Regional Spread of CTX-M-2-Producing Proteus mirabilis with the Identical Genetic Structure in Japan

Abstract

Aims:

In this study, we analyzed the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Proteus mirabilis isolates collected from the central region of Japan.

Materials and Methods:

Between 2005 and 2012, 820 clinical P. mirabilis isolates were obtained from ten acute care hospitals in Japan. We characterized ESBL confirmatory test-positive isolates by sequencing the ESBL genes and their flanking regions, detecting plasmid replicons, and performing pulsed-field gel electrophoresis (PFGE).

Results:

Ninety-six isolates (12%) were positive according to the ESBL confirmatory test; all these isolates possessed bla_CTX-M-2 with the same flanking structure of upstream ΔISEcp1 and a downstream region identical to downstream bla_KLUA-1. IncT was the prevalent, and only, replicon found in 63 isolates. PFGE analysis detected eight clusters with more than one isolate, among which three included 56 isolates and six included isolates from multiple hospitals.

Conclusion:

CTX-M-2-producing P. mirabilis with an identical genetic structure flanking bla_CTX-M-2 is dominant in this Japanese region, and there is evidence for the clonal spread of isolates.

Introduction

Proteus mirabilis is a common cause of urinary tract infections as well as an important cause of nosocomial infections such as respiratory tract and skin infections.¹ P. mirabilis-associated urinary tract infections may be difficult to treat because the bacterium persists with complications, including bladder and kidney stone formation.¹ Wild-type P. mirabilis is susceptible to β-lactams, aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole.² However, the emergence of resistance to β-lactams, such as penicillins and cephalosporins, has been reported in recent years in Japan (1998–2010),^3–5 Hong Kong (1999–2002),⁶ Taiwan (1999–2005),⁷ Korea (2008),⁸ Argentina (2000),⁹ and North America and Europe (2009–2010),¹⁰ which has challenged the treatment of infections.^11,12 The TEM,^13,14 VEB-1,¹⁵ and CTX-M⁶ types of extended-spectrum β-lactamases (ESBLs), as well as CMY-type plasmid-mediated β-lactamases (pAmpCs), are known to be determinants of resistance among clinical P. mirabilis isolates. Treatment failure and higher mortality are likely to occur in patients infected with ESBL-producing P. mirabilis due to inadequate empirical therapy.¹⁶ In Japan, previous studies found that CTX-M-2 was the dominant β-lactamase^3,4 and that CTX-M-2-producing P. mirabilis have been implicated in nosocomial infection outbreaks.¹⁷

Genes encoding the ESBL enzymes are often found in transferable elements, which may transfer the resistance genes and their flanking regions among isolates and species.¹⁸ Indeed, the genetic environment, including the resistance gene, may be associated with a specific plasmid or chromosomal location. Thus, to understand the epidemiology and contributors for the spread of ESBL-producing P. mirabilis in Japan, we examined the resistance mechanisms, genes, or plasmids related to resistance genes, and clonal relatedness of P. mirabilis clinical isolates collected in a regional surveillance program.

Materials and Methods

Bacterial isolates

This study was conducted at 10 acute care hospitals in the Kyoto and Shiga regions of Japan. The Kyoto and Shiga regions (combined population of four million people) are located adjacent to each other in central Japan. Ten of the 22 acute care hospitals in these regions with more than 400 beds and a microbiology laboratory participated in this study. Between January 2005 and December 2012, P. mirabilis clinical isolates were obtained from inpatients and outpatients. Nonduplicate isolates that tested positive according to an ESBL confirmation test were sent to a reference laboratory (Kyoto University) for further investigation. Collections were conducted every year; isolates were collected and stored anonymously, without any accompanying demographic data.

Identification and susceptibility testing

At each hospital, microbiological identification and susceptibility testing were performed using the Vitek 2 system (bioMérieux, Marcy l'Étoile, France) or the MicroScan system (Siemens Healthcare Diagnostics, Tokyo, Japan). Subsequently, the ESBL screening test was performed according to the Clinical and Laboratory Standards Institute (CLSI) microdilution method (cefotaxime, ceftriaxone, ceftazidime, cefpodoxime, and aztreonam) and the ESBL confirmation test was performed using cefotaxime and ceftazidime disks with or without clavulanate following the CLSI guidelines.¹⁹ In a reference laboratory, antibiotic susceptibility was re-evaluated by microdilution using the Dry Plate Eiken system (Eiken Chemical, Tokyo, Japan), which included testing with cefepime, cefmetazole, cefotaxime, ceftazidime, aztreonam, ampicillin–sulbactam, piperacillin, piperacillin–tazobactam, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim–sulfamethoxazole, and fosfomycin. Results were interpreted using 2014 CLSI breakpoints,¹⁹ and intermediate susceptibility was classified as resistant.

