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Antimicrob Agents Chemother. Feb 2012; 56(2): 1093–1096.
PMCID: PMC3264238

Chromosomal Integration and Location on IncT Plasmids of the blaCTX-M-2 Gene in Proteus mirabilis Clinical Isolates

Abstract

Analysis of five CTX-M-2-producing Proteus mirabilis isolates in Japan demonstrated that blaCTX-M-2 was located on the chromosome in four isolates and on IncT plasmids in three isolates, including two isolates that also carried the gene on the chromosome. In all four isolates with chromosomal blaCTX-M-2, ISEcp1 was responsible for the integration of the gene into the chromosome. Three different sites in the P. mirabilis genomic sequence were utilized as integration sites.

TEXT

In the past decade, CTX-M enzymes have become the most prevalent extended-spectrum β-lactamases (ESBLs) produced by members of the Enterobacteriaceae (3). In Japan, Toho-1, now renamed CTX-M-44, was initially identified in 1995 (14), and CTX-M enzymes have been prevalent among ESBLs since. Among Proteus mirabilis strain, all 71 CTX-M-producing clinical isolates collected from 2001 through 2003 in Japan carried blaCTX-M-2 (27). A nationwide surveillance study conducted in 2006 involving 54 health care facilities in Japan reported that 28 of 74 P. mirabilis clinical isolates (37.8%) produced ESBLs and that all of them except one harbored blaCTX-M-2 (16). Clonality of CTX-M-2-producing P. mirabilis isolates in Japan was not investigated in these studies. In this study, we characterized the location and genetic environment of the blaCTX-M-2 gene of P. mirabilis isolates in Japan to elucidate the genetic mechanisms involved in the acquisition of blaCTX-M-2 in this organism.

Seventy-six P. mirabilis clinical isolates from various culture samples were collected from 11 health care facilities in Japan through a nationwide surveillance of antimicrobial susceptibility between September 2005 and March 2006. Five of them (6.5%) were determined to be ESBL producers by the procedure described in the CLSI guidelines (7). The prevalence of ESBL-producing P. mirabilis strains was low compared with that in the above-mentioned study performed during the same time period (16). It is difficult to determine the true prevalence of ESBL-producing P. mirabilis in Japan because both studies involved only a limited number of isolates. These five isolates, TUM4653 to TUM4657, were subjected to further analysis (Table 1). While two of five isolates (TUM4655 and TUM4656) were collected from the same health care facility in Tokyo, the other three strains were isolated from geographically separated areas. MICs were determined by the broth dilution method according to CLSI guidelines (6) and then interpreted using CLSI criteria (7). All five isolates were resistant to cefotaxime and susceptible to cefoxitin and ceftazidime. Resistance to cefotaxime was reversed by the addition of clavulanic acid in all isolates (Table 1). Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) using CHEF-Mapper (Bio-Rad, Hercules, CA) with the genomic DNA of P. mirabilis clinical strains digested with SfiI and NotI (Bio-Rad) (8). Visual analysis of PFGE banding patterns according to the criteria of Tenover et al. showed that TUM4654 and TUM4656 were possibly related, while other isolates were unrelated to each other (Fig. 1) (29).

Table 1
Properties of ESBL-producing P. mirabilis clinical isolates and their transconjugants and transformantsa
Fig 1
PFGE profiles of SfiI- and NotI-digested fragments of the genomic DNA of P. mirabilis isolates producing CTX-M-2. The sizes of fragments were determined by comparison with a Lambda Ladder PFGE marker (New England BioLabs, Hertfordshire, United Kingdom). ...

Total genomic DNA was extracted from the five isolates and subjected to PCR amplification with the primer pairs CTX-M-2-Fw (5′-ACGCTACCCCTGCTATTT-3′)–CTX-M-2-Rv (5′-CCTTTCCGCCTTCTGCTC-3′) (27) and ISEcp1U3 (5′-ATTGGGTGAAAGAAAAGTGCTC-3′)–CTX-M-2-Rv, which yielded amplified products of the expected sizes. Sequencing of the PCR products revealed the presence of ISEcp1 upstream of blaCTX-M-2 and also an identical 49-bp sequence between ISEcp1 and blaCTX-M-2 in all isolates.

