime and ceftazidime); (ii) aminoglycosides (streptomycin, spectinomycin, kanamycin, gentamicin, tobramycin and amikacin); (iii) phenicols (chloramphenicol); (iv) tetracyclines (doxycycline); (v) folate inhibitors (sulfamethoxazole and trimethoprim); (vi) quinolones (nalidixic acid and ciprofloxacin); and (vii) rifampicin. DNA sequencing Genomic DNA was extracted using the DNeasy blood and tissue kit (QIAGEN). Class 1 integrons (sul1-type with primers L1-R1 and Tn402-type with L1-RH506) and class 2 integrons were searched and sequenced by the Sanger method.19 The whole genome was sequenced with an Illumina MiSeq sequencer using the Nextera XT DNA sample preparation kit, according to the manufacturer’s instructions. De novo assembly was performed with Velvet from the galaxy.prabi.fr web service (http://galaxy.prabi.fr/). All contigs were searched for resistance genes and potential foreign genetic structures using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). PCR linkage between genes belonging to non-repeated genetic elements and Sanger sequencing were performed to fill the gap between contigs of interest. The Illumina reads were mapped on the assembled sequences using the BWA (Burrows–Wheeler Aligner) read aligner (Galaxy) and visualized with the Integrative Genomics Viewer (IGV) (Broad Institute, http://software.broadinstitute.org/software/igv/) to identify potential base mismatches. Conjugation experiments and mobilization assays Conjugation experiments were carried out by a broth mating method using the rifampicin-resistant Escherichia coli K-12 C600 recipient strain. Transconjugants were selected on sorbitol MacConkey agar supplemented with rifampicin (50 mg/L) and gentamicin (4 mg/L) (for the transfer of GIPmi1) or with rifampicin (50 mg/L) and chloramphenicol (50 mg/L) (for the transfer of ICEPmiFra1). When transfer failed, mobilization assays were carried out using Enterobacter aerogenes 409 harbouring an A/C2 plasmid (pEA409TEM24 GenBank accession number MG764534) or its E. coli K-12 C600 transconjugant, as previously described, to mobilize SGI1 and PGI1.13 Nucleotide sequence accession number The nucleotide sequences of GIPmi1, ICEPmiFra1 and Tn7 were assigned GenBank accession numbers MF490433, MF490434 and MG701057, respectively. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PUXR00000000. The version described in this article is version PUXR01000000. Results and discussion Antibiotic susceptibility, integron content and genome analysis P. mirabilis PmPHI was resistant to: amoxicillin and ticarcillin; streptomycin, spectinomycin, kanamycin, gentamicin and tobramycin; chloramphenicol; doxycycline; sulfamethoxazole and trimethoprim; nalidixic acid and ciprofloxacin. Various cassette arrays were detected on two sul1-type class 1 integrons (aadB-aadA2 and dfrA32-ereA1-aadA2), one Tn402-type class 1 integron (estX-qacE), and one class 2 integron (dfrA1-sat2-aadA1). Genome analysis revealed that these integrons were embedded in three acquired genetic structures. The first one was a GI named GIPmi1 inserted at the 3′ end of trmE. The second one was an ICE named ICEPmiFra1 inserted within prfC. The last one was a Tn7 located downstream of glmS. Resistance to quinolones was due to the well-known mutations in the quinolone resistance-determining regions of gyrA (S83I) and parC (S84I). No plasmids were found. Characterization of GIPmi1 The 55.8 kb GI, GIPmi1, consisted of a backbone and an MDR region. Its backbone shared 93% identity with a structure detected in Enterobacter cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 (accession number CP017184). Both structures were inserted by site-specific recombination into the last 18 bp of trmE, as for SGI1 and PGI1. Unlike the GI from E. cloacae, GIPmi1 carried an MDR region close to a resolvase gene. The backbone of the GI (18.7 kb) contained 14 ORFs with a GC content equal to 43.5% (Figure 1a). The integrase gene was similar to those of SGI1, SGI2, PGI1 and AGI1 (64%–67% DNA identity). A higher similarity was found with other integrase genes: 95% identity with that of E. cloacae DSM 16690, 93% and 92% with an integrase gene found at the end of trmE in Shewanella putrefaciens 200 (CP002457) and S. putrefaciens CN-32 (CP000681), respectively; 98% with a partial integrase gene in S. enterica subsp. enterica serovar Senftenberg strain ATCC 43845 (CP019194). The resolvase gene and the nucleotide sequence of the three ORFs of unknown function at the right end of GIPmi1 shared 92%–99% identity with those found in these four strains. Other ORFs encoded products of putative function: a transcriptional regulator of the excisionase family, a helicase, a hydroxy acid dehydrogenase (virulence protein of the RhuM family), a DNA methyltransferase, an ATP-binding protein and a DEAD/DEAH-box helicase (ORFs RI02, RI03, RI05, RI08, RI09 and RI10, respectively). No reading frames encoding proteins with possible conjugative functions were found. Figure 1. View largeDownload slide Structure of GIPmi1. (a) Comparison of the backbone of GIPmi1 with the GI of E. cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 and (b) genetic organization of the MDR region of GIPmi1. Arrows indicate ORFs and genes, and their transcriptional orientation. (a) Black arrows represent chromosomal genes and white arrows the GI backbone: for E. cloacae, ORFs are indicated by the last three figures of the corresponding locus_tag (e.g. trmE: locus_tag = BFV67_22270). attL and attR sites are represented. (b) The 5′-CS and 3′-CS of the integrons are shown as filled arrows and the IRs (IRi and IRt) are indicated. Target site duplications are framed. Transposons and IS elements are shown as grey and hatched arrows, respectively, IRs are indicated by vertical bars. Antibiotic resistance genes are shown as dotted arrows. Figure 1. View largeDownload slide Structure of GIPmi1. (a) Comparison of the backbone of GIPmi1 with the GI of E. cloacae complex ‘Hoffmann cluster IV’ strain DSM 16690 and (b) genetic organization of the MDR region of GIPmi1. Arrows indicate ORFs and genes, and their transcriptional orientation. (a) Black arrows represent chromosomal genes and white arrows the GI backbone: for E. cloacae, ORFs are indicated by the last three figures of the corresponding locus_tag (e.g. trmE: locus_tag = BFV67_22270). attL and attR sites are represented. (b) The 5′-CS and 3′-CS of the integrons are shown as filled arrows and the IRs (IRi and IRt) are indicated. Target site duplications are framed. Transposons and IS elements are shown as grey and hatched arrows, respectively, IRs are indicated by vertical bars. Antibiotic resistance genes are shown as dotted arrows. The complex MDR region (37.1 kb) was flanked by a 5 bp duplication of the target sequence (CTGGT), as generated by insertion via transposition (Figure 1b). It was composed of two class 1 integrons separated by a structure of 22.9 kb. The Tn402-type class 1 integron at the left-hand end, with the tni module located adjacent to the resolvase gene, carried the estX-qacE cassette array. The four genes of the transposition module [87% identity with Tn402 (X72585)] were a structural chimera [tniR, tniQ, tniB and tniA sharing, respectively, 100%, 99%, 100% and 95% identities with their homologues in Tn402, Tn5053 (L40585), Tn502 (EU306743) and Tn502, respectively]. The sul1-type class 1 integron (In5-type) at the right-hand end contained the aadB-aadA2 cassette array. Both integrons were in inverse orientation compared with their classical position in other genetic structures such as SGI1 or PGI1. The mosaic structure between these two integrons was made of several parts of transposons and IS elements. It contained: (i) parts of transposons Tn1721, Tn5393 (strA-strB), Tn21 and the complete transposon Tn2 (blaTEM-1b); and (ii) the following IS elements: IS1133 harboured by Tn5393, IS26, IS4321, which targeted the IRtnp end of Tn21 and IS1R inserted 58 bp from the IRmer of Tn21 as in Tn2670. It is noteworthy that all components of this central region including the plasmid genes (topB and parB) have already been found in the MDR region of PGI1-PmCHA.9 However, they are not in the same arrangement. An extrachromosomal circular form of GIPmi1 was detected by a two-step PCR protocol, suggesting its potential mobility (primers used for PCRs are listed in Table S1, available as Supplementary data at JAC Online, and the attG site in the circular form of GIPmi1 is represented in Figure S1). However, the conjugative transfer of GIPmi1 and its mobilization by an A/C plasmid failed despite three independent attempts to select gentamicin-resistant transconjugants. This result is not surprising because GIPmi1 does not carry tra genes. Characterization of ICEPmiFra1 The ICEPmiFra1 of 104.2 kb belonging to the SXT/R391 family was inserted into the 5′ end of prfC. Its backbone (72.7 kb) shared high sequence identity (99%) with that of ICEPmiChn1 recently described in China.20 ICEPmiFra1 harboured floR and the sul2-strA-strB cluster in the variable region VRIII, as already described by Li et al.18 (Figure 2). A composite transposon flanked by two copies of a transporter ISPpu12 element (tISPpu12) (AY128707) composed of four ORFs (a transposase and three passenger genes) was inserted in the hot spot HS4. The right-hand copy shared 99% identity with AY128707, but the left-hand one had a 2 nt deletion in tnpA leading to a shortened transposase (tISPpu12 isoform). An 8 bp target site duplication (TAAAGAAA) was detected, characteristic of a transposition event. This composite transposon carried a partial tetA(C)-tetR(C) mobile unit flanked by two copies of IS26, one of them being truncated by the tISPpu12 isoform, the transposon Tn4352 (aphA1a) and a sul1-type class 1 integron (dfrA32-ereA1-aadA2) with truncated 5′-CS (conserved sequence) and 3′-CS. Tn4352 might have been generated by incorporation at an existing IS26 of a translocatable unit made up of aphA1a and an IS26 as recently described.21 Figure 2. View largeDownload slide Genetic organization of ICEPmiFra1. The white rectangles represent the backbone of ICEPmiFra1. The variable region VRIII and the hot spot HS4 are detailed. Antibiotic resistance genes and IS elements are shown as dotted arrows and hatched arrows, respectively. The 5′-CS of the integron is shown as filled arrows. Target site duplications are framed. Figure 2. View largeDownload slide Genetic organization of ICEPmiFra1. The white rectangles represent the backbone of ICEPmiFra1. The variable region VRIII and the hot spot HS4 are detailed. Antibiotic resistance genes and IS elements are shown as dotted arrows and hatched arrows, respectively. The 5′-CS of the integron is shown as filled arrows. Target site duplications are framed. The conjugative transfer of ICEPmiFra1 to E. coli K-12 C600 occurred at a frequency of 10−8 chloramphenicol-resistant transconjugants per donor. Conclusions The MDR P. mirabilis PmPHI harboured two large GIs including a new ICE of the SXT/R391 family (ICEPmiFra1) and a new genomic resistance island (GIPmi1). GIPmi1 is the first member of a new lineage of genomic resistance island belonging to the superfamily of GIs proposed by Hamidian et al.11 including SGI1, SGI2, PGI1 and AGI1. Indeed, it meets the criteria defining this superfamily: insertion at the 3′ end of trmE and similar integrases. Nevertheless, the gene synteny was not conserved between GIPmi1 and SGI1/SGI2/PGI1/AGI1 backbones. The presence of a resolvase gene within GIPmi1 allowed the integration of a class 1 integron. Multiple rearrangements involving fragments of known transposons and a IS26 element led to a very complex MDR region, which might easily incorporate genes conferring resistance to antibiotics of medical importance, as already described for SGI1 and PGI1. Funding This work was supported by the Association Dijonnaise des Bactériologistes (ADIBAC) and by the University Hospital Research Division (for genome sequencing). Transparency declarations None to declare. Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online. References 1 Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria . FEMS Microbiol Rev 2011 ; 35 : 820 – 55 . Google Scholar CrossRef Search ADS PubMed 2 Leverstein-van Hall MA , Blok HEM , Donders ART et al. Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin . J Infect Dis 2003 ; 187 : 251 – 9 . Google Scholar CrossRef Search ADS PubMed 3 Wei Q , Hu Q , Li S et al. A novel functional class 2 integron in clinical Proteus mirabilis isolates . J Antimicrob Chemother 2014 ; 69 : 973 – 6 . Google Scholar CrossRef Search ADS PubMed 4 Hansson K , Sundström L , Pelletier A et al. IntI2 integron integrase in Tn7 . J Bacteriol 2002 ; 184 : 1712 – 21 . Google Scholar CrossRef Search ADS PubMed 5 DeBoy RT , Craig NL. Target site selection by Tn7: attTn7 transcription and target activity . J Bacteriol 2000 ; 182 : 3310 – 3 . Google Scholar CrossRef Search ADS PubMed 6 Ahmed AM , Hussein AI , Shimamoto T. Proteus mirabilis clinical isolate harbouring a new variant of Salmonella genomic island 1 containing the multiple antibiotic resistance region . J Antimicrob Chemother 2007 ; 59 : 184 – 90 . 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Google Scholar CrossRef Search ADS PubMed 11 Hamidian M , Holt KE , Hall RM. Genomic resistance island AGI1 carrying a complex class 1 integron in a multiply antibiotic-resistant ST25 Acinetobacter baumannii isolate . J Antimicrob Chemother 2015 ; 70 : 2519 – 23 . Google Scholar CrossRef Search ADS PubMed 12 Doublet B , Boyd D , Mulvey MR et al. The Salmonella genomic island 1 is an integrative mobilizable element . Mol Microbiol 2005 ; 55 : 1911 – 24 . Google Scholar CrossRef Search ADS PubMed 13 Siebor E , de Curraize C , Amoureux L et al. 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 . J Antimicrob Chemother 2016 ; 71 : 2167 – 70 . Google Scholar CrossRef Search ADS PubMed 14 Hall RM. Salmonella genomic islands and antibiotic resistance in Salmonella enterica . Future Microbiol 2010 ; 5 : 1525 – 38 . Google Scholar CrossRef Search ADS PubMed 15 Harada S , Ishii Y , Saga T et al. Chromosomally encoded blaCMY-2 located on a novel SXT/R391-related integrating conjugative element in a Proteus mirabilis clinical isolate . Antimicrob Agents Chemother 2010 ; 54 : 3545 – 50 . Google Scholar CrossRef Search ADS PubMed 16 Mata C , Navarro F , Miró E et al. Prevalence of SXT/R391-like integrative and conjugative elements carrying blaCMY-2 in Proteus mirabilis . J Antimicrob Chemother 2011 ; 66 : 2266 – 70 . Google Scholar CrossRef Search ADS PubMed 17 Wozniak RA , Fouts DE , Spagnoletti M et al. Comparative ICE genomics: insights into the evolution of the SXT/R391 family of ICEs . PLoS Genet 2009 ; 5 : e1000786 . Google Scholar CrossRef Search ADS PubMed 18 Li X , Du Y , Du P et al. SXT/R391 integrative and conjugative elements in Proteus species reveal abundant genetic diversity and multidrug resistance . Sci Rep 2016 ; 6 : 37372 . Google Scholar CrossRef Search ADS PubMed 19 Post V , Recchia GD , Hall RM. Detection of gene cassettes in Tn402-like class 1 integrons . Antimicrob Agents Chemother 2007 ; 51 : 3467 – 8 . Google Scholar CrossRef Search ADS PubMed 20 Lei CW , Zhang AY , Wang HN et al. Characterization of SXT/R391 integrative and conjugative elements in Proteus mirabilis isolates from food-producing animals in China . Antimicrob Agents Chemother 2016 ; 60 : 1935 – 8 . Google Scholar CrossRef Search ADS PubMed 21 Harmer CJ , Hall RM. IS26-mediated precise excision of the IS26-aphA1a translocatable unit . MBio 2015 ; 6 : e01866 – 15 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 12, 2018

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