Molecular epidemiology of carbapenemase-producing Enterobacteriaceae in a primary care hospital in Japan, 2010–2013

J Infect Chemother 23 (2017) 224e229

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Original Article

Molecular epidemiology of carbapenemase-producing Enterobacteriaceae in a primary care hospital in Japan, 2010e2013 Yuki Ohno a, *, Akihiro Nakamura a, b, Eriko Hashimoto a, Hiroko Matsutani a, Noriyuki Abe a, Saori Fukuda a, Kohno Hisashi a, Masaru Komatsu b, Fumihiko Nakamura a a b

Department of Clinical Bacteriology, Clinical Laboratory Medicine, Tenri Hospital, Tenri, Japan Department of Clinical Laboratory Science, Faculty of Health Care, Tenri Health Care University, Tenri, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 April 2016 Received in revised form 12 December 2016 Accepted 22 December 2016 Available online 1 February 2017

Recently, carbapenemase-producing Enterobacteriaceae (CPE) have been spreading worldwide and have become a threat in healthcare systems. We investigated the isolation frequency and molecular epidemiological characteristics of CPE isolated from clinical samples collected at a primary care hospital over the four years of 2010e2013 in Japan. CPE were detected in 17 (0.34%) of 4875 isolates by the broth microdilution method, sodium mercaptoacetate inhibition test, and modified Hodge test using meropenem disks. The frequency of CPE isolates was 0.09% in 2010, 0.17% in 2011, 0.16% in 2012 and 0.82% in 2013. Isolates positive for carbapenemase included Klebsiella pneumoniae (0.92%), Escherichia coli (0.12%), Enterobacter cloacae (0.80%), Klebsiella oxytoca (0.55%), Enterobacter aerogenes (0.81%) and Proteus mirabilis (0.08%). Antimicrobial susceptibility testing showed low MICs for piperacillin-tazobactam, amikacin, ciprofloxacin and levofloxacin, and only one multidrug-resistant strain. The carbapenemase genotype of all strains was IMP-6, and 94% of the strains were simultaneous CTX-M-2 producers. Two K. pneumoniae and 3 E. coli isolates showed the same pulsed-field gel electrophoresis group. Multilocus sequence typing detected no international high-risk clone types. Plasmid replicon typing detected IncN from all CPE strains, and IncF and IncFIB were simultaneously detected in 24% and 18%, respectively. All patients with detected CPE were inpatients, and many were elderly long-term hospitalized patients or had a history of prior vancomycin or levofloxacin antibiotic administration. The rapid spread of CPE is a concern in Japan. Preventive measures must be implemented against the spread of CPE after considering the epidemiological trend of CPE detection, antibiograms, and risk factors. © 2017 Published by Elsevier Ltd on behalf of Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.

Keywords: Carbapenemase-producing Enterobacteriaceae Molecular epidemiology PFGE MLST Plasmid replicon type

1. Introduction Recently, carbapenemase-producing Enterobacteriaceae (CPE) have been spreading throughout the world and have now become a threat in healthcare systems [1]. The carbapenemase-encoding gene that is located on the R-plasmid of CPE is important in preventive measures against healthcare-acquired infection because it is frequently conjugally transferred to other bacterial species. In addition, carbapenemase-encoding genes simultaneously comprise several genes that tolerate antimicrobial agents other than beta-

* Corresponding author. Department of Clinical Bacteriology, Clinical Laboratory Medicine, Tenri Hospital, 200 Mishima-cho, Tenri-shi, Nara 632-8552, Japan. Fax: þ81 743 63 1530. E-mail address: [email protected] (Y. Ohno).

lactam antibiotics, such as fluoroquinolone or aminoglycoside, and often confer multidrug resistance. Therefore, CPE will often show multidrug resistance in antimicrobial susceptibility testing [2]. Consequently, the mortality rate from severe infections caused by CPE is very high [3]. The worldwide spread of CPE will be of great concern in the future. The main genotype of carbapenemase contains KPC, GES, IMP, VIM, NDM and OXA-48 and is different in each country around the world [4]. For example, the main carbapenemase genotype in the United States has been KPC since this type was discovered in 1996, whereas in India, it has been NDM since it was discovered in 2008. Furthermore, the carbapenemase genotype of Europe is not limited to one type but is distributed among various genotypes, particularly, OXA-48, VIM and KPC [4,5]. The carbapenemase genotype of CPE isolated in Japan is different from that of other countries. It is IMP for most of the CPE genotypes isolated in Japan, and the

http://dx.doi.org/10.1016/j.jiac.2016.12.013 1341-321X/© 2017 Published by Elsevier Ltd on behalf of Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.

Y. Ohno et al. / J Infect Chemother 23 (2017) 224e229

isolation of other types is rare [6]. However, there are few reports on isolation frequency and long-term annual change in CPE in Japan. In addition, molecular epidemiological characteristics such as CPE to be isolated in Japan by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and plasmid replicon typing have not yet been revealed. Therefore, the purpose of this study was to clarify the isolation frequency and molecular epidemiological characteristics of the CPE, including all of the carbapenemase genotypes in Japan, of a primary care hospital over the four years of 2010e2013.

225

2.5. Molecular epidemiological analysis

Specimens were collected from January 2010 to November 2013. A total of 4875 isolates of Enterobacteriaceae including Escherichia coli (3255 isolates), Klebsiella pneumoniae (870 isolates), Klebsiella oxytoca (179 isolates), Enterobacter cloacae (249 isolates), Enterobacter aerogenes (123 isolates) and Proteus mirabilis (199 isolates) were isolated from clinical specimens collected at Tenri Hospital (a 1001-bed primary care hospital in Nara, Japan). This study was approved by the ethics committee of Tenri Hospital (reference number 667), and informed consent was obtained from all patients.

The CPE identified as being of the same species were characterized by MLST, PFGE and plasmid replicon typing. MLST of K. pneumoniae was performed according to the MLST scheme of the Pasteur Institute (http://www.pasteur.fr/recherche/genopole/PF8/ mlst/Kpneumoniae.html). That of E. coli was performed according to the Achtman MLST scheme (http://mlst.warwick.ac.uk/mlst/dbs/ Ecoli), and that of E. cloacae was performed according to the pubMLST scheme (http://pubmlst.org/ecloacae/). PFGE analysis was performed using the restriction enzyme XbaI (Roche Diagnostics, Inc., Tokyo, Japan) on a CHEF-Dr II system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) [16]. The reaction conditions included the following running parameters: 6.0 V/cm, 5.3 s initial switching time, 49.9 s final switching time and 20 h of total running time at 14  C. The dendrograms were obtained using Fingerprinting Plus Version 1.12 (Bio-Rad Laboratories, Inc.) and calculated using Dice coefficients and the unweighted pair group method using arithmetic averages [16]. Isolates were considered to belong to the same PFGE group if their Dice similarity index was 80%. PCRbased replicon typing was performed on 837 strains as described by Carattoli et al. [17]. Eighteen primer pairs targeting the FIA, FIB, FIC, HI1, HI2, I1-Ic, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FII replicons were used in separate PCR reactions.

2.2. Identification and antimicrobial susceptibility testing

2.6. Characteristics of patients with detected CPE

All strains were identified with a MicroScan WalkAway 40 Plus System and the Neg Breakpoint Combo 6.23J measurement panel (both, Siemens Healthcare Diagnostics, Inc., Los Angeles, CA, USA). In addition, the strains identified as CPE were also identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry using MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany). Antimicrobial susceptibility tests were performed with the broth microdilution method using a dry plate ‘EIKEN’ (Eiken Chemical Co., Ltd., Tokyo, Japan) that conforms to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Quality control was managed by using E. coli ATCC 25922.

We searched the medical records of the patients in whom CPE was detected to extract data on inpatient background (sex, age, number of hospitalization days, underlying disease, antimicrobial dosage history, therapeutic drugs and outcome) and investigated the characteristics of these patients.