Conjugation experiments were performed with each P. mirabilis clinical isolate (all were non-lactose-fermenting isolates) as a donor and E. coli ML4909, a rifampin-resistant and lactose-fermenting derivative of E. coli K-12, as a recipient, using the filter mating method (13). Lactose-fermenting transconjugants were selected on bromothymol blue-lactose agar plates supplemented with rifampin (50 μg/ml) and cefotaxime (4 μg/ml). Transconjugants of TUM4653 and TUM4655 were obtained and were named TUM4683 and TUM4684, respectively (Table 1). For TUM4654, TUM4656, and TUM4657, transformation of E. coli DH5α Electro-Cells (Takara Bio Inc.) by electroporation with an E. coli Pulser (Bio-Rad) was attempted with plasmids extracted by alkaline lysis according to the procedure described by Kado and Liu (15), followed by selection on Luria-Bertani agar plates supplemented with cefotaxime (4 μg/ml). As a result, a transformant was obtained only for TUM4657 and was named TUM4685 (Table 1). The transconjugants and transformant showed reduced susceptibility to cefotaxime and were positive for blaCTX-M-2 by PCR. Resistance to gentamicin was also observed in TUM4683 and TUM4684, whereas reduced susceptibility to gentamicin and ciprofloxacin was noted in TUM4685, suggesting the copresence of other resistance genes on the plasmids harboring blaCTX-M-2 (Table 1).

PCR-based replicon typing as described by Carattoli et al. (5) was performed using extracted plasmids from TUM4653, TUM4655, TUM4657, TUM4683, TUM4684, and TUM4685. They were all positive for IncT, and the DNA sequences of the amplified products were identical to that of the Rts plasmid, a prototype for the IncT plasmid (21), for all plasmids. To estimate the sizes of plasmids carrying blaCTX-M-2, PFGE-separated, S1 nuclease-treated fragments of the genomic DNA of the clinical strains were transferred onto Hybond-N+ (GE Healthcare, Little Chalfont, United Kingdom) and hybridized with PCR-generated probes specific for blaCTX-M-2, tnpA of ISEcp1, and repA of IncT plasmids using a DIG High Prime I DNA labeling and detection starter kit (Roche Applied Science, Mannheim, Germany) (1, 11). The results showed that blaCTX-M-2 together with ISEcp1 was located on IncT plasmids of 145 kb, 170 kb, and 165 kb in TUM4653, TUM4655, and TUM4657, respectively (data not shown). Restriction of the extracted plasmids from TUM4683, TUM4684, and TUM4685 with HindIII, BamHI, and EcoRI (Takara Bio Inc.) yielded >10 visible bands by electrophoresis with 0.6% agarose (SeaKem Gold agarose; Takara Bio Inc.) with fewer than two fragment differences, and the probe for blaCTX-M-2 hybridized with the same fragment for each enzyme. These results suggested that the IncT plasmids with blaCTX-M-2 in TUM4653, TUM4655, and TUM4657 shared a similar genetic structure.

The I-CeuI technique was employed to establish the chromosomal location of blaCTX-M-2 (20, 22). Genomic DNA from the clinical strains was digested with I-CeuI (New England BioLabs, Hertfordshire, United Kingdom), separated by PFGE, transferred onto Hybond-N+, and hybridized with PCR-generated probes specific for blaCTX-M-2, tnpA of ISEcp1, and the 16S rRNA gene. The results showed that a copy of blaCTX-M-2 was integrated into the chromosomes of TUM4653, TUM4656, and TUM4657 and two copies of blaCTX-M-2 were integrated into the chromosome of TUM4654 and also that all blaCTX-M-2 copies were accompanied by ISEcp1 (data not shown). The location and copy number of blaCTX-M-2 did not appear to correlate with the level of resistance against expanded-spectrum cephalosporins (Table 1).

We then determined the nucleotide sequence surrounding the chromosomal copies of blaCTX-M-2 by inverse PCR (31). Whole-genomic DNA extracted from the P. mirabilis clinical isolates was digested with HindIII, and the resulting fragments were subjected to self-ligation using a TaKaRa Long DNA ligation kit (Takara Bio Inc.). The circular DNA obtained was amplified by PCR with the primers ISEcp1-Inv (5′-GCTTTTTGCATTCTCAAGGAGCAG-3′) and CTX-M-2-Inv (5′-TTACCCAACCGGAGCAGAAGG-3′), designed according to the nucleotide sequence around the 5′ end of ISEcp1 and the 3′ end of blaCTX-M-2, respectively. Platinum Taq DNA high-fidelity polymerase (Invitrogen, Carlsbad, CA) was used as the DNA polymerase. PCR products were purified and sequenced using the primer walking method. In the analysis of TUM4654 with multiple copies of blaCTX-M-2 on the chromosome, the inverse PCR products with different sizes were cloned into the pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen), and nucleotide sequences of the each cloned fragment were separately determined. If the presence of blaCTX-M-2 on both the plasmid and chromosome interrupted the procedure (TUM4653), the isolate was first cured of the plasmid by culturing the isolate at 42°C (21, 30), and the strain without plasmid was then subjected to extraction of genomic DNA and inverse PCR. The genetic environment surrounding each ISEcp1-blaCTX-M-2 locus is shown in Fig. 2. The ISEcp1-blaCTX-M-2 locus was inserted into the open reading frame (ORF) PMI2533, acs (encoding acetyl coenzyme A synthetase), and ORF PMI3202 in TUM4653, TUM4656, and TUM4657, according to the annotation of P. mirabilis HI4320 (24). In TUM4653, TUM4656, and TUM4657, 5-bp direct repeats, TATAA, AGTCA, and TATCA, respectively, were identified adjacent to the flanking left inverted repeat (IRL) or putative right inverted repeat (IRR). These findings suggested that ISEcp1-mediated transposition was responsible for the integration of blaCTX-M-2 into the chromosome. The 256-bp nucleotide sequence immediately downstream of all copies of chromosomal blaCTX-M-2 was identical to those found in the genomic sequence of Kluyvera ascorbata immediately downstream of kluA-1 (GenBank accession no. AJ272538). Although the 300-bp sequence immediately downstream of the K. ascorbata nucleotide sequence in TUM4657 was identical to that identified in TUM4656, a BLAST search identified no other nucleotide sequence with high similarity to this sequence. The sizes of all transposable units were approximately 3 kb, which is comparable to the sizes of ISEcp1-mediated transposable units reported in previous studies (17, 25, 26). The sequence surrounding the first copy of blaCTX-M-2 in TUM4654 was identical to that of blaCTX-M-2 in TUM4656. The sequence surrounding the other copy of blaCTX-M-2 in TUM4654 lacked the putative IRR and direct repeat downstream of the 256-bp K. ascorbata genomic fragment. A definitive mechanism for the transfer of this copy of blaCTX-M-2 was not evident.

Fig 2
Schematic representation of the genetic environment of blaCTX-M-2 located on the chromosomes of P. mirabilis clinical isolates. White arrows represent open reading frames. Black triangles represent 14-bp inverted repeats, including the left and right ...

To the best of our knowledge, this is the first study to report the presence of ESBL genes on IncT plasmids (4). The presence of IncT resistance plasmids, which mediate resistance to ampicillin and kanamycin, has mostly been reported in Proteus vulgaris and Providencia rettgeri in Asia (18, 21, 23). While comprehensive studies have not been performed on the prevalence of IncT plasmids in Enterobacteriaceae, including P. mirabilis, it is reasonable to infer that P. mirabilis is a potential host compatible with IncT plasmids under natural conditions because of the genetic similarity among members of the Proteeae (10).

Although chromosomal integration of blaCTX-M and other genes encoding extended-spectrum β-lactamases is regarded as a relatively uncommon event (11, 12), several studies have suggested that those events could be more common in P. mirabilis (2, 8, 9, 19, 28, 32). Of 28 CTX-M-producing P. mirabilis clinical isolates collected in Korea, 21 carried blaCTX-M on the chromosome (28). While members of the blaCTX-M-1 and blaCTX-M-9 groups were accompanied by ISEcp1, blaCTX-M-2 in two isolates was located on a complex class 1 integron. In our study, all copies of blaCTX-M-2 documented in five isolates were accompanied by ISEcp1, which is consistent with the findings obtained for 27 CTX-M-2-producing P. mirabilis isolates collected in another study in Japan (16; also our unpublished data).

A detailed molecular analysis involving 21 P. mirabilis isolates with chromosomally located blaCMY-2 group genes collected from Poland, Italy, Greece, and France revealed that Tn6093, an ISEcp1-mediated transposon associated with blaCMY-2-group, was integrated into the P. mirabilis pepQ gene in all these isolates (8). In four isolates with two copies of blaCMY-2-group, the second copy of ISEcp1-blaCMY-2-group locus was located on Tn6113, which was inserted into the P. mirabilis intergenic sequence between ORF PMI0120 and the ppiD gene. Although three different chromosomal integration sites for the ISEcp1-blaCTX-M-2 locus were observed in our study, it is difficult to determine whether hotspots for the integration of the ISEcp1-blaCTX-M-2 locus exist because of the limited number of the isolates analyzed.

In conclusion, this study demonstrated chromosomal integration mediated by ISEcp1 and location on IncT plasmids of blaCTX-M-2 in P. mirabilis clinical isolates. While it remains to be elucidated whether these mechanisms are related to the high prevalence of blaCTX-M-2 in P. mirabilis in Japan, these findings enhance our understanding of mobile genetic elements involved in the spread of genes conferring resistance to expanded-spectrum cephalosporins in P. mirabilis.

ACKNOWLEDGMENTS

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 19591185 and no. 22591113 to Y.I.). The collection of P. mirabilis isolates was supported by a grant from Toyama Chemical Co., Ltd., and Taisho Toyama Pharmaceutical Co., Ltd.

We thank Robert A. Bonomo, Case Western Reserve University School of Medicine, for his useful suggestions and comments.

Footnotes

Published ahead of print 21 November 2011

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