Abstract 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. Introduction In the early eighties, an MDR clone of Salmonella Typhimurium definitive type 104 (DT104) emerged from the UK and spread all over the world.1 Most of the isolates carry a genomic island called Salmonella genomic island 1 (SGI1). Since that time, SGI1 has been described in Salmonella enterica, Proteus mirabilis and Morganella morganii.1,2 SGI1 (42.4 kb) is an integrative mobilizable element made up of a backbone containing 28 ORFs (S001–S027 and S044) and an MDR region.3,4 The MDR region in SGI1 harbours a complex In4-type class 1 integron, which contains genes encoding multiresistance: ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamide and tetracycline resistance (ACSSuT).3 Since the first report, variants of SGI1 (SGI1-A to SGI1-Z) and related islands such as SGI2 and PGI1 have been described.1,5–9 The backbone is largely conserved among SGI1 variants, but some rearrangements have been reported. For instance, in SGI1-K, there is a deletion of the region spanning from S005 to S009 (2780 bp) that is replaced by an IS (IS1359, also named ISVch4) and S044 is truncated by an IS26.5 The MDR region is sometimes very complex as a result of various homologous recombinations, loss or exchange of gene cassettes, or IS-element-mediated rearrangements.1 The MDR region mainly contains genes conferring resistance to the old generation of antibiotics or mercuric ions.1 Nevertheless, rmtC, which encodes resistance to aminoglycosides, or qnrA1 and qnrB2, which encode resistance to fluoroquinolones, have been sporadically reported.6,8,10 Concerning genes conferring resistance to broad-spectrum β-lactams, only blaVEB-6 (ESBL) has been detected on SGI1.6 As SGI1 can be mobilized in vitro by plasmids IncA or IncC in Escherichia coli and other Enterobacteriaceae, it should be regarded as an important vehicle for genes conferring resistance to major antibiotics.4,11 Here, we describe two novel SGI1s that harbour resistance genes never before (to the best of our knowledge) detected in the MDR region. Materials and methods Bacterial strains In the context of our continuous survey of P. mirabilis with SGI1, the two Pm2CHAMA and Pm37THOMI isolates that harboured a penicillinase and an ESBL, respectively, were positively screened for the presence of S026 (sgiA gene), which is used to detect SGI1-bearing isolates.12 They were isolated from urine samples of hospitalized patients at the Dijon University Hospital (France) during 2013 and 2014. The API 20E system (bioMérieux, Marcy-l’Étoile, France) was used for the biochemical identification of these isolates. Susceptibility to antimicrobial agents Susceptibility to antimicrobial agents was tested using the disc diffusion method on Mueller–Hinton agar in accordance with the Antibiogram Committee of the French Society for Microbiology/EUCAST guidelines for the following antimicrobial agents: amoxicillin, amoxicillin/clavulanic acid, ticarcillin, ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, nalidixic acid, ofloxacin, chloramphenicol, kanamycin, spectinomycin, streptomycin, tobramycin, amikacin, gentamicin, sulphonamides, trimethoprim and doxycycline. Bacterial DNA preparation, gene amplification and sequencing Bacterial DNA was extracted with the Gentra® Puregene® Purification Kit (Qiagen, Courtaboeuf, France). DNA was then quantified using Qubit® (Life Technologies, Paisley, UK). The library was prepared with the Nextera® XT Kit (Illumina, Eindhoven, The Netherlands) following the manufacturer’s instructions. The samples were multiplexed using Illumina-supplied barcodes and paired-end sequencing was performed on the Illumina MiSeq with the 300 cycle (MiSeq® Reagent Kit v2) (Illumina). The reads were assembled using the Velvet tool from the Galaxy web-based platform (http://www.prabi.fr/spip.php?page=galaxy). Contigs were analysed with the Basic Local Alignment Search Tool (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. Nucleotide sequence accession number The nucleotide sequences of SGI1-Pm2CHAMA and SGI1-K7 were assigned GenBank accession numbers MF372716 and MF372717, respectively. Results and discussion Characterization of SGI1-Pm2CHAMA Pm2CHAMA had acquired resistance to penicillins, spectinomycin, streptomycin, sulphonamides and trimethoprim. It harboured SGI1-Pm2CHAMA of 53.6 kb, which was integrated into