Current status of extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan

doi:10.1016/j.jiac.2016.01.008

Journal of Infection and Chemotherapy

Volume 22, Issue 5, May 2016, Pages 281-286

https://doi.org/10.1016/j.jiac.2016.01.008 Get rights and content

Abstract

Enterobacteriaceae producing extended spectrum β-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types bla_CTX-M-1, _-3, _-15, _-2, _-14, _-27, and mutants of bla_SHV-1 were detected. Among them, bla_CTX-M-15 (33.3%), bla_CTX-M-14 (27.8%) and bla_CTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a bla_SHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of bla_CTX-M, and bla_SHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing bla_CTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

Introduction

Enterobacteriaceae producing extended spectrum β-lactamase (ESBL) have a worldwide distribution. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the major bacterial species producing CTX-M-, TEM- and SHV-type ESBLs [1]. Until now, more than 500 genetic variations of bla_CTX-M, bla_TEM and bla_SHV have been reported [2]. Among the ESBL-encoding genes, bla_CTX-M is being detected at a dramatically increasing rate in clinical and community settings in recent years [3], [4]. The CTX-M-type ESBL is classified into 5 major variant groups namely CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. Some regional differences have also been observed in the prevalence of the CTX-M groups detected, for example, CTX-M-9 group was detected at higher rate in Southeast Asian countries [3], [4], [5], [6], [7], [8].

Okinawa prefecture (known as The Ryukyu Kingdom until the 19th century) is located in the southwestern part of Japan and falls in the subtropical and tropical climatic region. Geographically, there is the Ryukyu arc including the Ryukyu Islands start from the Kyushu region of Japan and extend up to Taiwan (Fig. 1). The Okinawa region possesses a history and unique culture different from that in other parts of Japan. According to the statistics by the Okinawa prefectural government, more than 7 million domestic and 0.9 million international tourists visited the Okinawa region in the budget year 2014 and there are the US Forces bases, which comprise of approximately 75% of their bases in Japan. As many studies have reported, the distribution of ESBL-producing Enterobacteriaceae and their genetic composition in a certain region are affected by external factors associated with inbound travelers to the region [9], [10]. Even though international travel is one of the major risk factors for international dissemination of ESBL-producing Enterobacteriaceae [11], [12], other factors contributing to their transfer, dissemination and distribution have not been fully analyzed.

Prevalence of the ESBL-producing Enterobacteriaceae in various settings has been studied in Japan [13], [14], [15], [16], [17]. However, the distribution of ESBL-producing Enterobacteriaceae in Okinawa prefecture is still unclear. In this study, we have characterized and evaluated the ESBL-producing Enterobacteriaceae present in clinical isolates collected from an Okinawa prefectural hospital between June 2013 and July 2014.

Section snippets

Clinical isolates

Between June 2013 and July 2014, 1672 clinical isolates of Enterobacteriaceae were collected from the Okinawa prefectural Nanbu Medical Center & Children's Medical Center which is one of the tertiary hospital with 434 beds and which locates in the southern part of Okinawa prefecture (Fig. 1). They were isolated from several clinical specimens including feces, sputum, urine, and vaginal discharge of in- and out-patients. The VITEK2 system (bioMérieux, Marcy l'Etoile, France) was primarily used

Prevalence and antibiotic susceptibilities of ESBL-producing Enterobacteriaceae species in Okinawa prefecture, Japan

In this study, 121 ESBL-producing Enterobacteriaceae isolates including 90 E. coli (5.4%), 19 K. pneumoniae (1.1%), and 5 P. mirabilis (0.3%) were obtained from a sample set of 1672 Enterobacteriaceae clinical isolates examined. Overall, the prevalence of ESBL-producing bacteria in this study was 6.8%. Prevalence rate of each species was 11.5% in E. coli (90 of 783 isolates), 6.2% in K. pneumoniae (19 of 307 isolates), and 11.1% in P. mirabilis (5 of 45 isolates). Among the 90 ESBL-producing

Discussion

Prevalence of ESBL-producing Enterobacteriaceae in Japanese medical facilities has been reported: 2.9% at a Japanese tertiary hospital between 2008 and 2011 [22], 5.89% in the Kinki region in 2009 [15], 15.2% at a hospital in southwestern Japan in 2009 [23], and 19.6% at nursing homes in the Kinki region in 2010 [24]. The prevalence of ESBL-producing E. coli B2-O25b-ST131 has been rapidly increasing worldwide [25], [26]. In the case of Japanese medical facilities, prevalence rates (43% and

Conflict of interest

None.

Acknowledgment

This work was partly supported by the Japan Agency for Medical Research and Development/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (AMED/JICA, SATREPS).

