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In vitro activity of flomoxef and comparators against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis producing extended-spectrum β-lactamases in China
International Journal of Antimicrobial Agents 45 (2015) 485–490
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
In vitro activity of flomoxef and comparators against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis producing extended-spectrum -lactamases in China Qiwen Yang a , Hui Zhang a , Jingwei Cheng a , Zhipeng Xu a , Yingchun Xu a,∗ , Bin Cao b , Haishen Kong c , Yuxing Ni d , Yunsong Yu e , Ziyong Sun f , Bijie Hu g , Wenxiang Huang h , Yong Wang i , Anhua Wu j , Xianju Feng k , Kang Liao l , Dingxia Shen m , Zhidong Hu n , Yunzhuo Chu o , Juan Lu p , Jianrong Su q , Bingdong Gui r , Qiong Duan s , Shufang Zhang t , Haifeng Shao u a Department of Clinical Laboratory, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, 1 Shuaifuyuan, Wangfujing Street, Beijing 100730, China b Chaoyang Hospital of Capital Medical College, Beijing 100020, China c The First Affiliated Hospital of Zhejiang University, Hangzhou 310003, China d Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China e Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China f Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China g Zhongshan Hospital of Fu Dan University, Shanghai 200032, China h The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China i Shandong Provincial Hospital, Jinan 250021, China j Xiangya Hospital, Central Southern University, Changsha 410008, China k The First Affiliated Hospital of Zhengzhou University, Zhenzhou 450052, China l The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China m General Hospital of PLA, Beijing 100853, China n General Hospital of Tianjin Medical University, Tianjing 300052, China o The First Affiliated Hospital of Chinese Medical University, Shenyang 110001, China p The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China q Friendship Hospital of Capital Medical College, Beijing 100020, China r The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China s People’s Hospital of Jilin Province, Jilin 130021, China t People’s Hospital of Haikou City, Haikou 570208, China u General Hospital of Nanjing Military Command, Nanjing 210002, China
a r t i c l e
i n f o
Article history: Received 5 November 2014 Accepted 25 November 2014 Keywords: Flomoxef Extended-spectrum -lactamases Escherichia coli Klebsiella pneumoniae Proteus mirabilis
a b s t r a c t The objective of this study was to better understand the in vitro activity of flomoxef against clinical extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae. A total of 401 ESBL-producing isolates, including 196 Escherichia coli, 124 Klebsiella pneumoniae and 81 Proteus mirabilis, were collected consecutively from 21 hospitals in China in 2013. Minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. Phenotypic identification of ESBL production was detected as recommended by the Clinical and Laboratory Standards Institute (CLSI). ESBL genes were detected by PCR and sequencing. Flomoxef, doripenem, meropenem, ertapenem, cefmetazole and piperacillin/tazobactam exhibited good activity against ESBL-producing isolates, with susceptibility rates >90%. Tigecycline showed good activity against E. coli and K. pneumoniae (100% and 97.6%, respectively). Cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0–0.8% and 1.0–13.6%, respectively. blaCTX-M were the major ESBL genes, with occurrence in 99.5% of E. coli, 91.1% of K. pneumoniae and 97.5% of P. mirabilis. blaCTX-M-14 was the predominant ESBL gene, detected in 46.9% (188/401) of the isolates, followed by blaCTX-M-15 (21.4%), blaCTX-M-55 (17.2%), blaCTX-M-65 (12.7%) and blaCTX-M-3 (6.7%). Flomoxef exhibited excellent activity against the different CTX-M-type ESBL-producing
∗ Corresponding author. Tel.: +86 10 6915 9766/1391 130 3028; fax: +86 10 6915 9766. E-mail address: [email protected] (Y. Xu). http://dx.doi.org/10.1016/j.ijantimicag.2014.11.012 0924-8579/© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
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isolates, with MIC50 and MIC90 values of 0.064–0.125 g/mL and 0.25–0.5 g/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. In conclusion, flomoxef showed good activity against ESBLproducing Enterobacteriaceae and may be a choice to treat infections caused by these isolates in China. © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction A number of surveillance programmes exist to monitor the susceptibility of clinically important pathogens at national and international levels [1–3]. The Enterobacteriaceae are a major group of pathogens causing community- and hospital-acquired infections. With the increase in the prevalence of extendedspectrum -lactamases (ESBLs) in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, clinicians have a limited choice of antimicrobial agents to use, which in turn leads to an increase in the abuse of certain antimicrobial agents such as the carbapenems. ESBL rates in E. coli and K. pneumoniae in China have been reported as 60–70% and 30–40%, respectively [2,3], which has resulted in a challenge to Chinese clinicians in terms of selecting appropriate antimicrobial agents to treat infections caused by ESBL-producing isolates. Flomoxef is a unique -lactam antibiotic with oxygen substituted for the sulphur, a 7-␣-methoxy group in the cephalosporin core and a difluoromethylthio-acetamido group at position 7, which gives it better in vitro activity against ESBLproducing Enterobacteriaceae [4]. However, the activity of this drug in China has not been reported. In view of this, in this study consecutive community- and hospital-associated ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates were collected and antimicrobial susceptibility testing was conducted on these strains to evaluate the in vitro activity of flomoxef and other comparators. Although there have been some reports describing the activity of flomoxef [1–6], this is the first systematic report to provide an overview specifically of the results in China. 2. Materials and methods 2.1. Clinical isolates A total of 401 ESBL-producing isolates, including 196 E. coli, 124 K. pneumoniae and 81 P. mirabilis, were collected consecutively from 21 hospitals in China in 2013. Specimens included 147 urine, 101 abdominal fluid, 37 pus, 32 blood, 31 sputum, 25 bile, 17 liver tissue, 6 pancreas, 2 appendix and 3 other specimens. Bacteria were identified by standard methods used in each site and were re-identified in Peking Union Medical College Hospital (Beijing, China) by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). All organisms were deemed clinically significant according to local criteria. For the purpose of comparison of the activity of different antimicrobial agents against ESBL-producers, 244 non-ESBL-producing isolates were collected from the same hospitals during the same period, including 63 E. coli, 103 K. pneumoniae and 78 P. mirabilis isolates from 67 blood, 61 urine, 46 sputum, 29 gallbladder, 13 abdominal fluids, 9 liver tissue, 8 pus, 4 bile and 7 other specimens. 2.2. Antimicrobial susceptibility test method Minimum inhibitory concentrations (MICs) were determined using a broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [7].Fifteen
antimicrobial agents were tested, including flomoxef, ceftazidime, ceftazidime/clavulanic acid (fixed clavulanic acid concentration of 4 g/mL), cefotaxime, cefotaxime/clavulanic acid (fixed clavulanic acid concentration of 4 g/mL), cefepime, cefmetazole, piperacillin/tazobactam (TZP) (fixed tazobactam concentration of 4 g/mL), cefoperazone/sulbactam (fixed ratio of 2:1), ertapenem, meropenem, doripenem, levofloxacin, tigecycline and amikacin. MIC50 and MIC90 values (MICs at which 50% and 90% of isolates were inhibited, respectively) were calculated using WHONET software v.5.6 (http://whonet.software.informer.com/5.6/). Susceptibility interpretations were based on CLSI clinical breakpoints [8]. The CLSI breakpoints for latamoxef [susceptible (S) ≤8 g/mL, resistant (R) ≥64 g/mL for Enterobacteriaceae] were used for flomoxef. US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam. The reference strains E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and K. pneumoniae ATCC 700603 (positive ESBL control) were used as quality control strains for each batch of MIC testing. Results were included in the analysis only when corresponding quality control isolates tested within the acceptable range according to CLSI guidelines.
