蓮沼 裕也

蓮沼, 裕也

ハスヌマ, ユウヤ

Toin University of Yokohama Academic Repository

71「桐蔭論叢」第 34 号 2016 年 6 月Characteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students Hasunuma Yuya 桐蔭横浜大学医用工学部生命医工学科（2016 年 3 月 28 日　受理）1. Introduction Several antimicrobial resistant bacteria are spreading throughout the world. It is difficult to treat a patient infected with an immunity to antibiotics and to develope a novel antimicrobi-al agent. Antimicrobial resistant bacteria have typically been isolated from hospitals (inpa-tients, environments and medical staff). In 1969, moreover, “the Swann Report” drew attention to the potential for antibiotic-resistant bacte-ria to spread from treated animals to humans through consumption. Nevertheless, there was little response to this finding until the detection of vancomycin-resistant enterococci (VRE), a related glycopeptide avoparcin, in animals fed the enhanced feed 1). Recently, antimicrobial resistant bacteria such as extended-spectrum β-lactamase (ESBL) producing Enterobacte-riaceae and community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) have been pervaded in healthy humans 2). It has been increasingly considered that ESBL producing strains are spreading to healthy hu-mans through the consumption of livestock, processed with the use of a broiler like in the case of VRE 3).Although Enterobacteriaceae normally exists in the intestinal tracts of humans, it is also frequently caused not only various intes-tinal diseases but also extraintestinal diseases such as urinary tract infection, bacteremia, and pneumonia. Broad-spectrum oxyminocepha-losporins, such as cefotaxime, ceftazidime and ceftriaxone, have potent activity against En-terobacteriaceae, which is one of clinical iso-lates possessing resistance to narrow-spectrum β-lactam agents. On the other hand, ESBL is produced by Enterobacteriaceae effectively hy-drolyses the third generation cephalosporins but not cephamycins (cefmetazole, cefoxitin) or carbapenems (imipenem, meropenem) 4). Most ESBLs can be classified into three main groups: TEM, SHV, and CTX-M families. The number of natural variants found within these families has increased; 170, 130, and 100 types respectively had been identified by 2010, respec-tively 5). The CTX-M types of ESBL that can efficiently hydrolyze cefotaxime and ceftriaxone are spreading in Japan 6). Moreover, interna-tional attention is brought to the fact that a 〈医用工学部研究論文〉Laboratory of Clinical Microbiology, Faculty of Biomedical Engineering, Toin University of Yokohama, 1614 Kurogane-cho, Aoba-ku,Yokohama, Japan  225-8503 Hasunuma Yuya72strain of CTX-M-15 type ESBL producing bacteria could hydrolyze ceftazidime and is not susceptible to other drugs such as fluoroquinolone 7) 8). Other β-lactam agent resistant mechanisms are asso-ciated with  by upregulation of the chromosom-al AmpC gene, plasmid mediated AmpC, efflux pumps, decreased in the permeability of the outer membrane and a mutation of penicillin binding protein (PBP). The aim of this research is to investigate the characteristics and molec-ular epidemiology of cefotaxime resistant En-terobacteriaceae isolated from healthy college students.2. Materials and Methods2.1 Bacterial IsolatesOne hundred and fory-five stool swab sam-ples were collected from healthy college and nursing school students. Stool samples were cultured for 18–24 hours in BHI culture media (BD, Japan) with 2 mg/mL cefotaxime (Sigma, Japan). Growth samples were isolated Mac-Conkey ager (Eiken Chemical, Japan) including 2 mg/mL cefotaxime and incubated at 37 ℃ for 18–24 hours. Colonies were chosen and sub-cul-tured by the BTB lactose ager (Kokuto-Seiyaku, Japan) and identified by Api20E (biomerieux). 2.2 Antimicrobial Susceptibility TestsAntimicrobial susceptibility was evaluated by ager plate dilution methods with ampicillin (Wako, Japan), cefazolin (Sigma, Japan), cef-metazole (Sigma, Japan), ceftazidime (Sigma, Japan), cefotaxime (Sigma, Japan), gentamycin (Wako, Japan), amikacin (Wako, Japan), and ciprofloxacin (Wako, Japan). Susceptibilities to amoxicillin/clavulanate, ceftriaxone, imipen-em, and meropenem were evaluated using Etest (biomerieux). Breakpoints were determined ac-cording to the recommendations of the CLSI 9). Intermediate susceptibility to each antibiotic was defined as resistance. An ESBL confirming test was performed by double disk synergy test (DDST) used with cefotaxime (BD, Japan), ce-fotaxime/clavulanate (Eiken Chemical, Japan), ceftazidime (BD, Japan), and ceftazidime/clavu-lanate (Eiken Chemical, Japan).2.3 PCR & DNA Direct Sequence Analysis2.3.1 β-Lactamase IdentificationESBL and plasmid-mediated AmpC β-lactamase (pAmpC) genes were detected by PCR amplification of blaCTX-M, blaTEM, and blaSHV genes and six main groups (ACCM, CITM, DHAM, EBCM, FOXM, MOXM) of pAmpC- type genes, as described previously 10) 11) 12). The PCR was performed with Takara Ex taq (Taka-ra Bio, Japan). All PCR amplicons were verified by gel electrophoresis on a 1.8 % SeaKem LE agarose (Takara Bio, Japan).2.3.2 Phylogenetic Groups of Escherichia coli E. coli isolates were analyzed to determine their phylogenetic groups (A, B1, B2, and D) using the triplex PCR assay of chuA, yjaA, and tspE4.C2, as described previously 13). 2.3.3 Fluoroquinolone Resistant MechanismsThe quinolone resistance determining re-gions (QRDRs) of gyrA and parC genes were sequenced, and the correlating amino acids were compared with the corresponding regions of E. coli strain (GeneBank No. AF052244, AB083821). PCR was performed in a final vol-ume of 25 µL. Each reaction contained 2.0 mM MgCl, 0.2 mM dNTPs, 0.4 µM of each primer, and 1.0 U of Ex taq. The primers for PCR were used as described previously 14) 15). The PCR program consisted of an initial denaturation step at 95 ℃ for 5 minutes, followed by 30 cycles of DNA denaturation at 95 ℃ for 15 seconds, annealing at 56 ℃ for 20 seconds, and extension at 72 ℃ for 1 minute. After the last cycle, a final extension step at 72 ℃ for 5 minutes was add-ed. PCR products were purified by DNA RNA Characteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students73Table 1. All strains isolated and susceptibility testsHasunuma Yuya74and Protein Purification Kit  (Macherey- Na-gel, Germany). After PCR purification, all of them were subjected to direct sequencing by the dye-terminator method by using the BigDye Terminator v3.1 Cycle Sequencing Kit (Ther-moFisher Science, USA) with the ABI Prism 3100 Genetic Analyzer (ThermoFisher Science, USA). 3. Results3.1 Bacterial Isolates and Antimicrobial Sus-ceptibility ProfileCefotaxime resistant Enterobacteriaceae was isolated from 22 samples (15.2 %) and gathered 25 strains of the 16 isolates of Escherichia coli, 3 isolates of Enterobacter cloacae, 2 isolates of Citrobacter braakii, 2 isolates of Klebsiella oxy-toca, 1 isolate of Enterobacter aerogenes, and 1 isolate of Hafnia alvei. These antibiotic suscep-tibility are listed in Table 1. The resistant rate and MIC parameter is shown in Table 2. Amox-icillin/clavulanate, ceftriaxone and ciprofloxacin were less active with resistance rates of 36, 100, and 44 %. Ceftazidime, cefmetazole, and gen-tamycin had moderate activity, and their resis-tance rates were 32, 28, and 12 %, respectively. All the strains were susceptible to imipenem, meropenem, and amikacin. As shawn in Table 3, moreover 18 strains (16 E. coli and 2 K. oxy-toca) were positive of ESBL screening DDST. 3.2 Molecular Characteristic of Antimicrobial Resistance EnterobacteriaceaeAll of the ESBL-producing E. coli and K. oytoca were examined by PCR for the determi-nation of blaCTX-M genes. Five E. coli and one K. oxytoca carried blaTEM combined with blaCTX-M. One isolate for which the cefotaxime MIC val-ue was 2 mg/mL harbored blaSHV. Additionally, ACCM was positive in H. alvei. None of the other types of AmpC were detected (Table 3). Among 18 CTX-M-positive isolates, 4 isolates carried CTX-M-1 group ESBL genes, and 13 isolates carried CTX-M-2 group ESBL genes. Isolated E. coli of phylogenetic subgroup D was predominant, followed by subgroup B2, A, and B1  (Table 4). The qnrA and B of  fluoroquino-lone binding protein was not detected in any strain (data not shown). Table 2. Resistant rate and MIC parameterCharacteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students754. DiscussionIn this study, prevalent rates of the third generation cephalosporins resistance Entero-bacteriaceae in healthy college students were in-vestigated. The prevalent rate was 15.9 %, much higher than that in the previous study reported by Luvsansharav UO in Japan 16). One of the reasons may be that the fecal samples were col-lected from the students studying medical and nursing techniques in a college. Among the 25 isolated strains, 16 strains (64.0 %) were E. coli producing ESBLs. ESBL is produced by not only E. coli by but also K. oxytoca, which was susceptible to cefmetazole, ceftazidime, and carbapenems. Additionally, SH Choi reported that cefepime was susceptible to ESBL produc-ing strain and could still treat their infections 17). Carbapenem is the most commonly used anti-microbial agent, but it contributes to the initial selection of multi-drug resistant organisms.Ceftazidime resistant isolates were only En-terobacter sp., Citrobacter sp., and Hafnia sp., which were characterized by chromosomally, encoded AmpC β-lactamases. In these species, upregulation of chromosomal AmpC develop resistance to ceftazidime, cefmetazole, and oth-er third and forth generation cepharosporins. Pattarachai K. found that 17 % of non-Esch-erichia and non-Klebsiella Enterobacteriaceae had harboring pAmpC genes and that almost all isolates had a coexistence of ESBL genes in tertiary care university hospital patients in Thailand 18). This study suggested that pAmpC harboring Enterobacteriaceae did not emerge in healthy adults yet. On the other hand, Amber class C (ACC) β- lactamase which H. alvei has on its chromosome was detected by AmpC mul-tiplex PCR. David N reported that the trans-fer of the plasmid by ACC β-lactamase gene of H. alvei and the receipt of plasmids by K. pneumoniae occurred during the outbreak in their hospital 19). Therefore, the colonization Table 3. Characteristic of β-lactamaseTable 4. Relationship between CTX-M types and phylogenetic group of E. coliHasunuma Yuya76of AmpC overproducing strains is a risk factor for spreading plasmid to other species when the host initial flora is affected by antimicrobial se-lection pressure. The most prevalent ESBL type is the CTX-M-2 group at cefotaxime resistant strains in healthy adults. The CTX-M types are categorized into four groups; CTX-M-1, CTX-M-2, CTX-M-8, and CTX-M-9 groups. The CTX-M-2 group ESBLs are dominant in hospital patients in Japan 20). In a current study, CTX-M-15 type, which belongs to CTX-M-1 group of ESBL producing E. coli B2-O25:H4-ST131, developed a resistance to another cef-tazidime and was found to be emerging in Euro-pean countries 8). Furthermore, a current study suggested that the CTX-M-15 harboring E. coli B2-O25:H4-ST131 strain emerge in Japan 7) and that the strain could have multiple reservoirs from which they spread to different blaCTX-M types 21). Nevertheless, this potential strain was not isolated in this study because of the suscep-tibility of CTX-M type producing strains to ceftazidime. These strains are resistance to flu-oroquinolones, aminoglycosides, and other an-tibiotics. JANIS, a medical surveillance system in Japan, reported a drastic increase of fluoro-quinolone resistance E. coli as the result of iso-lation at medical facilities. However, this study suggests that fluoroquinolone resistant Entero-baceriaceae is found in ESBL producing strains in healthy adults, and this finding may be due to previous abuse of antibiotics. The high rates of resistance to fluoroquinolone is probably caused by chromosome-mediated resistance such as point mutations in the gyrase and to-poisomerase IV genes (Table 5). Quinolones are frequently used to treat infections among out-patients in many countries including Japan. But there is concern that these drugs will not be ef-fective and will apply selection pressure to these isolates. Continuous surveillance of antibiotic resistance is essential to detect the emergence and spread of broad-spectrum β-lactamase producing strains in Enterobacteriaceae.AcknowledgementThanks to Noriko Iida for her technical Table 5. Fluoroquinorone resistant stainsCharacteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students77support of direct sequencing analysis and to our laboratory students for their assistance. Reference1) Mary D. B. 2000. Antibiotic use in animal feed and its impact on human health. Nutr. Res. Rev. 13. 279–299. 2) Paul-Louis W., Charles B. et al. 2013. Trends in Human Fecal Carriage of Extended-Spectrum β-Lactamases in the Community: Toward the Globalization of CTX-M. Clin. Microbiol. 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Characteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students

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Characteristics and Molecular Epidemiology of Cefotaxime Resistant Enterobacteriaceae Isolated from Healthy College Students

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