alexa Clinical and Molecular Epidemiology of Extended-Spectrum β-lactamase- Producing Klebsiella pneumoniae and Escherichia Coli in a Japanese Tertiary Hospital
ISSN: 2161-0703
Journal of Medical Microbiology & Diagnosis
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Clinical and Molecular Epidemiology of Extended-Spectrum β-lactamase- Producing Klebsiella pneumoniae and Escherichia Coli in a Japanese Tertiary Hospital

Yosuke Harada1,2, Yoshitomo Morinaga1*, Koichi Yamada1,2, Yohei Migiyama1,2, Kentaro Nagaoka1,2, Naoki Uno1, Shigeki Nakamura2, Yoshifumi Imamura2, Taiga Miyazaki2, Hiroo Hasegawa1, Koichi Izumikawa2, Hiroshi Kakeya2, Katsunori Yanagihara1 and Shigeru Kohno2,3
1Department of Laboratory Medicine, Japan
2Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
3Global COE Program, Nagasaki University, Nagasaki, Japan
Corresponding Author : Yoshitomo Morinaga, MD, PhD
Department of Laboratory Medicine
Nagasaki University Graduate School of Biomedical Sciences
1-7-1 Sakamoto, Nagasaki 852-8501, Japan
Tel: +81-95-819-7574
Fax: +81-95-819-7422
E-mail: [email protected]
Received June 28, 2013; Accepted September 03, 2013; Published September 06, 2013
Citation: Harada Y, Morinaga Y, Yamada K, Migiyama Y, Nagaoka K, et al. (2013) Clinical and Molecular Epidemiology of Extended-Spectrum ß-lactamase- Producing Klebsiella pneumoniae and Escherichia Coli in a Japanese Tertiary Hospital. J Med Microb Diagn 2:127. doi:10.4172/2161-0703.1000127
Copyright: © 2013 Harada Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Keywords
Extended-spectrum β-lactamase; Escherichia coli; Klebsiella pneumonia; TEM; SHV; CTX-M
Introduction
The emergence of extended-spectrum β-lactamase (ESBL)- producing bacteria, particularly Escherichia coli and Klebsiella pneumoniae, is now a critical concern for the development of therapies against bacterial infection. Since the early 1980s, the number of nosocomial infections by ESBL-producing, gram-negative bacteria has been increasing worldwide, and β-lactamase production has become a major causative factor for increasing resistance to antibiotics [1-3]. The ESBL genes are mostly plasmid encoded [4], and most ESBLs can be divided into 3 genotypes: TEM, SHV, and CTX-M [5]. The major ESBL producer was K. pneumoniae before 2000, and the predominant ESBL genotypes were TEM and SHV [1]. E. coli has now become an important ESBL carrier in Western countries. In addition, a genotype CTX-M has become more prevalent worldwide compared to the TEM and SHV genotypes [1]. During the 1990s, ESBL-producing organisms were described mainly as members of the TEM- and SHV-β-lactamase families in E. coli and K. pneumoniae causing nosocomial outbreaks [6]. In the 1990s, a novel type of ESBL, the CTX-M enzyme, emerged worldwide [6]. The CTX-M types, now exceeding 50 different types, can be divided into 5 groups based on their amino acid identities: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 [7]. It is clear that various CTX-M type ESBLs have spread worldwide and that specific CTX-M subgroups have been localized to different geographic areas [1,8,9]. Thus, in Japan, it is known that the CTX-M-8 and CTX-M-25 groups of enzymes are rarely found [10]. In Japan, since the first isolate of ESBL-producing bacterium in 1993 [11], the detection rates of ESBL-producers have been much lower than those in the rest of Asia; however, the increase in the incidence of ESBL producers remains a common issue [12,13]. Because the current trend of both bacteria in Japan is unknown and few studies with longitudinal observations of the nosocomial spread of these bacteria have been reported [14], we investigated epidemiologic data on the ESBL-producing E. coli and K. pneumoniae in our hospital and the ESBL genotypes. In addition, the antimicrobial susceptibilities of the ESBL producers were examined.
Materials and Methods
Study design
The present study was conducted at Nagasaki University Hospital located in southwestern Japan, which has 861 beds. Hospital microbiology laboratory databases from January 2006 to December 2010 were reviewed, and clinical isolates of E. coli and K. pneumoniae from specimens, except for feces, were analyzed for bacteriological and molecular epidemiology. The bacteria were identified using the Vitek-2 system (bioMerieux Japan Ltd., Tokyo, Japan) or the BD PhoenixTM Automated Microbiology System (BD Diagnostic Systems, Sparks, MD). For the isolates identified by Vitek-2, additional susceptibility testing was performed. When several strains with ESBL were detected from the same patient, only 1 sample was counted as an ESBL isolate. Information on the isolated strains, including etiology and susceptibility to antibiotics, was also obtained.