2.3. Extended-spectrum ˇ-lactamase detection Phenotypic identification of ESBL production among E. coli, K. pneumoniae and P. mirabilis was detected according to the methods recommended by the CLSI [8]. If the cefotaxime or ceftazidime MIC was ≥2 g/mL, then the MIC of cefotaxime or ceftazidime was compared with the MIC of cefotaxime/clavulanic acid or ceftazidime/clavulanic acid. A positive test for ESBL production was defined as a ≥8-fold (i.e. three-fold doubling dilution) decrease in the MIC for cefotaxime or ceftazidime when tested in combination with clavulanic acid versus their MIC when either drug was tested alone.
2.4. PCR amplification and DNA sequence analysis of extended-spectrum ˇ-lactamase genes Template DNA of the ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates was obtained by placing several small colonies of each strain in 200 L of double-distilled water and boiling the sample for 10 min. After cooling on ice, 1–2 L of each lysate was used in the PCR. The primers used in this study have been described previously [9]. The reaction was conducted in a PTC-200 PCR system (MJ Research Inc., Watertown, MA). The amplified genes included blaTEM , blaSHV , blaCTX-M-1 group , blaCTX-M-2 group , blaCTX-M-8 group , blaCTX-M-9 group and blaCTX-M-25 group . A positive and negative control was followed in each batch of PCR. PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and were sequenced on an ABI PRISM 3730XL Sequencer Analyzer (Applied Biosystems, Foster City, CA). DNA sequencing data were analysed with the GenBank BLASTN tool (http://www.ncbi.nlm.nih.gov/blast).
0.5 1 0.25 0.032 4 4 64 2 8 1 32 256 256
487
R, resistant; S, susceptible; I, intermediate; MIC50/90 , minimum inhibitory concentration at which 50% and 90% of isolates were inhibited, respectively; CLSI, Clinical and Laboratory Standards Institute. a No breakpoints for flomoxef have been published by the CLSI. The CLSI breakpoints for latamoxef (S ≤8 g/mL, R ≥64 g/mL for Enterobacteriaceae) were used for flomoxef. b US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). c The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam.
0.125 0.5 0.064 0.016 2 1 4 1 4 0.25 8 128 256 98.8 98.8 100 100 80.2 98.8 86.4 98.8 96.3 92.6 28.4 13.6 0 0 1.2 0 0 17.3 1.2 2.5 1.2 1.2 0 16 9.9 0 1.2 0 0 0 2.5 0 11.1 0 2.5 7.4 55.6 76.5 100 0.25 0.125 0.125 0.25 2 4 4 16 64 128 64 256 256 0.064 0.064 0.032 0.064 0.5 2 2 4 16 8 1 256 256 98.4 99.2 99.2 95.2 97.6 98.4 95.2 91.9 57.3 42.7 58.9 6.5 0.8 0.8 0 0 2.4 2.4 0.8 0 3.3 26.6 8.9 7.2 12 0 0.8 0.8 0.8 2.4 0 0.8 4.8 4.8 16.1 48.4 33.9 81.5 99.2 0.5 0.064 0.064 0.5 0.5 8 8 8 64 128 32 256 256 0.125 0.032 0.032 0.032 0.25 2 2 2 16 16 8 256 256 97.4 100 100 92.9 100 97.4 95.9 94.4 66.4 37.2 22.4 1 0 1.6 0 0 4 0 1.6 0.5 3 21.3 9.2 9.2 12.8 0 1 0 0 3.1 0 1 3.6 2.6 12.3 53.6 68.4 86.2 100 Flomoxef Doripenem Meropenem Ertapenem Tigecyclineb Cefmetazole Amikacin Piperacillin/tazobactam Cefoperazone/sulbactamc Ceftazidime Levofloxacin Cefepime Cefotaxime
MIC50 %S %I %R
P. mirabilis (n = 81)
MIC90 MIC50 %S %R
%I
K. pneumoniae (n = 124)
MIC90 MIC50 %S
DNA of all of the ESBL-producing isolates was extracted and ESBL genotypes were determined by PCR and sequencing. blaCTX-M were the major ESBL genes, with occurrence in 99.5% of E. coli, 91.1% of K. pneumoniae and 97.5% of P. mirabilis. blaCTX-M-14 was the predominant ESBL gene, which was detected in 46.9% (188/401) of the isolates, followed by blaCTX-M-15 (21.4%; 86/401), blaCTX-M-55 (17.2%; 69/401), blaCTX-M-65 (12.7%; 51/401) and blaCTX-M-3 (6.7%; 27/401). The blaTEM-type ESBL gene was not detected, even though blaTEM-1 was found in 42.9% (172/401) of the total isolates. blaSHV-type ESBL genes were detected in 9.2% (37/401) of the isolates, which included blaSHV-2 , blaSHV-11 , blaSHV-12 , blaSHV-27 , blaSHV-28 , blaSHV-32 , blaSHV-33 , blaSHV-38 , blaSHV-132 , blaSHV-136 and blaSHV-137 . The occurrence of SHV-type ESBL in K. pneumoniae (26.6%; 33/124) was higher than in E. coli (1.0%; 2/196) and P. mirabilis (2.5%; 2/81). The genotype distribution between species was diverse. Among E. coli isolates, blaCTX-M-14 , blaCTX-M-55 and blaCTX-M-15 were the most frequent ESBL genes, which accounted for 50.5%, 28.6% and 20.9% of the isolates, respectively. However, blaCTX-M-14 (48.4%), blaCTX-M-15 (25.8%) and blaSHV (26.6%) were the major ESBL genes in K. pneumoniae. For P. mirabilis, blaCTX-M-65 (53.1%) and blaCTX-M-14 (35.8%) were the major ESBL genes (Fig. 1). A proportion of isolates harboured multiple ESBL genes. Among the E. coli isolates, 11.7% of the isolates carried multiple ESBL genes, which were mainly blaCTX-M-14 plus blaCTX-M-15 (or blaCTX-M-55 ). Among the K. pneumoniae isolates, the proportion of isolates
%R
3.2. Extended-spectrum ˇ-lactamase genotype distribution
E. coli (n = 196)
Against ESBL-producing E. coli and K. pneumoniae isolates, antimicrobial agents with susceptibility rates of >90% included flomoxef (97.4% and 98.4%, respectively), doripenem (100% and 99.2%), meropenem (100% and 99.2%), ertapenem (92.9% and 95.2%), tigecycline (100% and 97.6%), cefmetazole (97.4% and 98.4%), amikacin (95.9% and 95.2%) and TZP (94.4% and 91.9%). In contrast, the susceptibility rates to cefepime and cefotaxime were quite low (<7%). Levofloxacin showed higher activity against K. pneumoniae (58.9%) than against E. coli (22.4%). Against ESBL-producing P. mirabilis isolates, antimicrobial agents with susceptibility rates of >90% included flomoxef, doripenem, meropenem, ertapenem, cefmetazole, TZP, cefoperazone/sulbactam and ceftazidime. The susceptibility rates to levofloxacin (28.4%), cefepime (13.6%) and cefotaxime (0%) were low. Ceftazidime showed quite different activity (susceptibility rate of 92.6%) compared with other two cephalosporins (cefotaxime and cefepime) (Table 1).