2. Materials and methods 2.1. Bacterial isolates

2.3. Screening for CPE Among all target strains, for those which showed a MIC of 8 mg/L to any of the 3rd- or 4th-generation cephalosporins (ceftriaxone [CTRX], ceftazidime [CAZ] and cefepime [CFPM]) and a MIC of 2 mg/L to any of the carbapenems (imipenem [IPM], meropenem [MEPM], and doripenem [DRPM]) in the antimicrobial susceptibility testing, an inhibition assay using sodium mercaptoacetate and a modified Hodge test using meropenem disks were performed [7,8]. 2.4. Multiplex PCR amplification and sequencing for carbapenemase and ESBL Six carbapenemase-producing genotypes, comprising the IMP1, IMP-2, VIM, KPC, GES and NDM groups, and five ESBLproducing genotypes, comprising the SHV, TEM, CTX-M-1, CTXM-2 and CTX-M-9 groups, were searched for using multiplex PCR [4,9e15]. The PCR products were amplified using an Applied Biosystems Veriti 96 Thermal Cycler (Life Technologies, Tokyo, Japan). Moreover, PCR direct sequencing using an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific Inc., MA, USA) was performed for strains that were positive for one of the genotypes in the multiplex PCR assay. For sequence of IMP-1 group, forward primer 50 -ATGAGCAAGTTATCTG-30 and reverse primer 50 TTAGTTGCTTGGTTTTGATGG-30 were designed.

3. Results 3.1. Prevalence of CPE isolates The frequencies of isolation of CPE over 2010e2013 are shown in Table 1. The frequency of CPE isolation was 0.1% (1/1015) in 2010, 0.2% (2/1168) in 2011, 0.2% (2/1232) in 2012 and 0.8% (12/1460) in

Table 1 Number of isolates of CPE in each term. Organism

Number of isolates (%) 2010

Klebsiella pneumoniae Collected isolates 184 CPE 1 (0.54) Escherichia coli Collected isolates 664 CPE 0 (0) Enterobacter cloacae Collected isolates 43 CPE 0 (0) Klebsiella oxytoca Collected isolates 39 CPE 0 (0) Enterobacter aerogenes Collected isolates 37 CPE 0 (0) Proteus mirabilis Collected isolates 48 CPE 0 (0) Total Collected isolates 1015 CPE 1 (0.09)

Total

2011

2012

2013

206 1 (0.48)

213 1 (0.46)

267 5 (1.87)

870 8 (0.92)

770 0 (0)

833 0 (0)

988 4 (0.40)

3255 4 (0.12)

63 0 (0)

60 0 (0)

83 2 (2.40)

249 2 (0.80)

43 1 (2.32)

44 0 (0)

53 0 (0)

179 1 (0.55)

31 0 (0)

23 1 (4.34)

32 0 (0)

123 1 (0.81)

55 0 (0)

59 0 (0)

37 1 (2.70)

199 1 (0.50)

1168 2 (0.17)

1232 2 (0.16)

1460 12 (0.82)

4875 17 (0.34)

CPE carbapenemase-producing Enterobacteriaceae.

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Y. Ohno et al. / J Infect Chemother 23 (2017) 224e229

2013. The numbers of isolates positive for the carbapenemase gene were 8 (0.92%) for K. pneumoniae, 2 (0.80%) for E. cloacae, 1 (0.55%) for K. oxytoca, 1 (0.81%) for E. aerogenes and 1 (0.50%) for P. mirabilis. There were three cases in which two different species of CPE were detected in one patient. The rates of CPE detection in the sampled specimens were 59% (10/17) in urine, 18% (3/17) in catheters, 12% (2/17) in blood and 6% (1/17) each in sputum and in skin tissue.

ST1423 and ST2387; E. coli included 3 strains of ST95 and 1 strain of ST4508; and E. cloacae included 1 strain each of ST78 and ST721. Plasmid replicon typing detected the IncN group in all 17 strains along with the simultaneous presence of IncF in 24% (4/17) and IncFIB in 18% (3/17) of the strains.

3.2. Antimicrobial susceptibility testing

Characteristics of the 14 patients in whom CPE was detected are shown in Table 4. Of these 14 patients, 7 (50%) were men, 7 (50%) were women, and 86% (12/14) were older than 60 years of age. All of the patients in whom CPE was detected were hospitalized, and 93% (13/14) had been in the hospital for more than 30 days. In addition, 64% (9/14) of these patients had a history of prior antibiotics administration within the past two months: VCM was administered in 29% (4/14), LVFX þ trimethoprim-sulfamethoxazole (SXT) in 21% (3/14), single-agent LVFX in 7% (1/14) and LVFX þ VCM in 7% (1/14) of these patients. Therapeutic antibacterial agents administered against CPE included single-agent administration of LVFX in 43% (6/14), CPFX in 14% (2/14), SXT in 7% (1/14) and LVFX þ SXT administration in 7% (1/14) of the patients, whereas 29% (4/14) received no antibiotic therapy. Patient outcomes included death in 36% (5/14) and improvement in 64% (9/ 14) of the patients.