Antimicrobial susceptibility testing
Information on the susceptibility of the bacterial strains that had been performed according to the Clinical and Laboratory Standard Institute (CLSI) was obtained from the hospital microbiology laboratory databases [15]. The analyzed drugs were the following 11 agents: penicillin, ceftazidime, ceftriaxone, cefepime, imipenem, meropenem, aztreonam, gentamicin, minocycline, ciprofloxacin, and levofloxacin.
Detection of ESBL-producing E. coli and K. pneumonia
The isolates identified by BD Phoenix™ were analyzed with the integrated BDXpert™ System [16]. These processes were performed according to the original algorithm [17]. For the isolates identified by Vitek-2, the confirmatory testing for ESBL was performed by cephalosporin plus clavulanic acid (CA). Confirmation of ESBL isolates was based on 8-fold reduction with CA when combined with cefpodoxime, cefotaxime and ceftazidime. The proportional reduction in growth in the wells containing cephalosporin plus CA compared with those containing cephalosporin alone was considered indicative of ESBL production.
Extraction of plasmids
A few colonies were suspended in 700 μL of Tris-EDTA buffer (pH 8.0). The suspensions were boiled for 10 min and subsequently centrifuged for 5 min at 13000 rpm. The supernatant, containing DNA, was transferred to new tubes and stored at 4°C for subsequent PCR analysis.
Genotyping of ESBL
PCR was performed using 5 sets of previous published primers to amplify type-specific ESBL genes, including CTX-M-1, CTX-M-2, CTX-M-9, TEM and SHV [18]. For detecting TEM or SHV genes, initial denaturation at 95°C for 10 min, denaturation at 95°C for 1 min, primer annealing at 56°C for 1 min, and extension at 72°C for 1 min, was repeated for 40 cycles; with a final extension at 72°C for 7 min. For CTX-M gene, initial denaturation at 95°C for 10 min, denaturation at 95°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°C for 1 min, was repeated for 40 cycles; a final extension at 72°C for 7 min was carried out. The PCR products were analyzed using 2% agarose gel electrophoresis and visualized by staining with ethidium bromide.
Results
Identification of ESBL-producing E. coli strains
ESBL-producing E. coli accounted for 160 of 1359 isolates (11.8%) and ESBL-producing K. pneumoniae accounted for 20 of 725 isolates (2.8%). The number of ESBL-producing E. coli markedly increased from 17 isolates (6.8%) in 2006 to 68 isolates (22.7%) in 2010 (Figure 1). ESBL-producing K. pneumoniaewas not detected in 2006, but appeared after 2007 and peaked in 2009 with 9 isolates (6.4%). The majority of ESBL-producing E. coli were isolated from urine (59%), followed by respiratory specimens (14%), pus (12%), blood (9%) and others (6%) (Figure 2).
Molecular characterization of ESBL-producing strains
The phenotypically identified ESBL-producing E. coli and K. pneumoniae were molecularly analyzed. Of the 160 ESBL-producing E. coli isolates, 143 (89.4%) were positive for ESBL genes (Table 1). Fifty percent of the isolates had at least 2 ESBL genes. The number of ESBLproducing E. coli with TEM, SHV, CTX-M-1, CTX-M-2 and CTX-M-9 was 13 (8.1%), 5 (3.1%), 7 (4.4%), 3 (1.9%) and 35 (21.9%), respectively (Table 1). The number of ESBL-producing E. coli with TEM+SHV, TEM+CTX-M-1, TEM+CTX-M-2, TEM+CTX-M-9, TEM+CTX-M- 1+CTX-M-9, TEM+CTX-M-1+CTX-M-2 and SHV+CTX-M-9 was 2 (1.3%), 10 (6.3%), 3 (1.9%), 61 (38.1%), 2 (1.3%), 1 (0.6%) and 1 (0.6%), respectively. In addition, the detection number of ESBL-producing E. coli of the CTX-M-1 group increased from 1 to 11 from 2006 to 2010. For K. pneumoniae, 17 (85.0%) of the 20 isolates were positive for ESBL genes (Table 2). The number of ESBL-producing K. pneumoniae with TEM, SHV, TEM+SHV, SHV+CTX-M-1, SHV+CTX-M-2, SHV+CTX-M-9 and TEM+SHV+CTX-M-1 was 1 (5%), 5 (25%), 6 (30%), 1 (5%), 1 (5%), 2 (10%), and 1 (5%), respectively. ESBLproducing K. pneumoniae isolates carrying CTX-M alone were not detected.
Antimicrobial susceptibilities of ESBL-producing E. coli
We analyzed the antimicrobial susceptibilities of the ESBLproducing E. coli between genotypes (Table 3). The isolates were categorized according to the following genotypes: TEM/SHV (TEM and/or SHV), CTX-M (CTX-M-1, CTX-M-2, and CTX-M-9), and TEM/SHV+CTX-M (TEM/SHV and CTX-M), as previously reported [8]. The value of MIC50 of TEM/SHV in ceftriaxone and cefepime were 2 μg/mL and 1 μg/mL, respectively. The values of MIC50 of CTX-M or TEM/SHV+CTX-M in ceftriaxone and cefepime were ≥ 32 μg/ mL and 8 μg/mL respectively. All of the TEM/SHV- and CTX-Mproducing isolates retained favorable susceptibility to carbapenems. In all genotypes, the value of MIC50 in fluoroquinolones was 8 μg/mL.