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A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-bla<inf>CTX-M</inf> transposition unit and application of the UGS to classification of bacterial isolates possessing bla<inf>CTX-M</inf>
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Citation Excerpt :
IS species, such as ISEcp1 and IS26, exist upstream of blaCTX-M, and play important roles in expression of blaCTX-M and transport of the IS-blaCTX-M transposition unit among plasmids and chromosome [5–7]. Therefore, bacterial isolates producing CTX-M type ESBL could possess multiple copies of blaCTX-M on antimicrobial resistance plasmids and/or chromosomes [8–12]. We have previously analyzed the transposition of ISEcp1-blaCTX-M by ISEcp1.
Because bla_CTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of bla_CTX-M could be a good indicator for classifying bacterial isolates harboring bla_CTX-M in molecular epidemiology. However, determination of bla_CTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of bla_CTX-M location.
Extracted DNA samples obtained from 168 Escherichia coli isolates possessing bla_CTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer.
Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures.
Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-bla_CTX-M transposition unit was useful for classification of bacterial isolates harboring bla_CTX-M.
Characterization of CTX-M-type-extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolated from Indonesian undergraduate medical students of a university in Surabaya, Indonesia
2020, Journal of Infection and Chemotherapy
Citation Excerpt :
In this study, the other bacterial species was not included. PCR analysis were performed with four primer sets (Table 1) to amplify blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, and blaCTX-M-25 groups as described previously [22]. blaSHV and blaTEM were also detected by PCR analysis using primer sets (Table 1).
Enterobacteriaceae isolates producing CTX-M-type extended-spectrum β-lactamase (ESBL) has been found in hospitalized patients and healthy individuals in communities of the Southeast Asian countries. Medical students might have more risk of ESBL-producing Enterobacteriaceae contagion, because medical students who belong to communities have direct and indirect contacts with workers and patients in healthcare facilities. The aim of this study was to collect information for evaluation of the potential risk of ESBL-producing Enterobacteriaceae contagion in Indonesian undergraduate medical students by characterizing genotypic properties of Escherichia coli isolates-producing CTX-M-type ESBL. A total 141 fecal samples collected from 207 medical students of a university in Surabaya, Indonesia were subjected to PCR, XbaI and S1 nuclease-pulsed-field gel electrophoresis (PFGE), Southern blotting, and sequencing analysis. Eighty-two ESBL-producing Enterobacteriaceae, including 75 E. coli and 7 Klebsiella pneumoniae were isolated from 79 (56.0%) students. Among 75 ESBL-producing E. coli, bla_CTX-M-15 was the most prevalent type (44.0%). Although XbaI-PFGE results showed genetic background of the E. coli isolates producing CTX-M-type ESBL were diverse, five clonal spread cases of certain E. coli producing CTX-M-type ESBL isolates were observed among the medical students. Our results suggested that ESBL-producing Enterobacteriaceae might be circulating among the medical students through contaminated environment such as in a university or communities they belonged.
Characterisation of chromosomally-located bla <inf>CTX-M</inf> and its surrounding sequence in CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli isolates
2019, Journal of Global Antimicrobial Resistance
Citation Excerpt :
In this study, nine new chromosomal regions with insertion of a blaCTX-M transposition unit that has been transposed into clinical E. coli isolates producing CTX-M-type ESBLs were determined. A total of 81 ESBL-producing E. coli isolates obtained from specimens (faeces, sputum, urine and vaginal discharge) at a hospital in Okinawa Prefecture between July 2013 and July 2014 were examined [15]. Confirmation of chromosomally-located blaCTX-M was performed following a previously described method [7,8].
Although it has been regarded that the CTX-M-type extended-spectrum β-lactamase (ESBL) gene bla_CTX-M is mainly carried by antimicrobial resistance plasmids, Escherichia coli possessing chromosomally-located bla_CTX-M has been reported in previous studies. This study aimed to characterise the genetic structure of the chromosomally-located bla_CTX-M transposition unit and its surrounding sequence in ESBL-producing E. coli isolated in a Japanese hospital.
A total of 81 ESBL-producing E. coli isolates were studied. The existence of chromosomally-located bla_CTX-M was confirmed by S1 nuclease-digested pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation and by sequencing analysis of the PCR-amplified DNA fragments.
Chromosomally-located bla_CTX-M was confirmed in 22 (27.2%) of the 81 E. coli isolates examined; five and four location types of chromosomally-located bla_CTX-M-14 and bla_CTX-M-15 were determined, respectively. Among the 22 E. coli isolates, 15 (68.2%) possessed single chromosomally-located bla_CTX-M gene, probably due to single transposition of a plasmidic bla_CTX-M to the chromosome. In isolate N0057, the bla_CTX-M-15 transposition unit was transferred from a plasmid into two different chromosomal regions. In addition, ‘recurrent’ transposition of already existing chromosomally-located bla_CTX-M-14 to another chromosomal region was observed in isolates N0211, N0214, N01127, N1682 and N1753; consequently, these isolates possessed two copies of chromosomally-located bla_CTX-M-14.
Considering that isolates N0211, N0214, N01127, N1682 and N1753 in which the ‘recurrent’ transposition event occurred were genetically related according to PFGE, these data suggest the possibility of accumulation of bla_CTX-M on the chromosome in CTX-M-type ESBL-producing E. coli.
Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture, Japan
2019, Journal of Infection and Chemotherapy
This study was performed to characterize CTX-M type extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 μg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of bla_CTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred bla_CTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.
Frequent isolation of extended-spectrum beta-lactamase-producing bacteria from fecal samples of individuals with severe motor and intellectual disabilities
2018, Journal of Infection and Chemotherapy
Citation Excerpt :
Although ESBL-producing bacteria asymptomatically colonize and are not pathogenic to healthy individuals, they occasionally induce fatal outcomes by sepsis due to their resistance to most antibiotics. As a result, surveillance of ESBL in general hospitals and long-term care facilities (LTCF) has increased [6–12]. Residents of LTCF share similar characteristics with SMID individuals, and the rate of ESBL colonization in residents of LTCF has been shown to be highly varied, ranging from close to zero up to 64.0% [12].
Extended-spectrum beta-lactamase (ESBL) producing bacteria spread worldwide and became major concern for antibiotic treatment. Although surveillance reports in general hospitals and long-term care facilities are increasing, their frequencies in individuals with severe motor and intellectual disabilities (SMID) are so far unknown. In this study, we examined the frequency of ESBL in stool samples collected from 146 asymptomatic SMID subjects hospitalized in a single institution. With their clinical information, we evaluated possible risk factors for ESBL colonization. From 146 fecal samples, ESBL-producing bacteria were isolated in 45 cases (31%). Drug sensitivity testing showed that 82% of the isolates were resistant to levofloxacin but were sensitive to tazobactam/piperacillin and cefmetazole. The most frequent genotype was CTX-M-9 detected in 36/45 (80%). A high degree of disability, antibiotic use within three months before sampling and post-tracheostomy were statistically significant risk factors. Tube feeding was also strongly correlated with ESBL colonization (p < 0.001) and associated with lower micro-organismic diversities. Our findings are the first to reveal a high prevalence of ESBL in the fecal samples of SMID individuals and suggest possible relationships between high degree disability, tube feeding and latest histories of antibiotic use.
Proposed breakpoint of piperacillin/tazobactam against extended spectrum β-lactamases producing bacteria in bacteremia
2017, Journal of Infection and Chemotherapy
The isolation rate of extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing in Japan. While the efficacy of piperacillin/tazobactam (PIPC/TAZ) for ESBL-producing bacteria is controversial, carbapenems have generally been shown to be effective. The aim of this study was to determine whether the current Clinical and Laboratory Standards Institute susceptibility breakpoint of ≤16/4 μg/mL PIPC/TAZ predicts the clinical usefulness for bacteremia caused by ESBL-producing Enterobacteriaceae. We retrospectively investigated 35 patients with bacteremia caused by Enterobacteriaceae producing ESBLs treated with PIPC/TAZ monotherapy. The microbiological and clinical efficacy with PIPC/TAZ minimum inhibitory concentrations (MICs) of ≤16/4 μg/mL was better than that with MICs ≥ 32/4 μg/mL. In contrast, MICs ≤8/4 μg/mL showed significantly higher microbiological and clinical efficacy compared to that of MICs ≥16/4 μg/mL (P < 0.05). These results suggest that 8/4 μg/mL PIPC/TAZ MIC is recommended as a breakpoint for bacteremia caused by ESBL-producing Enterobacteriaceae in Japan, although the current CLSI breakpoint is also useful.