The results of the antimicrobial susceptibility testing of 17 strains of CPE are shown in Table 2. Piperacillin/tazobactam, amikacin (AMK), levofloxacin (LVFX) and ciprofloxacin (CPFX) showed the best sensitivity. Besides, b-lactam antibiotic drugs showed resistance to almost all types of drugs, whereas only MEPM and DRPM among the carbapenem antibiotics showed resistance in all strains. For IPM, 59% (10/17) of the strains showed a MIC of 1 mg/L. 3.3. Molecular epidemiological analysis The results of molecular epidemiological analysis are shown in Table 3. The carbapenemase gene type of all 17 strains was IMP-6, and no other carbapenemase gene types were detected. Also, for the ESBL gene type, CTX-M-2 co-producing strains were detected in 94% (16/17). The results of PFGE showed the same pattern in 2 K. pneumoniae and 3 E. coli strains, but other strains showed a different band pattern. The time course of 5 patients with E. coli and K. pneumoniae with the same PFGE patten is shown in Fig. 1. The 3 patients in whom E. coli was detected had been admitted to the same ward during the same period. In contrast, the 2 patients in whom K. pneumoniae was detected were hospitalized at different times. The results of MLST were as follows: K. pneumoniae included 2 strains of ST2388 and 1 strain each of ST12, ST23, ST37, ST466,

3.4. Characteristics of patients with detected CPE

4. Discussion We investigated the frequency of isolation of CPE and the associated molecular epidemiology over a 4-year period from 2010 to 2013 in a Japanese primary care hospital and found that the rate of CPE increased rapidly in 2013 in the hospitalized patients. Kurokawa et al. reported on the isolation frequency of metallobeta-lactamase (MBL)-producing Enterobacteriaceae bacteria in Japan and found the MBL production rate of Serratia marcescens in their survey of the strains that had been aggregated to the National

Table 2 Susceptibilities of CPE isolates to various antimicrobial agents. Species (no. of strains) and strain no. K. pneumoniae (8) THC3 THC6 THC7 THC9 THC11 THC13 THC18 THC26 E. coli (4) THC15 THC19 THC23 THC27 E. cloacae (2) THC10 THC17 K. oxytoca (1) THC5 E. aerogenes (1) THC8 P. mirabilis (1) THC25