β-lactamase identification

Bacterial DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The presence of ESBL or pAmpC genes was detected by polymerase chain reaction (PCR) amplification and sequencing of the CTX-M,^20,21 TEM,²² and SHV²³ genes, as well as the six main groups of pAmpC-type genes.²⁴ We used following strains with ESBL genes as controls: ATCC 700603 (bla_SHV-18), ATCC 35218 (bla_TEM-1), KUN-2012-1590 (bla_CTX-M-27, GenBank accession no. AB976590), KS128 (bla_CTX-M-15, GenBank accession no. AB976566), and SI34 (bla_CTX-M-2, GenBank accession no. AB976588). PCR amplification was performed with TaKaRa Ex Taq DNA polymerase (TaKaRa Bio, Inc., Otsu, Japan) using TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio) according to PCR conditions described in the above references. PCR products were analyzed using the QIAxcel DNA Screening Kit (Qiagen) on the QIAxcel capillary electrophoresis system (Qiagen). Purified PCR amplicons were directly sequenced. Sequencing reactions were performed in a Bio-Rad DNA Engine Dyad PTC-220 Peltier Thermal Cycler (Bio-Rad, Hercules, CA) using an ABI BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA). Sequencing reactions were read using an ABI 3730xl sequencer (ThermoFisher Scientific).

Analysis of bla_CTX-M-2 surrounding regions

To determine the genetic structure of the regions that flank the bla_CTX-M-2 gene, PCR amplification was conducted between bla_CTX-M-2 and ISEcp1, ISCR1, IS903, intI1, qacEΔ1-sul1, orf1, or aadA1 using previously described primers.²⁵ We used ISEcp1A, ISEcp1B,²⁶ and ISEcp1 U1²⁷ primers to identify the precise structure of ISEcp1. In addition, a new primer dubbed klu-r (5′-TGAAGCTAAGATATTTGGTCCAG-3′) was designed to amplify a downstream sequence of bla_CTX-M-2 using the sequence of P. mirabilis TUM4653 (GenBank accession no. AB543698, position 2795–2817) as a reference. Amplification was performed with 1× PCR buffer, 2.0 mM MgCl₂, 0.25 mM deoxynucleoside triphosphates, each primer, 0.75 U TaKaRa Ex Taq DNA polymerase (TaKaRa Bio), and 1 μL purified DNA in a total volume of 30 μL. The cycling protocol was as follows: 30 cycles of 98°C for 10 sec, 60°C for 20 sec, and 72°C for 180 sec.

Plasmid replicon typing

Plasmid replicon typing was performed using total DNA and a PCR-based method with 18 pairs of primers, as described by Carattoli et al.²⁸

Pulsed-field gel electrophoresis analysis

Pulsed-field gel electrophoresis (PFGE) was performed with NotI and SfiI-digested whole genomic DNA samples of P. mirabilis isolates to analyze their genetic relatedness. The PFGE patterns were interpreted based on the criteria proposed by Tenover et al.,²⁹ and profiles obtained were analyzed with GelCompar II version 4.6 (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis, using the unweighted pair-group method based on Dice coefficients, was used to quantify similarities, and the relatedness among isolates was defined based on PFGE profiles with ≥80% similarity.⁴

Results

During the study period, 820 nonduplicate P. mirabilis isolates were obtained from patients at ten hospitals. Of these, 96 P. mirabilis isolates from nine hospitals tested positive according to the ESBL confirmation test and they were included in this study. The annual proportions of ESBL-producing P. mirabilis were 2.0% in 2005, 25% in 2006, 31% in 2007, 9.1% in 2008, 7.4% in 2009, 8.2% in 2010, 4.5% in 2011, and 11% in 2012.

Susceptibility testing

Results of antimicrobial susceptibility testing are shown in Table 1; all 96 isolates were susceptible to cefmetazole, ceftazidime, aztreonam, piperacillin–tazobactam, meropenem, and amikacin, whereas less than 10% were susceptible to cefotaxime and piperacillin.

Table 1.

Antimicrobial Susceptibilities of the 96 CTX-M-2-Producing Proteus mirabilis Isolates

Antimicrobial agent	Total no. (%)
Cefepime	53 (55)
Cefmetazole	96 (100)
Cefotaxime	2 (2)
Ceftazidime	96 (100)
Aztreonam	96 (100)
Ampicillin–sulbactam	88 (92)
Piperacillin	3 (3)
Piperacillin–tazobactam	96 (100)
Imipenem	37 (39)
Meropenem^a	96 (100)
Amikacin	96 (100)
Gentamicin	91 (95)
Tobramycin	93 (97)
Ciprofloxacin^b	13 (14)
Levofloxacin^b	60 (63)
Trimethoprim–sulfamethoxazole	81 (84)
Fosfomycin	16 (17)

All isolates had minimum inhibitory concentrations (MICs) of less than 0.5 μg/mL.

Median MICs for ciprofloxacin and levofloxacin were 2 and 2 μg/mL, respectively. Forty-seven ciprofloxacin-non-susceptible and levofloxacin-susceptible isolates had MIC ranges of 2–4 μg/mL for ciprofloxacin, and 1–2 μg/mL for levofloxacin.

β-lactamase identification

All 96 isolates possessed bla_CTX-M-2 and had no other ESBL or pAmpC genes. Eight isolates possessed bla_TEM-1 and two other isolates possessed bla_TEM-135.

Analysis of bla_CTX-M-2 surrounding regions

All 96 isolates shared an identical structure for the ΔISEcp1-bla_CTX-M-2-downstream bla_KLUA-1 gene. This structure was identical to that found in P. mirabilis TUM4653 (GenBank accession no. AB543698, position 1707–2766). However, only the 3′-end of ISEcp1 was present, and PCR amplification was successful with only the ISEcp1 U primer, while it was unsuccessful with ISEcp1A1 and ISEcp1B primers that targeted the 5′-side of ISEcp1 or primers that targeted ISCR1 or intI1. The sequence downstream of bla_CTX-M-2 was identical to downstream Kluyvera ascorbata bla_KLUA-1 (GenBank accession no. AJ272538).

Plasmid replicon typing

IncT was the prevalent type (n = 63, 66%) and the only replicon (Fig. 1). The other isolates without the IncT replicon were regarded as nontypeable.

FIG. 1.

Dendrogram based on PFGE for NotI and SfiI-digested whole genomic DNA samples of the 96 CTX-M-2-producing Proteus mirabilis isolates. The vertical line indicates the ≥80% similarity score employed to assign isolates to the same cluster using the unweighted pair-group method and an arithmetic mean. The black circle indicates isolates with the IncT plasmid replicon. PFGE, pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis

PFGE classified the study isolates into 12 clusters designated as C1–C12 (Fig. 1). There were three major clusters, with C5 forming the largest group with 56 isolates (58%), followed by C1 (n = 15, 16%) and C6 (n = 9, 9.4%). The C5 cluster included 46 isolates from hospital F (82%), whereas the other ten isolates came from four other hospitals. Five other minor clusters (C3, C4, C7, C8, and C11) included more than one isolate, with the two major clusters (C1 and C6), and three minor clusters (C3, C4, and C7) including isolates from more than one hospital. The other four clusters included only one isolate, and IncT plasmids were detected from isolates in clusters C1, C3, C5, and C6.

Discussion

This 8-year study analyzed the spread of CTX-M-2-producing P. mirabilis in the Kyoto and Shiga regions of Japan, as well as the potential nosocomial outbreaks due to this pathogen. The prevalence of ESBL-producing P. mirabilis differs among countries or regions within a country, where the reported rates range from 0% to more than 52.5%.^6,7,30,31 In this study, 2.0–31% of isolates produced ESBL with variation between years. A previous surveillance study conducted at acute hospitals in the same Kinki region (to which Kyoto and Shiga belong) of Japan determined comparable prevalence rates of 2.8% in 2005 and 12.9% in 2009,³ whereas another Japanese study that analyzed isolates collected in a commercial clinical testing laboratory between 2009 and 2010 found that as many as 45.6% of isolates were ESBL-producing P. mirabilis.⁴ These Japanese studies, and those from other countries such as China,⁸ have indicated an increasing trend in ESBL-producing P. mirabilis. However, the trend could not be evaluated in the present study due to the presence of a large number of isolates from potential outbreaks (as discussed below).