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Original articleCurrent status of extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan

Abstract

Introduction

Section snippets

Clinical isolates

Prevalence and antibiotic susceptibilities of ESBL-producing Enterobacteriaceae species in Okinawa prefecture, Japan

Discussion

Conflict of interest

Acknowledgment

Int J Med Microbiol

J Infect Chemother

Diagn Microbiol Infect Dis

J Infect

Determining a clinical framework for use of cefepime and beta-lactam/beta-lactamase inhibitors in the treatment of infections caused by extended-spectrum-beta-lactamase-producing Enterobacteriaceae

J Antimicrob Chemother

Trends in human fecal carriage of extended-spectrum beta-lactamases in the community: toward the globalization of CTX-M

Clin Microbiol Rev

High prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand

J Antimicrob Chemother

Prevalence of and risk factors associated with faecal carriage of CTX-M beta-lactamase-producing Enterobacteriaceae in rural Thai communities

J Antimicrob Chemother

Analysis of risk factors for a high prevalence of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in asymptomatic individuals in rural Thailand

J Med Microbiol

Wide dissemination of extended-spectrum beta-lactamase-producing Escherichia coli in community residents in the Indochinese peninsula

Infect Drug Resist

Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors

J Antimicrob Chemother

Antimicrobials increase travelers' risk of colonization by extended-spectrum betalactamase-producing Enterobacteriaceae

Clin Infect Dis

Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers

Antimicrob Agents Chemother

Colonization of returning travelers with CTX-M-producing Escherichia coli

J Travel Med

Community spread of extended-spectrum beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis: a long-term study in Japan

J Med Microbiol

Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum beta-lactamases from clinical samples in the Kinki Region of Japan

Am J Clin Pathol

Epidemiology of extended-spectrum beta-lactamase producing Escherichia coli in the stools of returning Japanese travelers, and the risk factors for colonization

PLoS ONE

Original article
Current status of extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan