Bareum Gwon; Eun-Jeong Yoon; Dokyun Kim; Hyukmin Lee; Jong Hee Shin; Jeong Hwan Shin; Kyeong Seob Shin; Young Ah Kim; Young Uh; Hyun Soo Kim; Young Ree Kim; Seok Hoon Jeong

doi:10.5145/ACM.2019.22.1.1

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Gwon, Yoon, Kim, Lee, Shin, Shin, Shin, Kim, Uh, Kim, Kim, and Jeong: Differences in Antimicrobial Resistance Phenotypes by the Group of CTX-M Extended-Spectrum β-Lactamase

Original Article

Annals of Clinical Microbiology 2019; 22(1): 1-8.

Published online: 21 March 2019

DOI: https://doi.org/10.5145/ACM.2019.22.1.1

Differences in Antimicrobial Resistance Phenotypes by the Group of CTX-M Extended-Spectrum β-Lactamase

Bareum Gwon^1,², Eun-Jeong Yoon², Dokyun Kim², Hyukmin Lee², Jong Hee Shin³, Jeong Hwan Shin⁴, Kyeong Seob Shin⁵, Young Ah Kim⁶, Young Uh⁷, Hyun Soo Kim⁸, Young Ree Kim⁹, Seok Hoon Jeong²

¹Department of Clinical Pathology, Sangji University College of Science, Wonju, Korea.

²Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea.

³Department of Laboratory Medicine, Chonnam National University School of Medicine, Gwangju, Korea.

⁴Department of Laboratory Medicine, Inje University Busan Paik Hospital, Busan, Korea.

⁵Department of Laboratory Medicine, College of Medicine, Chungbuk National University, Cheongju, Korea.

⁶Department of Laboratory Medicine, National Health Insurance Service Ilsan Hospital, Goyang, Korea.

⁷Department of Laboratory Medicine, Wonju Severance Christian Hospital, Yonsei University Wonju College of Medicine, Wonju, Korea.

⁸Department of Laboratory Medicine, Hallym University College of Medicine, Hwaseong, Korea.

⁹Department of Laboratory Medicine, School of Medicine, Jeju National University, Jeju, Korea.

Correspondence: Eun-Jeong Yoon, Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 06273, Korea. (Tel) 82-2-2019-3783, (Fax) 82-2-2019-3784, ejyoon@yuhs.ac

Received 26 July 2018 Revised 11 September 2018 Accepted 12 September 2018

The Korean Society of Clinical Microbiology

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Escherichia coli and Klebsiella pneumoniae clinical isolates producing CTX-M extended-spectrum β-lactamases (ESBLs) were assessed for antimicrobial resistance phenotypes varied by group of enzymes.

Methods

A total of 1,338 blood isolates, including 959 E. coli and 379 K. pneumoniae, were studied. All the strains were collected between January and July 2017 from eight general hospitals in South Korea. The species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibilities were determined by disk diffusion methods and ESBL phenotypes by double-disk synergy tests using disks containing cefotaxime, ceftazidime, cefepime, aztreonam, and clavulanic acid (CA). The genes for β-lactamases were identified by PCR and sequencing.

Results

Of total microbes, 31.6% (303/959) E. coli and 24.0% (91/379) K. pneumoniae were resistant to cefotaxime and 28.1% (269/959) E. coli and 20.1% (76/379) K. pneumoniae were CTX-M-type ESBL producers. Among the detected CTX-M ESBLs, 58.0% (156/269) in E. coli and 86.8% (66/76) in K. pneumoniae belonged to group 1, 46.8% (126/269) in E. coli and 14.5% (11/76) in K. pneumoniae were group 9. Ten E. coli and one K. pneumoniae isolates co-produced both groups of CTX-M ESBL. The group 1 CTX-M producers had a higher level of resistance to cefotaxime, ceftazidime, cefepime, and aztreonam and exhibited stronger synergistic activities when combined with CA compared to group 9.

Conclusion

ESBL phenotypes differ by CTX-M ESBL group and phenotype testing with drugs including 4^th generation cephalosporins and monobactams is critical for screening CTX-M-producers with better sensitivity.

Keywords: CTX-M, Escherichia coli, Extended-spectrum β-lactamase, Klebsiella pneumoniae

INTRODUCTION

Extended-spectrum cephalosporins are one of the preferred choice for empirical therapy for infections by Gram-negative bacteria. However, dissemination of the extended-spectrum β-lactamases (ESBLs) conferring resistance to wide-range of β-lactams including the 3rd and 4th generation cephalosporins and monobactams, complicates antimicrobial therapy in the clinical settings [1].

Among over 10 families of ESBLs documented to date [2], the plasmid-mediated cefotaximase CTX-M is the most rapidly growing family with a significant clinical impact [3]. The CTX-M ESBL was first identified in Japan [4] and designated in Germany [5]. The growing family consists with heterogeneous groups of enzymes. The amino acid sequence alignment of CTX-M variants categorized the enzymes into five groups, i.e., 1, 2, 8, 9, and 25, sharing >94% amino acid identity within a group and ≤90% amino acid identity between groups [6]. The groups are also differed by the hydrolytic property. The first cefotaximase CTX-M-3, renamed from FEC-1, did not confer resistance to ceftazidime [4] and the following CTX-M variants also confer resistance to cefotaxime. The C7 β-amino thiazol-oxyimino-amide side chain protected the ceftazidime against majority of the CTX-M ESBLs [7]. However, some CTX-M ESBLs, mostly belonging to the group 1, had broader substrate-spectrum including ceftazidime resulting in varied resistance phenotypes. For instance, the dominant CTX-M ESBL CTX-M-15 a representative group 1 enzyme confers resistance to ceftazidime, while the other dominant CTX-M-14 a representative group 9 CTX-M ESBL does not.

Clavulanic acid (CA) is an effective inhibitor for ESBL including CTX-M, and the combination with amoxicillin and ticarcillin, which are both good substrates for the ESBLs, are used in the clinical settings for the infection treatment [8]. By using this activity, CA is widely used for synergy test to detect ESBL producers in the laboratory [9]. CA induces the AmpC production, which can mask the synergistic effects of cephalosporins with CA by inhibiting ESBLs when a bacterial host carries both an ESBL and an AmpC [10].

The differed activity and spectrum between the groups of CTX-M ESBLs are empirical facts, however the phenotypic differences are indeed controversial. Thereby, this study was designed to evaluate the differences in resistance phenotypes by the group of CTX-M ESBLs.

MATERIALS AND METHODS

1. Bacterial strains

Non-duplicate 1,338 blood isolates including 959 E. coli and 379 K. pneumoniae were collected between January and July 2017 from eight general hospitals participating in Korea GLobal Antimicrobial resistance Surveillance System. The species were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry using MALDI Biotyper® (Bruker Daltonik GmbH, Bremen, Germany) and/or 16S rDNA sequencing.

2. Antimicrobial susceptibility and double-disk synergy testing

Antimicrobial susceptibilities to cefotaxime, ceftazidime, cefepime, and aztreonam were tested by the disk diffusion method on Mueller-Hinton agar (Difco Laboratories, Detroit, MI, USA), following the Clinical and Laboratory Standards Institute guidelines [11]. Further ESBL-testing was conducted for the isolates non-susceptible to both cefotaxime and/or ceftazidime, following the EUCAST guidelines [12] with slight modification. Both E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used for quality control of each batch of experiment. Double-disk synergy test (DDST) was performed using disks (Oxoid Ltd., Basingstoke, UK) containing cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), aztreonam (30 µg), and CA (10 µg). The CA disk was freshly made using 6 mm-sized filter disks (Adventec Toyo Kaisha, Ltd., Tokyo, Japan). The inhibition zone diameters of each drug and enlarged zone of inhibition toward a CA disk were recorded and the synergy by CA (SC) was determined by dividing the enlarged zone diameter toward a CA disk with the original zone diameter.

3. DNA manipulation and PCR

Total DNA was extracted by using MG Cell genomic DNA extraction kit (MGmed Inc., Seoul, South Korea) and, by using the extracted total DNA, PCR and sequencing were carried out to detect the genes for carbapenemases, i.e., bla_KPC, bla_NDM, bla_IMP, bla_VIM, bla_GES, and bla_OXA-48 [13]; ESBLs, i.e. for bla_CTX-M-1-like, bla_CTX-M-9-like, and bla_{CTX-M-25-like}; and plasmid-mediated AmpCs, i.e., bla_CMY, bla_DHA, and bla_ACC [14 15].

4. Statistical analysis

Differences existed between groups were calculated by chi-square or t-test [16] and the correlation coefficient (r) for simple linear regression was calculated using Pearson's correlation [16] by the parametric method, both using SPSS statistics (version 23, IBM Corp., Armonk, NY, USA). All tests were two-tailed, and P value of <0.05 was determined to represent statistical significance.

RESULTS

1. Resistance to extended-spectrum cephalosporins and aztreonam in E. coli and K. pneumoniae clinical isoaltes

Of total, 31.6% (303/959) E. coli and 24.0% (91/379) K. pneumoniae were resistant to cefotaxime and 19.7% (n=189) E. coli and 19.5% (n=74) K. pneumoniae were resistant to cefepime (Table 1). Resistance rates for ceftazidime and aztreonam in E. coli were 11.0% (n=106) and 18.8% (n=180), respectively, and those in K. pneumoniae were 20.0% (n=76) and 21.4% (n=81), respectively. A total of 407 isolates including 315 (32.8%) E. coli and 92 (24.3%) K. pneumoniae met criteria for further ESBL-testing [12] and 282 E. coli and 88 K. pneumoniae exhibited ESBL phenotypes by DDST.

For the DDST-positive isolates, zone diameters of cefotaxime and cefepime were equivalent by bacterial species, presenting median values of 9 mm and 14 mm, respectively, while those of ceftazidime (17 mm vs. 12 mm) and aztreonam (12 mm vs. 6 mm) were greater in E. coli than in K. pneumoniae (Fig. 1A).

2. CTX-M-type ESBL-producing E. coli and K. pneumoniae

Of the DDST-positive isolates, 95.4% (269/282) E. coli and 86.4% (76/88) K. pneumoniae were identified as CTX-M ESBL producers by PCR (Table 2). In both enterobacterial species, the group 1 CTX-M ESBL was more identified than the group 9: 146:113 in E. coli and 65:10 in K. pneumoniae. Ten E. coli and one K. pneumoniae co-produced both groups of CTX-M ESBL. Co-carrying β-lactamase genes were identified in 6.7% (23/345) CTX-M ESBL producers, including 25.0% (19/76) K. pneumoniae and 1.5% (4/269) E. coli: bla_KPC-2 in one K. pneumoniae, blaSHV for ESBL in three K. pneumoniae, both blaSHV for ESBL and bla_DHA in two K. pneumoniae, and bla_DHA in 13 K. pneumoniae, and either of blaCMY or blaDHA in four E. coli. The CTX-M-negative isolates with ESBL-phenotype included one KPC-2-producing K. pneumoniae and three SHV-12-producing K. pneumoniae isolates and the rest 21 isolates were negative for the known ESBLs.

The 222 CTX-M group 1 ESBLs identified were mainly composed with CTX-M-15 (78.4%, n=174) and CTX-M-55 (14.0%, n=31) and CTX-M-28 (3.2%, n=7), CTX-M-3 (2.7%, n=6), CTX-M-1 (0.9%, n=2), CTX-M-79 (0.5%, n=1), and CTX-M-123 (0.5%, n=1) were followed. The CTX-M group 9 ESBLs mostly comprised CTX-M-14 (50.0%, 67/134), CTX-M-27 (24. 6%, n=33), CTX-M-17 (15.7%, n=21), and CTX-M-24 (6.0%, n=8) and, as minor, CTX-M-98 (1.5%, n=2), CTX-M-9 (0.7%, n=1), CTX-M-113 (0.7%, n=1), and CTX-M-129 (0.7%, n=1) were included in the group. Of the 174 CTX-M-15 producers, eleven isolates co-produced group 9 CTX-M ESBLs, such as CTX-M-14 (n=3), CTX-M-27 (n=3), CTX-M-17 (n=2), CTXM-24 (n=2), and CTX-M-98 (n=1).

3. Resistance phenotypes conferred by CTX-M ESBL

By the group of CTX-M enzymes, the zone diameters by the disks containing extended-spectrum cephalosporins or monobactams were smaller in group 1 than those in group 9 exhibiting the median values of 6 mm vs. 9 mm for cefotaxime, 14 mm vs. 22 mm for ceftazidime, 13 mm vs. 17 mm for cefepime, and 9 mm vs. 17 mm for aztreonam (Fig. 1C). The zone diameters of 11 Enterobacteriaceae producing both group 1 and group 9 CTX-M ESBLs were close to those in group 1 CTX-M ESBL producers rather than those in the group 9 producer: median values at 6 mm for cefotaxime, 12 mm for ceftazidime, 11 mm for cefepime, and 9 mm for aztreonam. Co-production of either SHV ESBL or AmpC in K. pneumoniae retained the inhibition zone diameters: median values of zone diameter at 9 mm for cefotaxime, 10 mm for ceftazidime, 14 mm for cefepime, and 6 mm for aztreonam in SHV ESBL co-producers and 9 mm for cefotaxime, 11 mm for ceftazidime, 14 mm for cefepime, and 6 mm for aztreonam in AmpC co-producers. The KPC-2 carbapenemase co-producer exhibited the reduced inhibition zones, 6 mm for cefotaxime, 9 mm for ceftazidime, 9 mm for cefepime, and 6 mm for aztreonam.

The synergistic effect with CA was evaluated using SC values (Fig. 1B–D). The cefotaxime SC was the greatest (median, 3.66) and followed by those of aztreonam (2.50), cefepime (2.14), and ceftazidime (1.63). The median SC for cefotaxime was greater in E. coli than in K. pneumoniae (3.78:3.33), while those of the other three drugs were less in E. coli, 1.56:2.00 for ceftazidime, 2.08:2.19 for cefepime, and 2.36:3.50 for aztreonam. By the group of CTX-M ESBLs, the SCs were always greater in the group 1 CTX-M ESBL compared to those in the group 9: 4.44:3.11 for cefotaxime, 1.83:1.30 for ceftazidime, 2.23:1.83 for cefepime, and 2.91:1.75 for aztreonam.

The group 9 CTX-M producers displayed stronger linear relationships between cefotaxime SC and either SCs of ceftazidime, cefepime, and aztreonam (Fig. 2) compared to the group 1 CTX-M ESBL producers having correlation coefficients (r): 0.5409:0.4988 for ceftazidime SC, 0.6945:0.4131 for cefepime SC, and 0.7012:0.5753 for aztreonam SC. Slopes were calculated by comparing the SCs for ceftazidime, cefepime, and aztreonam with the cefotaxime SC. The group 1 CTX-M ESBL producers exhibited steeper slopes than the group 9 producers for ceftazidime SC (0.3394:0.1929) and for aztreonam SC (0.6209:0.4989), while no slope difference between the two groups was observed for cefepime SC (0.2783:0.3091).

DISCUSSION

Continuing increase in CTX-M-type ESBL-producing Enterobacteriaceae, mainly led by the predominant CTX-M-15 of group 1 and CTX-M-14 of group 9 [17], triggers more usage of last options for the treatment resulting in vicious antimicrobial resistance. In South Korea, cefotaxime resistance reached at 38% for E. coli and at 35% for K. pneumoniae clinical isolates in 2015 [18] and it was associated with dominance of both CTX-M-15 and -14 since the mid-2010s [19]. The enterobacterial blood isolate collection of seven-month-period in 2017 presented cefotaxime resistance rates at 31.6% for E. coli and at 24.0% for K. pneumoniae, which were less than those in 2015, probably due to the differed specimens and surveillance systems. The dominant type of group 1 was CTX-M-15 (78.4%) and that of group 9 was CTX-M-14 (50.0%).

The rate of human carriages of CTX-M producers are increasing [17] and the dissemination is issuing not only among human beings but also for animals [20 21] and environments [22]. The resistance rates for cefotaxime (5.0%) and cefepime (1.4%) in E. coli from food animals [23] and in Enterobacteriaceae from edible vegetables (10.1% for cefotaxime and 1.1% for cefepime) [24] were comparably lower than those in clinical isolates. However, the prevalence of CTX-M ESBLs (78.6% of cefotaxime-resistant E. coli from food animals and 73.7% of cefotaxime-resistant Enterobacteriaceae from vegetables) and the dominant types of the enzyme (CTX-M-14 and CTX-M-15) corresponded to those of clinical isolates, highlighting the One-Health concepts.

Inhibition zone sizes of the tested drugs were always smaller for the group 1 CTX-M ESBL than those for the group 9 confirming that the group 1 enzyme confers higher level of resistance to the drugs than the group 9 [25]. As well, the SCs, indicating the hydrolytic activity inhibited by CA, were greater for the tested drugs in the group 1 CTX-M ESBL compared to those in the group 9 supporting that the group 1 enzymes hydrolyzed more the drugs than those belonging to the group 9. It is noteworthy that the ceftazidime SC in group 9 CTX-M ESBL is close to 1, agreeing the point that ceftazidime resistance, which has been used in practice as an indicator of ESBL producers, frequently leads to fail recognizing CTX-M-producers [6]. The higher prevalence of CTX-M ESBLs, which is the case of this study, may elevate the false-negative error rates [26]. Masked synergistic activity with CA by induced AmpC [10] was not observed in this study, doubtlessly because intrinsic AmpC producing enterobacterial species, including Enterobacter spp., Serratia marcescens, and Citrobacter freundii, were not included in the study.

The SCs for ceftazidime and aztreonam compared with that for cefotaxime pronounced well the differed range of substrates by group of CTX-M ESBL. The linear correlation of SCs for ceftazidime and aztreonam with that for cefotaxime in group 1 CTX-M ESBL presented steeper slope than those in group 9 CTX-M ESBL verifying the hydrolytic activity of the group 9 restricted to cefotaxime, while the group 1 has expanded range of the substrate. It correlates well to the biochemical properties demonstrated by kinetic constants [27 28].

Bacterial host itself seemed having little impact on the level of resistance to the tested drugs. CTX-M-producing K. pneumoniae presented higher levels of resistance to ceftazidime and aztreonam compared to E. coli. Since ceftazidime and aztreonam presented greater differences in the group 1 CTX-M compared to cefotaxime and cefepime, it is likely that the higher proportion of group 1 CTX-M in K. pneumoniae than in E. coli led the difference. The SCs differed by the bacterial species were spotted in aztreonam, which is also explained by the predominance of group 1 CTX-M ESBL in K. pneumoniae.

Finally, this study confirmed that the phenotype testing with drugs including 4^th generation cephalosporins and monobactams is critical for screening the CTX-M-producers with better sensitivity [16]. From 0.9% to 2.9% CTX-M ESBL producers had SCs at 1 for at least one drugs, which means no obvious synergistic effect with CA was obtained for cefotaxime (n=5), ceftazidime (n=10), cefepime (n=3), and aztreonam (n=5). Moreover, four strains (1.2%) had both cefotaxime SC and ceftazidime SC at 1, and the simplified DDST using only the two drugs could have missed the CTX-M ESBL producers. Estimating the group of CTX-M ESBL by DDST was unsound, however evaluating the composition of CTX-M ESBL producer population would be possible somehow by using linear correlation for SCs of cefotaxime SC-ceftazidime SC and cefotaxime SC-aztreonam SC.

This study had aware imperfections: i) the restricted bacterial species, ii) only isolations from blood specimens of patients in one country, and iii) lack of other groups of CTX-M ESBLs, such as the group 2 and the group 25. However, at least, the most clinically relevant panel of ESBL producers in the world, group 1 and 9 CTX-M-producing E. coli and K. pneumoniae, were covered and the necessity of complete set of testing drugs for DDST in the medical laboratory was emphasized. The not-yet-determined ESBLs in the strains exhibiting ESBL phenotypes are going to be further studied.

Figures and Tables

Fig. 1

Zone diameter of the four drugs (A and C) and the synergy with clavulanic acid (SC) (B and D) by bacterial species (A and B) and by groups of CTX-M (C and D). A total of 269 E. coli (ECO) and 76 K. pneumoniae (KPN) CTX-M producers (A and B) including 211 group 1 CTX-M producers and 123 group 9 CTX-M producers (C and D) were plotted. Boxplots present the 1 and 3 quartiles with whiskers showing either the maximum and minimum values. The thick horizontal line indicates median values, black lines for E. coli or group 1 CTX-M and gray lines for K. pneumoniae or group 9 CTX-M ESBL producers. The SC value was calculated by dividing the enlarged zone diameter near the CA disk by inhibition zone diameter of each drug. The statistical significance was calculated using Pearson's chi-square test [16] by using SPSS statistics (version 23, IBM Corp., Armonk, NY, USA) and the significance (P) was indicated by using asterisks: ^**P<0.01; ^*P<0.05.

Fig. 2

Correlation between synergy with clavulanic acid (SC) of cefotaxime and ceftazidime SC (A), cefepime SC (B), and aztreonam SC (C). The SC was calculated by dividing the enlarged zone diameter near the clavulanic acid disk by inhibition zone diameter of each drug. The correlation coefficient (r) was calculated using Pearson's correlation [16] by the parametric method using SPSS statistics and the significance (P) was determined by a two-tailed method using the correlation value and the sample size. Each dot indicates one strain, gray triangles and black dots indicate strains producing CTX-M group 1 and group 9, respectively.

Table 1

The 3^rd and 4^th generation cephalosporins and monobactam susceptibilities of Escherichia coli and Klebsiella pneumoniae blood isolates^*

^*The breakpoints were applied according to the Clinical and Laboratory Standards Institute guideline [11].

Abbreviations: R, resistant; I, intermediate; S, susceptible.

Table 2

CTX-M-type ESBL-producing Escherichia coli and Klebsiella pneumoniae

ACKNOWLEDGMENTS

This research was supported by a fund (2017E4400100#) from the Research Program of Korea Centers for Disease Control and Prevention.

References

1. Pitout JD. Extended-spectrum beta-lactamaseproducing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis. 2008; 8:159–166.

2. Paterson DL. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev. 2005; 18:657–686.

3. D'Andrea MM, Arena F, Pallecchi L, Rossolini GM. CTX-M-type β-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol. 2013; 303:305–317.

4. Matsumoto Y, Ikeda F, Kamimura T, Yokota Y, Mine Y. Novel plasmid-mediated beta-lactamase from Escherichia coli that inactivates oxyimino-cephalosporins. Antimicrob Agents Chemother. 1988; 32:1243–1246.

5. Bauernfeind A, Grimm H, Schweighart S. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection. 1990; 18:294–298.

6. Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004; 48:1–14.

7. Delmas J, Leyssene D, Dubois D, Birck C, Vazeille E, Robin F, et al. Structural insights into substrate recognition and product expulsion in CTX-M enzymes. J Mol Biol. 2010; 400:108–120.

8. Livermore DM. Determinants of the activity of beta-lactamase inhibitor combinations. J Antimicrob Chemother. 1993; 31:Suppl A. 9–21.

9. Livermore DM, Hope R, Mushtaq S, Warner M. Orthodox and unorthodox clavulanate combinations against extended-spectrum beta-lactamase producers. Clin Microbiol Infect. 2008; 14:Suppl 1. 189–193.

10. Livermore DM, Akova M, Wu PJ, Yang YJ. Clavulanate and beta-lactamase induction. J Antimicrob Chemother. 1989; 24:Suppl B. 23–33.

11. CLSI. CLSI document M100-S25. Performance standards for antimicrobial susceptibility testing: twenty-fifth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute;2015.

12. European Committee on Antimicrobial Susceptibility Testing. EUCAST Guideline for the Detection of Resistance Mechanisms and Specific Resistances of Clinical and/or Epidemiological Importance. Version 2.0. Vasel: EUCAST;2017.

13. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011; 70:119–123.

14. Ryoo NH, Kim EC, Hong SG, Park YJ, Lee K, Bae IK, et al. Dissemination of SHV-12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea. J Antimicrob Chemother. 2005; 56:698–702.

15. Pérez-Pérez FJ. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002; 40:2153–2162.

16. Campbell MJ. Statistics at Square Two. London: BMJ Books;2006.

17. Bevan ER, Jones AM, Hawkey PM. Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype. J Antimicrob Chemother. 2017; 72:2145–2155.

18. Kim D, Ahn JY, Lee CH, Jang SJ, Lee H, Yong D, et al. Increasing resistance to extended-spectrum cephalosporins, fluoroquinolone, and carbapenem in Gram-negative bacilli and the emergence of carbapenem non-susceptibility in Klebsiella pneumoniae: analysis of Korean Antimicrobial Resistance Monitoring System (KARMS) data from 2013 to 2015. Ann Lab Med. 2017; 37:231–239.

19. Park SH, Byun JH, Choi SM, Lee DG, Kim SH, Kwon JC, et al. Molecular epidemiology of extended-spectrum β-lactamaseproducing Escherichia coli in the community and hospital in Korea: emergence of ST131 producing CTX-M-15. BMC Infect Dis. 2012; 12:149.

20. Stedt J, Bonnedahl J, Hernandez J, Waldenström J, McMahon BJ, Tolf C, et al. Carriage of CTX-M type extended spectrum β-lactamases (ESBLs) in gulls across Europe. Acta Vet Scand. 2015; 57:74.

21. Timofte D, Maciuca IE, Williams NJ, Wattret A, Schmidt V. Veterinary hospital dissemination of CTX-M-15 extended-spectrum beta-lactamase-producing Escherichia coli ST410 in the United Kingdom. Microb Drug Resist. 2016; 22:609–615.

22. Ojer-Usoz E, González D, Vitas AI. Clonal diversity of ESBL-producing Escherichia coli isolated from environmental, human and food samples. Int J Environ Res Public Health. 2017; 14:E676.

23. Shin SW, Jung M, Won HG, Belaynehe KM, Yoon IJ, Yoo HS. Characteristics of transmissible CTX-M- and CMY-Type β-lactamase-producing Escherichia coli isolates collected from pig and chicken farms in South Korea. J Microbiol Biotechnol. 2017; 27:1716–1723.

24. Kim HS, Chon JW, Kim YJ, Kim DH, Kim MS, Seo KH. Prevalence and characterization of extended-spectrum-β-lactamaseproducing Escherichia coli and Klebsiella pneumoniae in readyto-eat vegetables. Int J Food Microbiol. 2015; 207:83–86.

25. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966; 45:493–496.

26. Maurer FP, Courvalin P, Böttger EC, Hombach M. Integrating forecast probabilities in antibiograms: a way to guide antimicrobial prescriptions more reliably? J Clin Microbiol. 2014; 52:3674–3684.

27. Bonnet R, Recule C, Baraduc R, Chanal C, Sirot D, De Champs C, et al. Effect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother. 2003; 52:29–35.

28. Poirel L, Gniadkowski M, Nordmann P. Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum beta-lactamase CTX-M-15 and of its structurally related beta-lactamase CTX-M-3. J Antimicrob Chemother. 2002; 50:1031–1034.