In terms of antimicrobial susceptibility, the studied isolates exhibited major differences between ciprofloxacin and levofloxacin (14% and 63%, respectively), which may be explained by the fact that most isolates had minimum inhibitory concentrations close to their breakpoints (Table 1); one previous study obtained similar results in China (27% for ciprofloxacin and 53% for levofloxacin).³² P. mirabilis is known to possess an intrinsic nonsusceptibility to imipenem,¹³ which is consistent with our results. All of the studied isolates had low minimum inhibitory concentrations for meropenem, which were below the carbapenemase screening cutoff (CLSI gudelines¹⁹ and EUCAST guidelines³³), thereby supporting the absence of carbapenemases. The studied isolates had 84% susceptibility to trimethoprim–sulfamethoxazole, which is similar to that found for a non-ESBL isolate from Canada (84%).

CTX-M-2 was the only ESBL type found in our study, and is almost exclusively dominant among P. mirabilis-associated ESBL enzymes in Japan^3,17 and in Argentina, where this enzyme has been initially reported.^9,34 However, CTX-M-2 is in the minority in Korea (7.1%)⁸ and absent in Italy (0%).³⁵

Harada et al. investigated Japanese P. mirabilis isolates and found that bla_CTX-M-2 was present on the IncT plasmid and/or on chromosome.²⁵ Regardless of the location of bla_CTX-M-2, the isolates all possessed a similar ISEcp1-bla_CTX-M-2-downstream bla_KLUA-1 structure, which is consistent with our determination that IncT was prevalent, and that an identical genetic structure was present. The only difference was that the studied isolates lacked the full upstream ISEcp1. These results suggest that bla_CTX-M-2 was spread among the studied isolates through a specific genetic structure with some contribution from IncT plasmids.

Outbreaks of specific strains contributed to the increases in ESBL-producing isolates because the PFGE analysis detected the intrahospital and interhospital dissemination of specific clones (especially clusters C5, C1, and C6; Fig. 1). The temporal increase of ESBL-producing P. mirabilis in 2006 and 2007 was mainly related to the outbreak of the cluster C5 strain in hospital F. Interhospital spread of a specific clone might be explained by exposure to a common source that was contaminated with the outbreak strain or transfer of patients with the outbreak strain between hospitals, but we do not have any supporting evidence due to lack of clinical or epidemiological information.

This study had several limitations. In particular, we could not determine the location of the bla_CTX-M-2 gene (due to lack of hybridization or transformation/conjugation experiments) and the molecular mechanisms that promote the dissemination of its genetic structure. It may be harbored by the IncT plasmid or IncT-negative isolates may carry it in their chromosome.²⁵ In addition, we could not investigate the epidemiology of outbreaks because of lack of clinical information.

In conclusion, CTX-M-2-producing P. mirabilis, with an identical genetic structure flanking bla_CTX-M-2, exclusively dominated in the acute care hospitals in our study region, and there was evidence for the clonal spread of isolates within each hospital and between hospitals. Further analysis is required to elucidate the mechanisms responsible for this spread and the specific epidemiological aspects of outbreaks. Nevertheless, this study provides insights into the regional epidemiology of ESBL-producing P. mirabilis, and our findings may help to minimize further dissemination of drug-resistant bacteria, as well as guiding hospital and public health infection control policies.

Footnotes

Acknowledgments

Members of the Kyoto–Shiga Clinical Microbiology Study Group included Y.M., M.Y., M.N., S.T., S.I., Naohisa Fujita, Toshiaki Komori, Yukiji Yamada, Tsunehiro Shimizu, Akihiko Hayashi, Tamotsu Ono, Harumi Watanabe, Naoko Fujihara, Takeshi Higuchi, Kunihiko Moro, Masayo Shigeta, Kaneyuki Kida, Hiromi Terada, Fusayuki Tsuboi, Yoshihisa Sugimoto, and Chiyoko Fukumoto.

Author Disclosure Statement

No competing financial interests exist.

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Regional Spread of CTX-M-2-Producing Proteus mirabilis with the Identical Genetic Structure in Japan

Abstract

Aims:

Materials and Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Bacterial isolates

Identification and susceptibility testing

β-lactamase identification

Analysis of blaCTX-M-2 surrounding regions

Plasmid replicon typing

Pulsed-field gel electrophoresis analysis

Results

Susceptibility testing

β-lactamase identification

Analysis of blaCTX-M-2 surrounding regions

Plasmid replicon typing

Pulsed-field gel electrophoresis

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Analysis of bla_CTX-M-2 surrounding regions

Analysis of bla_CTX-M-2 surrounding regions