Table 1 In vitro activity of antimicrobial agents against extended-spectrum -lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
3.1. In vitro activity of antimicrobial agents against extended-spectrum ˇ-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolates
Antimicrobial agent
3. Results
%I
Fig. 1. Occurrence of major extended-spectrum -lactamase (ESBL) genes among Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
a
MIC90
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Table 2 Extended-spectrum -lactamase (ESBL) genotype distribution among ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Rank
E. coli ESBL genotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Total
CTX-M-14 CTX-M-55 CTX-M-15 CTX-M-15 + CTX-M-14 CTX-M-55 + CTX-M-14 CTX-M-3 CTX-M-65 CTX-M-123 CTX-M-64 CTX-M-15 + CTX-M-27 CTX-M-55 + CTX-M-65 SHV-132 + CTX-M-14 CTX-M-27 SHV-11 – – – – –
K. pneumoniae N 80 45 30 9 9 6 5 3 2 2 2 1 1 1 – – – – –
P. mirabilis
ESBL genotype
N
CTX-M-14 CTX-M-15 SHV CTX-M-3 SHV + CTX-M-14 CTX-M-55 SHV + CTX-M-15 SHV + CTX-M-3 CTX-M-15 + CTX-M-14 CTX-M-55 + CTX-M-14 SHV + CTX-M-3 + CTX-M-14 CTX-M-64 SHV-132 + CTX-M-125 SHV-11 + CTX-M-15 + CTX-M-14 CTX-M-3 + CTX-M-14 CTX-M-15 + CTX-M-27 SHV-11 + CTX-M-15 + CTX-M-65 CTX-M-15 + CTX-M-65 CTX-M-22
196
harbouring multiple ESBL genes was 25.0%, mainly including blaSHV plus blaCTX-M . Among P. mirabilis, 18.5% of the isolates carried multiple ESBL genes, which were mainly blaCTX-M-15 plus blaCTX-M-65 (Table 2). 3.3. In vitro activity of flomoxef and comparators against extended-spectrum ˇ-lactamase (ESBL)-producing strains with different ESBL genotypes Flomoxef, doripenem, meropenem, ertapenem, cefmetazole, TZP, tigecycline and amikacin exhibited excellent activity (susceptibility rates of >90%) against isolates producing a single CTX-M-type enzyme (CTX-M-14, -15, -55, -3 or -65), except ertapenem and TZP against CTX-M-15-producing isolates (susceptibility rate of 88.2% and 84.3%, respectively), and ertapenem against CTX-M-3-producing isolates (susceptibility rate of 83.3%). The MIC90 values of these antimicrobial agents against ESBLproducing isolates were similar (same or with two-fold increase) to ESBL-negative isolates. The MIC90 of flomoxef against isolates solely producing CTX-M-14, -15, -55, -3 or -65 was 0.5, 0.5, 0.25, 0.5 and 0.5 g/mL, respectively, which is similar to that against ESBL-negative isolates (MIC90 = 0.25 g/mL). Ceftazidime showed distinct activity against isolates producing different ESBL types. It showed a susceptibility rate of 97.4% against solely CTX-M-65 producing isolates, 71.4% against solely CTX-M-14-producing isolates, 66.7% against solely CTX-M-3-producing isolates, 7.8% against solely CTX-M-15-producing isolates and 5.7% against solely CTXM-55-producing isolates. Cefepime and cefotaxime showed very low activity against isolates producing different enzymes (0–18.4%) (Table 3). 4. Discussion Cephamycins (i.e. cefmetazole and cefotetan), characterised by their 7-␣-methoxy -lactam, have been reported to be highly active against ESBL-producing Enterobacteriaceae. Furthermore, flomoxef has a unique structure in the cephalosporin core with oxygen substituted for sulphur giving it better stability to -lactamases, and a difluoromethylthio-acetamido group at position 7 giving it better in vitro activity against ESBL-producing Enterobacteriaceae [4]. Some research has suggested that flomoxef may be as clinically effective as a carbapenem in treating
43 20 11 9 7 7 5 5 3 3 2 2 1 1 1 1 1 1 1 124
ESBL genotype
N
CTX-M-65 CTX-M-14 CTX-M-15 + CTX-M-65 CTX-M-15 + CTX-M-14 CTX-M-3 SHV-12 CTX-M-3 + CTX-M-14 CTX-M-55 + CTX-M-14 CTX-M-27 CTX-M-55 + CTX-M-65 CTX-M-15 CTX-M-22 CTX-M-55 – – – – – –
33 24 9 3 3 2 1 1 1 1 1 1 1 – – – – – – 81
flomoxef-susceptible ESBL-producing K. pneumoniae bacteraemia [10]. Unfortunately, few reports on the antimicrobial activity of flomoxef against ESBL-producing isolates have been published in China. The objective of this study was to better understand the in vitro activity of flomoxef against clinical ESBL-producing Enterobacteriaceae. ESBL production is the predominant resistance mechanism to cephalosporins among Enterobacteriaceae. In this study, cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0–0.8% and 1.0–13.6%, respectively, whereas ceftazidime showed susceptibility rates of 37.2%, 42.7% and 92.6% against ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates, respectively. These results indicate that cefotaxime and cefepime may not be ideal choices for empirical therapy of infections caused by ESBL-producers. However, carbapenems (doripenem, meropenem and ertapenem), flomoxef, cefmetazole, TZP, tigecycline and amikacin retained high activity against ESBL-producing isolates. Flomoxef has been shown to have potential activity against clinically important Enterobacteriaceae, such as E. coli and K. pneumoniae [4–6]. In this study, flomoxef showed excellent activity against ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates, with susceptibility rates of 97.4%, 98.4% and 98.8%, and MIC90 values of 0.5, 0.25 and 0.5 g/mL, respectively. The Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project revealed that the percentage of ESBLpositive E. coli isolates from intra-abdominal infections in China increased from 20.8% in 2002 to 64.9% in 2009, whilst this rate increased somewhat more slowly among K. pneumoniae, ranging from 24.0% to 46.8% during the 8 years [2,3]. Researchers in China have also determined that the ESBL genotypes in Beijing, Guangdong and Hangzhou were mainly CTX-M types [11–13], which preferentially hydrolyse cefotaxime and ceftriaxone over ceftazidime. Unlike TEM and SHV variants, CTX-M-type ESBLs are not mutants of old plasmid-mediated penicillinases. Rather, their genes originated in the chromosomes of Kluyvera spp., a genus of no direct clinical importance, and were mobilised to plasmids by insertion sequences, notably ISEcp1 [14,15]. Plasmids encoding these CTX-M enzymes then reached human opportunists, where they have proliferated in community E. coli and hospital Klebsiella spp. Gene escape has occurred repeatedly, with five CTX-M families (groups 1, 2, 8, 9 and 25) circulating. Different CTX-M families
S, susceptible; MIC50/90 , minimum inhibitory concentration at which 50% and 90% of isolates were inhibited, respectively; CLSI, Clinical and Laboratory Standards Institute. a No breakpoints for flomoxef have been published by the CLSI. The CLSI breakpoints for latamoxef (S ≤8 g/mL, R ≥64 g/mL for Enterobacteriaceae) were used for flomoxef. b US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). c The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam.