MIC (mg/mL) P/T

CEZ

CTRX

CAZ

CFPM

IPM

DRPM

MEPM

AMK

TOB

MINO

SXT

CPFX

LVFX

8 4 4 16 16 >16 2 2

>8 >8 >8 >8 >8 >8 >8 >8

>8 >8 >8 >8 >8 >8 >8 >8

>8 >8 >8 >8 >8 >8 8 >8

2 >8 >8 >8 >8 >8 >8 >8

4 1 1 8 0.5 4 0.5 2

>4 >4 >4 >4 >4 >4 >4 >4

>4 >4 >4 >4 >4 >4 >4 >4

1 1 2 2 2 2 1 2

8 >8 8 >8 8 8 8 >8

>4 >4 >4 >4 >4 >4 <¼4 >4

2 2 >2 2 >2 2 2 2

0.12 0.03 0.5 0.12 0.5 0.12 0.06 0.03

0.25 0.5 1 0.12 1 0.25 0.12 0.06

2 2 2 2

>8 >8 >8 >8

>8 >8 >8 >8

>8 >8 >8 >8

>8 >8 >8 >8

1 1 1 1

>4 >4 >4 >4

>4 >4 >4 >4

2 2 >8 2

>8 >8 >8 >8

<¼4 <¼4 <¼4 <¼4

2 2 2 2

0.03 0.03 0.03 0.03

0.06 0.06 0.12 0.06

16 2

>8 >8

>8 >8

>8 >8

>8 >8

1 0.5

>4 4

>4 4

4 1

>8 8

>4 <¼4

>2 2

>2 0.06

>2 0.06

0.5

>8

>8

>8

>8

2

>4

>4

1

>8

>4

2

1

1

8

>8

>8

>8

>8

2

>4

>4

2

>8

>4

2

0.12

0.5

0.25

>8

>8

0.5

>8

8

>4

>4

4

8

>4

2

0.06

0.12

Abbreviations: CPE carbapenemase-producing Enterobacteriaceae, antimicrobial agents (tested range), P/T piperacillin-tazobactam (0.25e16 mg/ml), CEZ cefazolin (0.06e8 mg/ ml), CTRX ceftriaxone (0.06e8 mg/ml), CAZ ceftazidime (0.06e8 mg/ml), CFPM cefepime (0.06e8 mg/ml), IPM imipenem (0.12e8 mg/ml), MEPM meropenem (0.03e4 mg/ml), DRPM doripenem (0.03e4 mg/ml), AMK amikacin (0.5e8 mg/ml), TOB tobramycin (0.5e8 mg/ml), MINO minocycline (4 mg/ml), SXT trimethoprim-sulfamethoxazole (2 mg/ml), CPFX ciprofloxacin (0.03e2 mg/ml), LVFX levofloxacin (0.03e2 mg/ml).

Y. Ohno et al. / J Infect Chemother 23 (2017) 224e229

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Table 3 Clinical characteristics of patients infected with carbapenemase-producing Enterobacteriaceae. Species (no. of strains) and strain no. K. pneumoniae (8) THC3 THC6 THC7 THC9 THC11 THC13 THC18 THC26 E. coli (4) THC15 THC19 THC23 THC27 E. cloacae (2) THC10 THC17 K. oxytoca (1) THC5 E. aerogenes (1) THC8 P. mirabilis (1) THC25

Patient No.

PFGE

MLST

Plasmid replicon type

b-lactamase type

Source

Isolation date (mo/yr)

P-1 P-3 P-4 P-6 P-8 P-9 P-11 P-14

Kp-1 Kp-2 Kp-3 Kp-4 Kp-5 Kp-4 Kp-6 Kp-7

ST12 ST466 ST2387 ST2388 ST23 ST2388 ST37 ST1423

IncN IncN IncN IncN IncN, IncF IncN IncN IncN

IMP-6 IMP-6, IMP-6, IMP-6, IMP-6, IMP-6, IMP-6, IMP-6,

CTX-M-2 CTX-M-2 CTX-M-2 CTX-M-2 CTX-M-2 CTX-M-2 CTX-M-2

Urine Urine Sputum Catheter Urine Skin tissue Catheter Urine

12/10 9/11 1/12 1/13 4/13 5/13 9/13 12/13

P-10 P-12 P-13 P-14

Es-1 Es-2 Es-2 Es-2

ST4508 ST95 ST95 ST95

IncN IncN, IncF, IncFIB IncN, IncF, IncFIB IncN, IncF, IncFIB

IMP-6, IMP-6, IMP-6, IMP-6,

CTX-M-2 CTX-M-2 CTX-M-2 CTX-M-2

Urine Urine Urine Blood

6/13 10/13 11/13 12/13

P-7 P-11

En-1 En-2

ST78 ST721

IncN IncN

IMP-6, CTX-M-2 IMP-6, CTX-M-2

Urine Catheter

4/13 9/13

P-2

NT

NT

IncN

IMP-6, CTX-M-2

Urine

4/11

P-5

NT

NT

IncN

IMP-6, CTX-M-2

Urine

8/12

P-12

NT

NT

IncN

IMP-6, CTX-M-2

Blood

11/13

PFGE pulsed-field gel electrophoresis, MLST multilocus sequence typing, NT not tested.

Fig. 1. The time course of 5 patients with E. coli (n ¼ 3) and K. pneumoniae (n ¼ 2) of the same PFGE pattern. The figure shows the time period of hospitalization for each individual patients at ward B (white), ward G (light gray), ward H (gray) and ICU (black). The asterisk in figure showed the date of detecting CPE.

Table 4 Epidemiological characteristics of the patients with isolated CPE. Patient no. (Strain no.)

Age (years)

Sex

Underlying condition

Ward

Hospitalization (days)

Antibiotic historya

Antimicrobial therapy

Outcome

P-1 (THC3) P-2 (THC5) P-3 (THC6) P-4 (THC7) P-5 (THC8) P-6 (THC9) P-7 (THC10) P-8 (THC11) P-9 (THC13) P-10 (THC15) P-11 (THC17, 18) P-12 (THC19, 25) P-13 (THC23) P-14 (THC26, 27)

88 84 85 89 28 81 61 82 86 69 80 78 53 66

F F F M F M M F M M M F M F

Aspiration pneumonia Mitral valve regurgitation Diabetes mellitus COPD Pulmonary hypertension Acute pancreatitis Malignant lymphoma Aortic dissection Iliac aneurysm Aortic dissection Aortic dissection Abdominal aortic aneurysm Chronic heart failure Chronic heart failure

A B C D E B F B B B B B B B

134 170 343 83 8 68 35 150 159 47 138 141 >400 156

VCM LVFX None None None None LVFX, VCM None LVFX, VCM VCM LVFX, LVFX,

None LVFX LVFX CPFX None None SXT LVFX CPFX LVFX None LVFX LVFX LVFX, SXT

Cure Cure Cure Death Cure Death Cure Death Cure Death Death Cure Cure Cure

SXT

VCM

SXT SXT

CPE carbapenemase-producing Enterobacteriaceae, COPD chronic obstructive pulmonary disease, VCM vancomycin, LVFX levofloxacin, CPFX ciprofloxacin, SXT trimethoprimsulfamethoxazole. a History of antibiotics administration within the last two months.

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Institute of Infectious Diseases in 1999 to be 4.4% [18]. The MBL production rate of Enterobacteriaceae in a multicenter study of hospitals in the Kinki district of Japan in 2000e2002 was 0.42% [19]. A 2009 multicenter study done at Hiroshima University of ESBLproducing E. coli and K. pneumoniae collected from 17 medical facilities in Hiroshima Prefecture showed the rate of IMP-6-producing bacteria to be 0.23% (5/2113) [20]. We showed an isolation frequency greater than that of these two previous reports and revealed that CPE was spreading in a Japanese a primary care hospital. The carbapenemase genotype of all of the CPE strains detected in this study was IMP-6, and no other types were detected. The various carbapenemase genotypes distributed in Europe mainly comprise KPC and OXA-48 [21]. In the United States, KPC has been distributed more in Klebsiella spp. In multicenter surveillance conducted in the United States in 2007e2009, of the K. pneumoniae collected from 42 facilities, 5.5% were KPC-producing strains [22]. Furthermore, among Asian strains of carbapenem non-susceptible Enterobacteriaceae in China, 18% were KPC-producing strains and 10% were IMP-4-producing strains, indicating that KPC is the more widely distributed strain [23]. In the present study, KPC and OXA, which are often detected in other countries, were not detected. However, in recent years, KPC, NDM and OXA-48 have been reported sporadically even in Japan [24e26]. In the future, it will be necessary to pay attention to the trend of CPE carrying genotypes other than IMP in Japan. The antimicrobial susceptibility of CPE often shows multidrug resistance such as that to the fluoroquinolones and aminoglycosides [2]. The CPE in this study showed resistance to many b-lactam agents but showed good sensitivity to other antimicrobial agents such as LVFX, CPFX, AMK and SXT, and only a few of the CPE strains exhibited multidrug resistance. In addition, the carbapenem antibiotics MEPM and DRPM showed high MICs, whereas the MIC of IPM was low. This is a general feature of IMP-6 previously reported, and similar features were confirmed in this study. The present molecular epidemiological analysis showed the same PFGE cluster group in 3 strains of E. coli and 2 strains of K. pneumoniae. These strains also showed the same sequence type in MLST, which suggests horizontal transmission. However, according to the time course of these patients, the 2 patients in whom K. pneumoniae was detected were hospitalized at different times. Therefore, we surmised that these strains spread through other carrier patients, medical staff and/or the ward surroundings. Sequence types of globally prevalent CPE including ST258 and ST11 of K. pneumoniae clonal complex 258 and E. coli ST131 are listed as international high-risk clones [27]. However, clonal complex 258 was not detected in the present study. Nevertheless, the present worldwide spread of this international high-risk clone is a concern, and it will be necessary to monitor the trend of high-risk clones in the future in Japan. We also detected an IncN plasmid replicon in all of the CPE strains, and IncF and IncFIB were simultaneously detected in E. coli ST95. Plasmid replicon types comprise a narrow-host-range group, in which plasmids transfer easily within the same bacterial species, and a broad-host-range group, in which plasmids are easily transmitted in different bacterial species. The IncN detected from all of the CPE strains belongs to the broad-host-range group, whereas the IncF and IncFIB simultaneously detected from a portion of the E. coli belong to the narrow-host-range group. Because E. coli ST95 strains in which IncN, IncF and IncFIB were simultaneously detected indicate horizontally infected cases, it will be important to monitor future trends. CPE that were isolated from Hiroshima, Japan contained mostly IncN plasmids encoding IMP-6 and CTX-M-2 [28], and the present study showed a similar result. Therefore, we suppose that the same plasmid type is prevalent in other species as well as in the same species in Japan.

All of the CPE in the present study were detected in hospital inpatients with risk factors associated with a history of antimicrobial therapy, old age, and long-term hospitalization. In an investigation of the risk factors of carbapenemase-resistant Enterobacteriaceae performed from 2007 through 2009 in an Israeli tertiary care teaching hospital, risk factors included an increase in the number of hospitalization days and a history of vancomycin or fluoroquinolone antibiotic administration [29]. Similar risk factors were extracted in the present study. Therefore, we should perform point-prevalence culture surveillance for the patient with these risk factors and then should implement preventive measures. The present study has two limitations. First, the investigation was restricted to a single facility. Second, the initial screening of CPE in the present study was performed with the conditions of a MIC of 8 mg/L for any of the 3rd- or 4th-generation cephalosporins and a MIC of 2 mg/L for any of the carbapenems based on the CLSI M100-S19 document. Therefore, we might have overlooked latent CPE. In particular, a part of the carbapenemase genotype such as OXA-48 and IMP-6 shows a low MIC of carbapenems and 3rd- or 4th-generation cephalosporins. Besides, in the EUCAST guidelines, the MIC for screening cut-off for CPE is set as a MIC of 0.25 mg/L for MEPM (or ETPM) or a MIC of 2 mg/L for IPM. We might be able to detect latent CPE when using these guidelines. However, the EUCAST guidelines were established in 2013. Therefore, we have referred to the CLSI documents in the present study starting from 2010. It is also necessary to recognize the presence of the latent CPE. In conclusion, the identity of the clones in some strains of CPE was confirmed, whereas other clones revealed various sequence types, and PFGE suggested the dispersal of a wide variety of clonal types. We also clarified that the sequence types, which were not international high-risk clone types, increased in the last year of the study period. In the future, it will be important to continuously collect data from various regions in Japan. Conflict of interest None. Acknowledgements This study was supported by the Charitable Trust Laboratory Medicine Research Foundation of Japan. References €fmark S, Sjo € stro € m K, M€ [1] Lo akitalo B, Edquist P, Tegmark Wisell K, Giske CG. Carbapenemase-producing Enterobacteriaceae in Sweden 2007e2013: experiences from seven years of systematic surveillance and mandatory reporting. Drug Resist Updat 2015;20:29e38. [2] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 2012;15:133e48. [3] Mouloudi E, Protonotariou E, Zagorianou A, Iosifidis E, Karapanagiotou A, Giasnetsova T, et al. Bloodstream infections caused by metallo-b-lactamase Klebsiella pneumoniae carbapenemase-producing K. pneumoniae among intensive care unit patients in Greece: risk factors for infection and impact of type of resistance on outcomes. Infect Control Hosp Epidemiol 2010;31: 1250e6. [4] Queenan AM, Bush K. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev 2007;20:440e58. [5] Pillai DR, McGeer A, Low DE. New Delhi metallo-b-lactamase-1 in Enterobacteriaceae: emerging resistance. CMAJ 2011;183:59e64. [6] Koyano S, Saito R, Nagai R, Tatsuno K, Okugawa S, Okamura N, et al. Molecular characterization of carbapenemase-producing clinical isolates of Enterobacteriaceae in a teaching hospital. Jpn J Med Microbiol 2013;62:446e50. [7] Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K, et al. PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003;41:5407e13.

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