Discussion
In this study, we examined the current trend of ESBL-producing E. coli and K. pneumoniae in our hospital, as well as the ESBL genotypes and the antimicrobial susceptibilities. Our data, based on the clinical isolates collected over 5 years, suggested that the incidence of ESBLproducing E. coli and K. pneumoniae have increased. The prevalence of ESBL-producing bacteria has been on the rise, particularly in Asia compared to other regions. A study conducted in 2007 reported that the frequencies of ESBL-producing K. pneumoniae and E. coli isolates exceeded 30% in both bacterial populations [11]. The proportion of ESBL-producing isolates in Japan was 4.3% in E. coli and 3.1% in K. pneumoniae in 2006 [13]. Thus, the geographical distribution can vary according to countries and institutions, although the prevalence of ESBL-producing bacteria is a global problem [19]. The prevalence of ESBL-producers in our study was lower than that reported by global surveillances [20] and in other Asian countries [21,22] but it was found to increase, implying that ESBL producers could become common drug-resistant bacteria in the near future. In addition, our study suggested that urine can be an important source of ESBL-producing E. coli as previously reported [23]. Worldwide, the predominant genotype of ESBL-producing E. coli has changed from TEM and/or SHV (TEM/ SHV) to CTX-M [1,24], and the detection rate of CTX-M has increased dramatically [25]. In this study, CTX-M was found most frequently in ESBL-producing E. coli isolates, in particular, the CTX-M-9 group whose prevalence increased during the observation period. Our results were similar to previously reported Japanese trends on the increase of CTX-M type ESBL [10,26], but different in that the increase of prevalence of CTX-M-9 was in combination with an increase in the prevalence of the TEM genotype. An ESBL-producing E. coli clone O25:H4-ST131 that often carries CTX-M-1-type ESBL has been spreading worldwide. Because of its resistance to fluoroquinolones and aminoglycosides, in addition to β-lactams, the increase of E. coli clone O25:H4-ST131 has become a serious problem [23]. We did not investigate any specific clones including E. coli clone O25:H4-ST131 in this study; however, the mild increase of CTX-M-1-type ESBL may imply the spreading of O25:H4-ST131. In fact, this clone has previously been found in Japan [26]; therefore, further examination will be required. In the present study, about one-half of the ESBL-producing E. coli isolates were molecularly confirmed to have 2 or more ESBL genes. The incidence of TEM+CTX-M-9 has increased remarkably in these last 2 years. These findings suggest that E. coli carrying multiple ESBL genes may be increasing. In contrast, those isolates from which these specific genes were not detected likely produce other types of enzymes that have yet to be investigated. Alternatively, some identification errors may be involved in our results because a high false-positivity rate of Phoenix identification system had been reported [27]. The most common ESBL genotype among the K. pneumoniae isolates was TEM+SHV, as opposed to a previous report from Japan [8] which showed that SHV+CTX-M was the most common. Because of the limited number of isolates in our hospital, it is difficult to place TEM+SHV as the predominant genotype of ESBL-producing K. pneumoniae. The potential for hydrolysis of β-lactams can vary according to the type of ESBL enzymes. CTX-M β-lactamase can elevate the MIC values of ceftriaxone, cefepime, and ceftazidime [28,29]. In the present study, the MIC50 of ceftriaxone and cefepime in E. coli with CTX-M or TEM/SHV+CTX-M was higher than in those E. coli with TEM/SHV. Among the E. coli with CTX-M, the MIC90 and MIC50 of ceftazidime in E. coli with TEM+CTX-M-9, the predominant ESBL in this study, were lower than other E. coli with TEM/ SHV+CTX-M (data not shown). CTX-M ESBLs can be also resistant to fluoroquinolones [30], but there were no significant differences in the MICs of fluoroquinolones between ESBL genotypes. Thus, ESBLs showed different hydrolysis potentials and some plasmids can possess other drug-resistant genes in addition to a β-lactamase gene. Therefore, ESBL genotyping can help in monitoring the important ESBLs that can lead to treatment failure and contribute to the appropriate use of antimicrobial agents and the infection control. In conclusion, ESBLproducing E. coli and K. pneumoniae were less frequent compared to the global trend but were found to be increasing in a tertiary hospital. Two or more ESBLs were detected in many isolates by molecular analysis, and MIC of some cephalosporin’s was elevated in the isolates with CTX-M-type ESBL. Considering regional variations and some easy-expanding clones, constant and careful surveillance is needed.
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