the specific attB attachment site at the 3′-end of the chromosomal trmE gene. The backbone shared 99.9% identity with that of SGI1 (AF261825) (differences in 41 nt and 20 amino acids). The MDR region of 26.3 kb was adjacent to the res gene. A 175 bp deletion between the 3′-end of the MDR region and the right hand of the backbone led to the loss of the 5 bp duplication usually flanking the MDR region and a 5′-truncated S044 (34 bp deletion). The MDR region consisted of two class 1 integrons (Figure 1). The first, an In2-type integron, contained the qacH-aadA1 cassette array. The qacH gene is known to confer resistance to quaternary ammonium compounds by increasing the efflux activity.13 It might therefore help Pm2CHAMA to survive in the hospital environment, where quaternary ammonium compounds are widely used as disinfectants. The second class 1 integron was an In4-type integron containing only dfrA15. Figure 1. View largeDownload slide Schematic view of the MDR region of SGI1-Pm2CHAMA. Arrows indicate genes and ORFs with their transcriptional orientation. Black arrows represent 5′-CSs and 3′-CSs of integrons. IRs of integrons and transposons are indicated by thick vertical bars. Resistance genes are shown as dotted arrows and ovals represent attC sites. ISs are represented by hatched arrows and their IRs by thin vertical bars. Target site duplications are framed and indicated by small vertical arrows. Figure 1. View largeDownload slide Schematic view of the MDR region of SGI1-Pm2CHAMA. Arrows indicate genes and ORFs with their transcriptional orientation. Black arrows represent 5′-CSs and 3′-CSs of integrons. IRs of integrons and transposons are indicated by thick vertical bars. Resistance genes are shown as dotted arrows and ovals represent attC sites. ISs are represented by hatched arrows and their IRs by thin vertical bars. Target site duplications are framed and indicated by small vertical arrows. These two class 1 integrons in direct orientation were separated by a 10.6 kb IS26 flanked region. It contained Tn4352 (aphA1a) and parts of Tn5393 (strA and strB) disrupted by an isoform of ISAba14 [94% identity with ISAba14 (CP001921)]. ISAba14 was previously reported in PGI1-PmPEL.14 This ISAba14 isoform was interrupted by the new ISPmi2 of the IS3 family. These ISs have been both acquired by transposition as indicated by the presence of DRs (CAC for ISAba14 and GTCG for ISPmi2). The strB gene of Tn5393 was followed by blaCARB-4 (87% identity with blaPSE-1) and a part of the tns module of a Tn7-like transposon [90% identity with Tn7 (DQ176869)]. The blaCARB-4 is usually a gene cassette, but, in this case, the first 9 bp at the beginning of the cassette and the 119 bp of the attC site were missing.15 This complex region was characterized by the presence of three IS26 in the same orientation. This suggested that DNA rearrangements within the region have been generated by IS26 movements. An IS26 next to the In4-type integron might have promoted the incorporation of a translocatable unit with an IS26 resulting in a cointegrate.16 The unusual inner location of 8 bp DRs (CTTCCGGC) of this transposon suggested that these DRs were probably originally present on either side of this IS26. Tn4352 might have been generated by incorporation at an existing IS26 of a translocatable unit made of aphA1a and an IS26. The formation of Tn4352 mediated by IS26 was recently reported in vitro by Harmer and Hall.17 Characterization of SGI1-K7 Pm37THOMI harboured an ESBL phenotype and was resistant to amoxicillin, amoxicillin/clavulanic acid, ticarcillin, piperacillin, cefotaxime and cefepime. Other acquired resistances were observed: fluoroquinolones, spectinomycin, streptomycin, tobramycin, gentamicin, sulphonamides and trimethoprim. Pm37THOMI harboured a 55.1 kb SGI1 variant, which was located in a specific site at the 3′-end of the chromosomal trmE gene, which we propose to call SGI1-K7. Indeed, its backbone was identical to that of SGI1-K (100% identity).5 Moreover, its MDR region (29.7 kb) was derived from that of SGI1-K and resulted from three events (Figure 2). Figure 2. View largeDownload slide Schematic view of the MDR region of SGI1-K7. Arrows indicate genes and ORFs with their transcriptional orientations. Black arrows represent 5′- CSs and 3′-CSs of integrons. IRs of integrons and transposons are indicated by thick vertical bars. Resistance genes are shown as dotted arrows and ovals represent attC sites. ISs are represented by hatched arrows and their IRs by thin vertical bars. Figure 2. View largeDownload slide Schematic view of the MDR region of SGI1-K7. Arrows indicate genes and ORFs with their transcriptional orientations. Black arrows represent 5′- CSs and 3′-CSs of integrons. IRs of integrons and transposons are indicated by thick vertical bars. Resistance genes are shown as dotted arrows and ovals represent attC sites. ISs are represented by hatched arrows and their IRs by thin vertical bars. The first was the inversion of a large part of the MDR region between the integrase gene of the integron containing aadA7-aacCA5 and strA of Tn5393. This inversion was probably due to an intramolecular replicative transposition mediated by the IS26 adjacent to Tn5393.18 Indeed, a second IS26 in opposite orientation was detected at the left of the MDR region. Another event was the deletion of part of Tn5393 (part of strA, IS1133 and tnpR) and part of intI1. This deletion also likely affected target site duplication, which was absent.18 Finally, another structure that had previously been detected on plasmids from prevalent and successful multiresistant clones of Enterobacteriaceae such as E. coli ST131 or Klebsiella pneumoniae ST307 clones was inserted on the right-hand side of the MDR region.19,20 It contained aacC2, tmrB (tunicamycin resistance) and blaCTX-M-15.21 The structure harboured a variant of the genetic environment of blaCTX-M-15 (257 bp) corresponding to 48 bp upstream of blaCTX-M-15 plus 209 bp of the remnant ISEcp1 as previously described. The promoter of ISEcp1 was present.22 There was also a partial ISKpn11-like element [86% identity with ISKpn11 (EU780013)]. This structure in the MDR region contained three copies of IS26 in the same orientation including one at each end. No 8 bp target site duplication was detected around the IS26. Therefore, one might suppose that two translocatable units were incorporated successively because of the presence of IS26 next to S044.17 This very first (to the best of our knowledge) detection of the blaCTX-M-15 gene on an SGI1 is alarming. Indeed, CTX-M-15 is the most widely encountered ESBL among E. coli isolates.23 SGI1 might contribute to its dissemination among Enterobacteriaceae. Conclusions This study confirmed the great plasticity of the MDR region and suggested the key role of ISs, particularly for IS26, which is widely distributed. These ISs promote the accumulation of various resistance genes within the MDR region and lead to a worrisome reduction of therapeutic options. Acknowledgements We thank Professor Michael Chandler for his advice on IS nomenclature and Suzanne Rankin for English corrections. Funding This work was supported by the ADIBAC (Association Dijonnaise des Bactériologistes). Transparency declarations None to declare. References 1 Hall RM. Salmonella genomic islands and antibiotic resistance in Salmonella enterica. Future Microbiol 2010; 5: 1525– 38. Google Scholar CrossRef Search ADS PubMed 2 Schultz E, Barraud O, Madec J-Y et al. Multidrug resistance Salmonella genomic island 1 in a Morganella morganii subsp. morganii human clinical isolate from France. mSphere 2017; 2: e00118-17. Google Scholar CrossRef Search ADS PubMed 3 Boyd D, Peters GA, Cloeckaert A et al. Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. J Bacteriol 2001; 183: 5725– 32. Google Scholar CrossRef Search ADS PubMed 4 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 5 Hamidian M, Holt KE, Hall RM. 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The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance. FEMS Microbiol Lett 1998; 163: 49– 56. Google Scholar CrossRef Search ADS PubMed 14 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 15 Sanschagrin F, Bejaoui N, Levesque RC. Structure of CARB-4 and AER-1 carbenicillin-hydrolyzing β-lactamases. Antimicrob Agents Chemother 1998; 42: 1966– 72. Google Scholar PubMed 16 Harmer CJ, Moran RA, Hall RM. Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. mBio 2014; 5: e01801-14. Google Scholar CrossRef Search ADS PubMed 17 Harmer CJ, Hall RM. 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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)
Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Apr 11, 2018
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