MIC90
0.25 0.5 0.125 0.016 2 4 4 8 0.5 1 8 0.125 0.125 0.064 0.064 0.032 0.008 1 2 0.25 2 0.125 0.25 0.125 0.064 0.064
MIC50 S (n)
240 239 244 244 240 243 209 239 244 244 202 243 244 0.5 1 0.125 0.032 2 4 2 8 2 8 32 >128 >128 0.125 0.25 0.064 0.016 1 1 2 4 0.25 2 4 16 128 37 37 38 38 38 38 35 36 37 37 10 7 0 0.5 0.5 0.125 1 4 16 2 8 64 64 32 >128 >128 0.125 0.064 0.032 0.032 2 2 0.5 2 4 16 2 >128 >128 18 18 18 15 18 18 17 18 12 11 10 0 0 0.25 0.125 0.064 0.25 4 8 0.5 8 128 32 32 >128 >128 0.064 0.032 0.032 0.064 2 2 0.25 4 32 8 8 >128 >128 52 53 53 52 53 51 53 49 3 44 13 1 0 0.5 0.125 0.125 1 8 32 1 8 >128 64 64 >128 >128 0.125 0.064 0.032 0.064 2 4 0.25 2 64 16 16 >128 >128 0.5 0.5 0.125 0.25 4 8 2 16 32 64 32 >128 >128 Flomoxefa Doripenem Meropenem Ertapenem Cefmetazole Piperacillin/tazobactam Tigecyclineb Amikacin Ceftazidime Cefoperazone/sulbactamc Levofloxacin Cefepime Cefotaxime
145 147 147 144 144 141 140 135 105 103 52 6 1
0.125 0.032 0.032 0.032 2 2 0.25 2 2 16 8 128 >128
50 51 51 45 49 43 48 50 4 29 15 1 0
MIC50 S (n) MIC50 S (n) MIC90 MIC50 S (n)
CTX-M-55 (n = 53)
MIC90 MIC50
CTX-M-15 (n = 51)
S (n) MIC90 MIC50 S (n)
CTX-M-14 (n = 147) Antimicrobial agent
Table 3 In vitro activity of flomoxef and comparators against isolates with different extended-spectrum -lactamase (ESBL) genotypes.
CTX-M-3 (n = 18)
MIC90
CTX-M-65 (n = 38)
MIC90
ESBL-negative (n = 244)
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dominate in different regions: CTX-M-15 (group 1) is predominant in most of Europe, North America, the Middle East and India, whereas CTX-M-14 (group 9) is most common in China, Southeast Asia and Spain, and CTX-M-2 (group 2) is predominant in Argentina, Israel and Japan [16,17]. In this study, blaCTX-M-14 was the predominant ESBL gene and was detected in 46.9% (188/401) of the total isolates, followed by blaCTX-M-15 (21.4%; 86/401), blaCTX-M-55 (17.2%; 69/401), blaCTX-M-65 (12.7%; 51/401) and blaCTX-M-3 (6.7%; 27/401). CTX-M-14 has been reported as the most abundant genotype in China, although the detection rate of CTX-M-15 has shown a continuously increasing trend in recent years among E. coli strains [18–20]. In this study, ESBL genotypes among different species were diverse. CTX-M-14, CTX-M-15 and CTX-M-55 were the main ESBL types in E. coli, whilst CTX-M-14, CTX-M-15 and SHV were the main ESBL types in K. pneumoniae isolates. In contrast, CTX-M-65 and CTX-M-14 were the predominant ESBL types in P. mirabilis isolates. Previous studies have revealed that these CTX-M-type ESBLs can hydrolyse cefotaxime, ceftriaxone and cefepime effectively. However, ceftazidime cannot be effectively hydrolysed by CTX-M-65 [21–25]. This may explain why ceftazidime showed higher activity against ESBLproducing P. mirabilis, in which species CTX-M-65 was the main ESBL genotype. CTX-M-65 has been reported in food animals several times [23–25], whilst the CTX-M-65-producing clinical E. coli isolates in China were reported by Yin et al. in 2009 [21]. The current study is the first report revealing the prevalence of this enzyme in clinical P. mirabilis isolates in China. However, continued surveillance of blaCTX-M genotype distribution is needed to determine whether blaCTX-M-65 is increasing among other species. Flomoxef exhibited excellent activity against the different CTXM-type ESBL-producing isolates, with MIC50 and MIC90 values of 0.064–0.125 g/mL and 0.25–0.5 g/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. The excellent activity of flomoxef against ESBL-producing isolates may come from its unique structure in the cephalosporin core with oxygen substituted for sulphur and a difluoromethylthio-acetamido group at position 7. A limitation of this study is that carbapenemase genes were not sought in the carbapenem-resistant isolates, which may be important in revealing the resistance mechanism to most of the antimicrobial agents of the isolates. In conclusion, flomoxef is a cephamycin showing excellent activity against ESBL-producing E. coli, K. pneumoniae and P. mirabilis. Although some evidence [26] suggests that cephamycins may select for resistant mutants producing AmpC or with porin loss, flomoxef may still be a good choice to treat infections caused by flomoxef-susceptible bacteria at a time when the problem of antimicrobial resistance continues to increase. However, proper use of flomoxef and continuous monitoring of susceptibility of clinical isolates to this antibiotic are necessary. Acknowledgments The authors thank all of the investigators for their participation in this project. Funding: This study was sponsored by Shionogi & Co., Ltd. and was supported by the National Natural Science Foundation of China [grant 81101287]. Competing interests: None declared. Ethical approval: Not required. References [1] Baquero F, Hsueh PR, Paterson DL, Rossi F, Bochicchio GV, Gallagher G, et al. In vitro susceptibilities of aerobic and facultatively anaerobic Gram-negative
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[14] Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extendedspectrum -lactamases. Clin Microbiol Infect 2008;14(Suppl. 1):33–41. Erratum in: Clin Microbiol Infect 2008;14(Suppl. 5):21–4. [15] Naseer U, Sundsfjord A. The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb Drug Resist 2011;17:83–97. [16] Hawkey PM. Prevalence and clonality of extended-spectrum -lactamases in Asia. Clin Microbiol Infect 2008;14(Suppl. 1):159–65. [17] Bush K. Extended-spectrum -lactamases in North America, 1987–2006. Clin Microbiol Infect 2008;14(Suppl. 1):134–43. [18] An S, Chen J, Wang Z, Wang X, Yan X, Li J, et al. Predominant characteristics of CTX-M-producing Klebsiella pneumoniae isolates from patients with lower respiratory tract infection in multiple medical centers in China. FEMS Microbiol Lett 2012;332:137–45. [19] Zhuo C, Li XQ, Zong ZY, Zhong NS. Epidemic plasmid carrying blaCTX-M-15 in Klebsiella pneumoniae in China. PLOS ONE 2013;8:e52222. [20] Kim S, Sung JY, Cho HH, Kwon KC, Koo SH. Characterization of CTX-M-14and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae isolates from urine specimens in a tertiary-care hospital. J Microbiol Biotechnol 2014;24:765–70. [21] Yin J, Cheng J, Sun Z, Ye Y, Gao YF, Li JB, et al. Characterization of two plasmidencoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87. J Med Microbiol 2009;58:811–5. [22] Liu W, Chen L, Li H, Duan H, Zhang Y, Liang X, et al. Novel CTX-M -lactamase genotype distribution and spread into multiple species of Enterobacteriaceae in Changsha, Southern China. J Antimicrob Chemother 2009;63: 895–900. [23] Rao L, Lv L, Zeng Z, Chen S, He D, Chen X, et al. Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003–2012. Vet Microbiol 2014;172:534–41. [24] Lv L, Partridge SR, He L, Zeng Z, He D, Ye J, et al. Genetic characterization of IncI2 plasmids carrying blaCTX-M-55 spreading in both pets and food animals in China. Antimicrob Agents Chemother 2013;57:2824–7. [25] Zheng H, Zeng Z, Chen S, Liu Y, Yao Q, Deng Y, et al. Prevalence and characterisation of CTX-M -lactamases amongst Escherichia coli isolates from healthy food animals in China. Int J Antimicrob Agents 2012;39: 305–10. [26] Lee CH, Chu C, Liu JW, Chen YS, Chiu CJ, Su LH. Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of blaDHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 -lactamases. J Antimicrob Chemother 2007;60:410–3.
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In vitro activity of flomoxef and comparators against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis producing extended-spectrum -lactamases in China Qiwen Yang a , Hui Zhang a , Jingwei Cheng a , Zhipeng Xu a , Yingchun Xu a,∗ , Bin Cao b , Haishen Kong c , Yuxing Ni d , Yunsong Yu e , Ziyong Sun f , Bijie Hu g , Wenxiang Huang h , Yong Wang i , Anhua Wu j , Xianju Feng k , Kang Liao l , Dingxia Shen m , Zhidong Hu n , Yunzhuo Chu o , Juan Lu p , Jianrong Su q , Bingdong Gui r , Qiong Duan s , Shufang Zhang t , Haifeng Shao u a Department of Clinical Laboratory, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, 1 Shuaifuyuan, Wangfujing Street, Beijing 100730, China b Chaoyang Hospital of Capital Medical College, Beijing 100020, China c The First Affiliated Hospital of Zhejiang University, Hangzhou 310003, China d Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China e Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China f Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China g Zhongshan Hospital of Fu Dan University, Shanghai 200032, China h The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China i Shandong Provincial Hospital, Jinan 250021, China j Xiangya Hospital, Central Southern University, Changsha 410008, China k The First Affiliated Hospital of Zhengzhou University, Zhenzhou 450052, China l The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China m General Hospital of PLA, Beijing 100853, China n General Hospital of Tianjin Medical University, Tianjing 300052, China o The First Affiliated Hospital of Chinese Medical University, Shenyang 110001, China p The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China q Friendship Hospital of Capital Medical College, Beijing 100020, China r The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China s People’s Hospital of Jilin Province, Jilin 130021, China t People’s Hospital of Haikou City, Haikou 570208, China u General Hospital of Nanjing Military Command, Nanjing 210002, China
a r t i c l e
i n f o
Article history: Received 5 November 2014 Accepted 25 November 2014 Keywords: Flomoxef Extended-spectrum -lactamases Escherichia coli Klebsiella pneumoniae Proteus mirabilis
a b s t r a c t The objective of this study was to better understand the in vitro activity of flomoxef against clinical extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae. A total of 401 ESBL-producing isolates, including 196 Escherichia coli, 124 Klebsiella pneumoniae and 81 Proteus mirabilis, were collected consecutively from 21 hospitals in China in 2013. Minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. Phenotypic identification of ESBL production was detected as recommended by the Clinical and Laboratory Standards Institute (CLSI). ESBL genes were detected by PCR and sequencing. Flomoxef, doripenem, meropenem, ertapenem, cefmetazole and piperacillin/tazobactam exhibited good activity against ESBL-producing isolates, with susceptibility rates >90%. Tigecycline showed good activity against E. coli and K. pneumoniae (100% and 97.6%, respectively). Cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0–0.8% and 1.0–13.6%, respectively. blaCTX-M were the major ESBL genes, with occurrence in 99.5% of E. coli, 91.1% of K. pneumoniae and 97.5% of P. mirabilis. blaCTX-M-14 was the predominant ESBL gene, detected in 46.9% (188/401) of the isolates, followed by blaCTX-M-15 (21.4%), blaCTX-M-55 (17.2%), blaCTX-M-65 (12.7%) and blaCTX-M-3 (6.7%). Flomoxef exhibited excellent activity against the different CTX-M-type ESBL-producing
∗ Corresponding author. Tel.: +86 10 6915 9766/1391 130 3028; fax: +86 10 6915 9766. E-mail address: [email protected] (Y. Xu). http://dx.doi.org/10.1016/j.ijantimicag.2014.11.012 0924-8579/© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
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isolates, with MIC50 and MIC90 values of 0.064–0.125 g/mL and 0.25–0.5 g/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. In conclusion, flomoxef showed good activity against ESBLproducing Enterobacteriaceae and may be a choice to treat infections caused by these isolates in China. © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction A number of surveillance programmes exist to monitor the susceptibility of clinically important pathogens at national and international levels [1–3]. The Enterobacteriaceae are a major group of pathogens causing community- and hospital-acquired infections. With the increase in the prevalence of extendedspectrum -lactamases (ESBLs) in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, clinicians have a limited choice of antimicrobial agents to use, which in turn leads to an increase in the abuse of certain antimicrobial agents such as the carbapenems. ESBL rates in E. coli and K. pneumoniae in China have been reported as 60–70% and 30–40%, respectively [2,3], which has resulted in a challenge to Chinese clinicians in terms of selecting appropriate antimicrobial agents to treat infections caused by ESBL-producing isolates. Flomoxef is a unique -lactam antibiotic with oxygen substituted for the sulphur, a 7-␣-methoxy group in the cephalosporin core and a difluoromethylthio-acetamido group at position 7, which gives it better in vitro activity against ESBLproducing Enterobacteriaceae [4]. However, the activity of this drug in China has not been reported. In view of this, in this study consecutive community- and hospital-associated ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates were collected and antimicrobial susceptibility testing was conducted on these strains to evaluate the in vitro activity of flomoxef and other comparators. Although there have been some reports describing the activity of flomoxef [1–6], this is the first systematic report to provide an overview specifically of the results in China. 2. Materials and methods 2.1. Clinical isolates A total of 401 ESBL-producing isolates, including 196 E. coli, 124 K. pneumoniae and 81 P. mirabilis, were collected consecutively from 21 hospitals in China in 2013. Specimens included 147 urine, 101 abdominal fluid, 37 pus, 32 blood, 31 sputum, 25 bile, 17 liver tissue, 6 pancreas, 2 appendix and 3 other specimens. Bacteria were identified by standard methods used in each site and were re-identified in Peking Union Medical College Hospital (Beijing, China) by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). All organisms were deemed clinically significant according to local criteria. For the purpose of comparison of the activity of different antimicrobial agents against ESBL-producers, 244 non-ESBL-producing isolates were collected from the same hospitals during the same period, including 63 E. coli, 103 K. pneumoniae and 78 P. mirabilis isolates from 67 blood, 61 urine, 46 sputum, 29 gallbladder, 13 abdominal fluids, 9 liver tissue, 8 pus, 4 bile and 7 other specimens. 2.2. Antimicrobial susceptibility test method Minimum inhibitory concentrations (MICs) were determined using a broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [7].Fifteen
antimicrobial agents were tested, including flomoxef, ceftazidime, ceftazidime/clavulanic acid (fixed clavulanic acid concentration of 4 g/mL), cefotaxime, cefotaxime/clavulanic acid (fixed clavulanic acid concentration of 4 g/mL), cefepime, cefmetazole, piperacillin/tazobactam (TZP) (fixed tazobactam concentration of 4 g/mL), cefoperazone/sulbactam (fixed ratio of 2:1), ertapenem, meropenem, doripenem, levofloxacin, tigecycline and amikacin. MIC50 and MIC90 values (MICs at which 50% and 90% of isolates were inhibited, respectively) were calculated using WHONET software v.5.6 (http://whonet.software.informer.com/5.6/). Susceptibility interpretations were based on CLSI clinical breakpoints [8]. The CLSI breakpoints for latamoxef [susceptible (S) ≤8 g/mL, resistant (R) ≥64 g/mL for Enterobacteriaceae] were used for flomoxef. US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam. The reference strains E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and K. pneumoniae ATCC 700603 (positive ESBL control) were used as quality control strains for each batch of MIC testing. Results were included in the analysis only when corresponding quality control isolates tested within the acceptable range according to CLSI guidelines.
2.3. Extended-spectrum ˇ-lactamase detection Phenotypic identification of ESBL production among E. coli, K. pneumoniae and P. mirabilis was detected according to the methods recommended by the CLSI [8]. If the cefotaxime or ceftazidime MIC was ≥2 g/mL, then the MIC of cefotaxime or ceftazidime was compared with the MIC of cefotaxime/clavulanic acid or ceftazidime/clavulanic acid. A positive test for ESBL production was defined as a ≥8-fold (i.e. three-fold doubling dilution) decrease in the MIC for cefotaxime or ceftazidime when tested in combination with clavulanic acid versus their MIC when either drug was tested alone.
2.4. PCR amplification and DNA sequence analysis of extended-spectrum ˇ-lactamase genes Template DNA of the ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates was obtained by placing several small colonies of each strain in 200 L of double-distilled water and boiling the sample for 10 min. After cooling on ice, 1–2 L of each lysate was used in the PCR. The primers used in this study have been described previously [9]. The reaction was conducted in a PTC-200 PCR system (MJ Research Inc., Watertown, MA). The amplified genes included blaTEM , blaSHV , blaCTX-M-1 group , blaCTX-M-2 group , blaCTX-M-8 group , blaCTX-M-9 group and blaCTX-M-25 group . A positive and negative control was followed in each batch of PCR. PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and were sequenced on an ABI PRISM 3730XL Sequencer Analyzer (Applied Biosystems, Foster City, CA). DNA sequencing data were analysed with the GenBank BLASTN tool (http://www.ncbi.nlm.nih.gov/blast).
0.5 1 0.25 0.032 4 4 64 2 8 1 32 256 256
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R, resistant; S, susceptible; I, intermediate; MIC50/90 , minimum inhibitory concentration at which 50% and 90% of isolates were inhibited, respectively; CLSI, Clinical and Laboratory Standards Institute. a No breakpoints for flomoxef have been published by the CLSI. The CLSI breakpoints for latamoxef (S ≤8 g/mL, R ≥64 g/mL for Enterobacteriaceae) were used for flomoxef. b US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). c The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam.
0.125 0.5 0.064 0.016 2 1 4 1 4 0.25 8 128 256 98.8 98.8 100 100 80.2 98.8 86.4 98.8 96.3 92.6 28.4 13.6 0 0 1.2 0 0 17.3 1.2 2.5 1.2 1.2 0 16 9.9 0 1.2 0 0 0 2.5 0 11.1 0 2.5 7.4 55.6 76.5 100 0.25 0.125 0.125 0.25 2 4 4 16 64 128 64 256 256 0.064 0.064 0.032 0.064 0.5 2 2 4 16 8 1 256 256 98.4 99.2 99.2 95.2 97.6 98.4 95.2 91.9 57.3 42.7 58.9 6.5 0.8 0.8 0 0 2.4 2.4 0.8 0 3.3 26.6 8.9 7.2 12 0 0.8 0.8 0.8 2.4 0 0.8 4.8 4.8 16.1 48.4 33.9 81.5 99.2 0.5 0.064 0.064 0.5 0.5 8 8 8 64 128 32 256 256 0.125 0.032 0.032 0.032 0.25 2 2 2 16 16 8 256 256 97.4 100 100 92.9 100 97.4 95.9 94.4 66.4 37.2 22.4 1 0 1.6 0 0 4 0 1.6 0.5 3 21.3 9.2 9.2 12.8 0 1 0 0 3.1 0 1 3.6 2.6 12.3 53.6 68.4 86.2 100 Flomoxef Doripenem Meropenem Ertapenem Tigecyclineb Cefmetazole Amikacin Piperacillin/tazobactam Cefoperazone/sulbactamc Ceftazidime Levofloxacin Cefepime Cefotaxime
MIC50 %S %I %R
P. mirabilis (n = 81)
MIC90 MIC50 %S %R
%I
K. pneumoniae (n = 124)
MIC90 MIC50 %S
DNA of all of the ESBL-producing isolates was extracted and ESBL genotypes were determined by PCR and sequencing. blaCTX-M were the major ESBL genes, with occurrence in 99.5% of E. coli, 91.1% of K. pneumoniae and 97.5% of P. mirabilis. blaCTX-M-14 was the predominant ESBL gene, which was detected in 46.9% (188/401) of the isolates, followed by blaCTX-M-15 (21.4%; 86/401), blaCTX-M-55 (17.2%; 69/401), blaCTX-M-65 (12.7%; 51/401) and blaCTX-M-3 (6.7%; 27/401). The blaTEM-type ESBL gene was not detected, even though blaTEM-1 was found in 42.9% (172/401) of the total isolates. blaSHV-type ESBL genes were detected in 9.2% (37/401) of the isolates, which included blaSHV-2 , blaSHV-11 , blaSHV-12 , blaSHV-27 , blaSHV-28 , blaSHV-32 , blaSHV-33 , blaSHV-38 , blaSHV-132 , blaSHV-136 and blaSHV-137 . The occurrence of SHV-type ESBL in K. pneumoniae (26.6%; 33/124) was higher than in E. coli (1.0%; 2/196) and P. mirabilis (2.5%; 2/81). The genotype distribution between species was diverse. Among E. coli isolates, blaCTX-M-14 , blaCTX-M-55 and blaCTX-M-15 were the most frequent ESBL genes, which accounted for 50.5%, 28.6% and 20.9% of the isolates, respectively. However, blaCTX-M-14 (48.4%), blaCTX-M-15 (25.8%) and blaSHV (26.6%) were the major ESBL genes in K. pneumoniae. For P. mirabilis, blaCTX-M-65 (53.1%) and blaCTX-M-14 (35.8%) were the major ESBL genes (Fig. 1). A proportion of isolates harboured multiple ESBL genes. Among the E. coli isolates, 11.7% of the isolates carried multiple ESBL genes, which were mainly blaCTX-M-14 plus blaCTX-M-15 (or blaCTX-M-55 ). Among the K. pneumoniae isolates, the proportion of isolates
%R
3.2. Extended-spectrum ˇ-lactamase genotype distribution
E. coli (n = 196)
Against ESBL-producing E. coli and K. pneumoniae isolates, antimicrobial agents with susceptibility rates of >90% included flomoxef (97.4% and 98.4%, respectively), doripenem (100% and 99.2%), meropenem (100% and 99.2%), ertapenem (92.9% and 95.2%), tigecycline (100% and 97.6%), cefmetazole (97.4% and 98.4%), amikacin (95.9% and 95.2%) and TZP (94.4% and 91.9%). In contrast, the susceptibility rates to cefepime and cefotaxime were quite low (<7%). Levofloxacin showed higher activity against K. pneumoniae (58.9%) than against E. coli (22.4%). Against ESBL-producing P. mirabilis isolates, antimicrobial agents with susceptibility rates of >90% included flomoxef, doripenem, meropenem, ertapenem, cefmetazole, TZP, cefoperazone/sulbactam and ceftazidime. The susceptibility rates to levofloxacin (28.4%), cefepime (13.6%) and cefotaxime (0%) were low. Ceftazidime showed quite different activity (susceptibility rate of 92.6%) compared with other two cephalosporins (cefotaxime and cefepime) (Table 1).
Table 1 In vitro activity of antimicrobial agents against extended-spectrum -lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
3.1. In vitro activity of antimicrobial agents against extended-spectrum ˇ-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolates
Antimicrobial agent
3. Results
%I
Fig. 1. Occurrence of major extended-spectrum -lactamase (ESBL) genes among Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
a
MIC90
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Table 2 Extended-spectrum -lactamase (ESBL) genotype distribution among ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Rank
E. coli ESBL genotype
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Total
CTX-M-14 CTX-M-55 CTX-M-15 CTX-M-15 + CTX-M-14 CTX-M-55 + CTX-M-14 CTX-M-3 CTX-M-65 CTX-M-123 CTX-M-64 CTX-M-15 + CTX-M-27 CTX-M-55 + CTX-M-65 SHV-132 + CTX-M-14 CTX-M-27 SHV-11 – – – – –
K. pneumoniae N 80 45 30 9 9 6 5 3 2 2 2 1 1 1 – – – – –
P. mirabilis
ESBL genotype
N
CTX-M-14 CTX-M-15 SHV CTX-M-3 SHV + CTX-M-14 CTX-M-55 SHV + CTX-M-15 SHV + CTX-M-3 CTX-M-15 + CTX-M-14 CTX-M-55 + CTX-M-14 SHV + CTX-M-3 + CTX-M-14 CTX-M-64 SHV-132 + CTX-M-125 SHV-11 + CTX-M-15 + CTX-M-14 CTX-M-3 + CTX-M-14 CTX-M-15 + CTX-M-27 SHV-11 + CTX-M-15 + CTX-M-65 CTX-M-15 + CTX-M-65 CTX-M-22
196
harbouring multiple ESBL genes was 25.0%, mainly including blaSHV plus blaCTX-M . Among P. mirabilis, 18.5% of the isolates carried multiple ESBL genes, which were mainly blaCTX-M-15 plus blaCTX-M-65 (Table 2). 3.3. In vitro activity of flomoxef and comparators against extended-spectrum ˇ-lactamase (ESBL)-producing strains with different ESBL genotypes Flomoxef, doripenem, meropenem, ertapenem, cefmetazole, TZP, tigecycline and amikacin exhibited excellent activity (susceptibility rates of >90%) against isolates producing a single CTX-M-type enzyme (CTX-M-14, -15, -55, -3 or -65), except ertapenem and TZP against CTX-M-15-producing isolates (susceptibility rate of 88.2% and 84.3%, respectively), and ertapenem against CTX-M-3-producing isolates (susceptibility rate of 83.3%). The MIC90 values of these antimicrobial agents against ESBLproducing isolates were similar (same or with two-fold increase) to ESBL-negative isolates. The MIC90 of flomoxef against isolates solely producing CTX-M-14, -15, -55, -3 or -65 was 0.5, 0.5, 0.25, 0.5 and 0.5 g/mL, respectively, which is similar to that against ESBL-negative isolates (MIC90 = 0.25 g/mL). Ceftazidime showed distinct activity against isolates producing different ESBL types. It showed a susceptibility rate of 97.4% against solely CTX-M-65 producing isolates, 71.4% against solely CTX-M-14-producing isolates, 66.7% against solely CTX-M-3-producing isolates, 7.8% against solely CTX-M-15-producing isolates and 5.7% against solely CTXM-55-producing isolates. Cefepime and cefotaxime showed very low activity against isolates producing different enzymes (0–18.4%) (Table 3). 4. Discussion Cephamycins (i.e. cefmetazole and cefotetan), characterised by their 7-␣-methoxy -lactam, have been reported to be highly active against ESBL-producing Enterobacteriaceae. Furthermore, flomoxef has a unique structure in the cephalosporin core with oxygen substituted for sulphur giving it better stability to -lactamases, and a difluoromethylthio-acetamido group at position 7 giving it better in vitro activity against ESBL-producing Enterobacteriaceae [4]. Some research has suggested that flomoxef may be as clinically effective as a carbapenem in treating
43 20 11 9 7 7 5 5 3 3 2 2 1 1 1 1 1 1 1 124
ESBL genotype
N
CTX-M-65 CTX-M-14 CTX-M-15 + CTX-M-65 CTX-M-15 + CTX-M-14 CTX-M-3 SHV-12 CTX-M-3 + CTX-M-14 CTX-M-55 + CTX-M-14 CTX-M-27 CTX-M-55 + CTX-M-65 CTX-M-15 CTX-M-22 CTX-M-55 – – – – – –
33 24 9 3 3 2 1 1 1 1 1 1 1 – – – – – – 81
flomoxef-susceptible ESBL-producing K. pneumoniae bacteraemia [10]. Unfortunately, few reports on the antimicrobial activity of flomoxef against ESBL-producing isolates have been published in China. The objective of this study was to better understand the in vitro activity of flomoxef against clinical ESBL-producing Enterobacteriaceae. ESBL production is the predominant resistance mechanism to cephalosporins among Enterobacteriaceae. In this study, cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0–0.8% and 1.0–13.6%, respectively, whereas ceftazidime showed susceptibility rates of 37.2%, 42.7% and 92.6% against ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates, respectively. These results indicate that cefotaxime and cefepime may not be ideal choices for empirical therapy of infections caused by ESBL-producers. However, carbapenems (doripenem, meropenem and ertapenem), flomoxef, cefmetazole, TZP, tigecycline and amikacin retained high activity against ESBL-producing isolates. Flomoxef has been shown to have potential activity against clinically important Enterobacteriaceae, such as E. coli and K. pneumoniae [4–6]. In this study, flomoxef showed excellent activity against ESBL-producing E. coli, K. pneumoniae and P. mirabilis isolates, with susceptibility rates of 97.4%, 98.4% and 98.8%, and MIC90 values of 0.5, 0.25 and 0.5 g/mL, respectively. The Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project revealed that the percentage of ESBLpositive E. coli isolates from intra-abdominal infections in China increased from 20.8% in 2002 to 64.9% in 2009, whilst this rate increased somewhat more slowly among K. pneumoniae, ranging from 24.0% to 46.8% during the 8 years [2,3]. Researchers in China have also determined that the ESBL genotypes in Beijing, Guangdong and Hangzhou were mainly CTX-M types [11–13], which preferentially hydrolyse cefotaxime and ceftriaxone over ceftazidime. Unlike TEM and SHV variants, CTX-M-type ESBLs are not mutants of old plasmid-mediated penicillinases. Rather, their genes originated in the chromosomes of Kluyvera spp., a genus of no direct clinical importance, and were mobilised to plasmids by insertion sequences, notably ISEcp1 [14,15]. Plasmids encoding these CTX-M enzymes then reached human opportunists, where they have proliferated in community E. coli and hospital Klebsiella spp. Gene escape has occurred repeatedly, with five CTX-M families (groups 1, 2, 8, 9 and 25) circulating. Different CTX-M families
S, susceptible; MIC50/90 , minimum inhibitory concentration at which 50% and 90% of isolates were inhibited, respectively; CLSI, Clinical and Laboratory Standards Institute. a No breakpoints for flomoxef have been published by the CLSI. The CLSI breakpoints for latamoxef (S ≤8 g/mL, R ≥64 g/mL for Enterobacteriaceae) were used for flomoxef. b US Food and Drug Administration (FDA)-approved breakpoints for tigecycline were used (S ≤2 g/mL, R ≥8 g/mL for Enterobacteriaceae). c The cefoperazone CLSI breakpoints were used for cefoperazone/sulbactam.
MIC90
0.25 0.5 0.125 0.016 2 4 4 8 0.5 1 8 0.125 0.125 0.064 0.064 0.032 0.008 1 2 0.25 2 0.125 0.25 0.125 0.064 0.064
MIC50 S (n)
240 239 244 244 240 243 209 239 244 244 202 243 244 0.5 1 0.125 0.032 2 4 2 8 2 8 32 >128 >128 0.125 0.25 0.064 0.016 1 1 2 4 0.25 2 4 16 128 37 37 38 38 38 38 35 36 37 37 10 7 0 0.5 0.5 0.125 1 4 16 2 8 64 64 32 >128 >128 0.125 0.064 0.032 0.032 2 2 0.5 2 4 16 2 >128 >128 18 18 18 15 18 18 17 18 12 11 10 0 0 0.25 0.125 0.064 0.25 4 8 0.5 8 128 32 32 >128 >128 0.064 0.032 0.032 0.064 2 2 0.25 4 32 8 8 >128 >128 52 53 53 52 53 51 53 49 3 44 13 1 0 0.5 0.125 0.125 1 8 32 1 8 >128 64 64 >128 >128 0.125 0.064 0.032 0.064 2 4 0.25 2 64 16 16 >128 >128 0.5 0.5 0.125 0.25 4 8 2 16 32 64 32 >128 >128 Flomoxefa Doripenem Meropenem Ertapenem Cefmetazole Piperacillin/tazobactam Tigecyclineb Amikacin Ceftazidime Cefoperazone/sulbactamc Levofloxacin Cefepime Cefotaxime
145 147 147 144 144 141 140 135 105 103 52 6 1
0.125 0.032 0.032 0.032 2 2 0.25 2 2 16 8 128 >128
50 51 51 45 49 43 48 50 4 29 15 1 0
MIC50 S (n) MIC50 S (n) MIC90 MIC50 S (n)
CTX-M-55 (n = 53)
MIC90 MIC50
CTX-M-15 (n = 51)
S (n) MIC90 MIC50 S (n)
CTX-M-14 (n = 147) Antimicrobial agent
Table 3 In vitro activity of flomoxef and comparators against isolates with different extended-spectrum -lactamase (ESBL) genotypes.
CTX-M-3 (n = 18)
MIC90
CTX-M-65 (n = 38)
MIC90
ESBL-negative (n = 244)
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dominate in different regions: CTX-M-15 (group 1) is predominant in most of Europe, North America, the Middle East and India, whereas CTX-M-14 (group 9) is most common in China, Southeast Asia and Spain, and CTX-M-2 (group 2) is predominant in Argentina, Israel and Japan [16,17]. In this study, blaCTX-M-14 was the predominant ESBL gene and was detected in 46.9% (188/401) of the total isolates, followed by blaCTX-M-15 (21.4%; 86/401), blaCTX-M-55 (17.2%; 69/401), blaCTX-M-65 (12.7%; 51/401) and blaCTX-M-3 (6.7%; 27/401). CTX-M-14 has been reported as the most abundant genotype in China, although the detection rate of CTX-M-15 has shown a continuously increasing trend in recent years among E. coli strains [18–20]. In this study, ESBL genotypes among different species were diverse. CTX-M-14, CTX-M-15 and CTX-M-55 were the main ESBL types in E. coli, whilst CTX-M-14, CTX-M-15 and SHV were the main ESBL types in K. pneumoniae isolates. In contrast, CTX-M-65 and CTX-M-14 were the predominant ESBL types in P. mirabilis isolates. Previous studies have revealed that these CTX-M-type ESBLs can hydrolyse cefotaxime, ceftriaxone and cefepime effectively. However, ceftazidime cannot be effectively hydrolysed by CTX-M-65 [21–25]. This may explain why ceftazidime showed higher activity against ESBLproducing P. mirabilis, in which species CTX-M-65 was the main ESBL genotype. CTX-M-65 has been reported in food animals several times [23–25], whilst the CTX-M-65-producing clinical E. coli isolates in China were reported by Yin et al. in 2009 [21]. The current study is the first report revealing the prevalence of this enzyme in clinical P. mirabilis isolates in China. However, continued surveillance of blaCTX-M genotype distribution is needed to determine whether blaCTX-M-65 is increasing among other species. Flomoxef exhibited excellent activity against the different CTXM-type ESBL-producing isolates, with MIC50 and MIC90 values of 0.064–0.125 g/mL and 0.25–0.5 g/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. The excellent activity of flomoxef against ESBL-producing isolates may come from its unique structure in the cephalosporin core with oxygen substituted for sulphur and a difluoromethylthio-acetamido group at position 7. A limitation of this study is that carbapenemase genes were not sought in the carbapenem-resistant isolates, which may be important in revealing the resistance mechanism to most of the antimicrobial agents of the isolates. In conclusion, flomoxef is a cephamycin showing excellent activity against ESBL-producing E. coli, K. pneumoniae and P. mirabilis. Although some evidence [26] suggests that cephamycins may select for resistant mutants producing AmpC or with porin loss, flomoxef may still be a good choice to treat infections caused by flomoxef-susceptible bacteria at a time when the problem of antimicrobial resistance continues to increase. However, proper use of flomoxef and continuous monitoring of susceptibility of clinical isolates to this antibiotic are necessary. Acknowledgments The authors thank all of the investigators for their participation in this project. Funding: This study was sponsored by Shionogi & Co., Ltd. and was supported by the National Natural Science Foundation of China [grant 81101287]. Competing interests: None declared. Ethical approval: Not required. References [1] Baquero F, Hsueh PR, Paterson DL, Rossi F, Bochicchio GV, Gallagher G, et al. In vitro susceptibilities of aerobic and facultatively anaerobic Gram-negative
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