Clonal Distribution of Clindamycin-Resistant Erythromycin-Susceptible (CRES) Streptococcus agalactiae in Korea Based on Whole Genome Sequences
2020; 40(5): 370-381
Ann Lab Med 2021; 41(3): 285-292
Published online May 1, 2021 https://doi.org/10.3343/alm.2021.41.3.285
Copyright © Korean Society for Laboratory Medicine.
1Department of Laboratory Medicine, National Health Insurance Service Ilsan Hospital, Goyang, Korea; 2Department of Laboratory Medicine, National Police Hospital, Seoul, Korea; 3Research Institute of Bacterial Resistance, 4Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea; 5Seoul Clinical Laboratories Academy, Yongin, Korea
Correspondence to: Kyungwon Lee, M.D.
Department of Laboratory Medicine and Research Institute of Bacterial Resistance Severance Hospital, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Background: One health is a flexible concept with many facets, including the environment, community, and the nosocomial super-bacteria resistance network. We investigated the molecular prevalence of extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) in workers, livestock, and the farm environment in Korea.
Methods: ESBL-EC isolates were obtained from samples from 19 swine farms, 35 retail stores, seven slaughterhouses, and 45 related workers throughout Korea from August 2017 to July 2018, using ChromID ESBL (BioMérieux, Marcy l’Etoile, France) agar and enrichment broth. The presence of ESBL and mobilized colistin resistance (mcr) genes and antimicrobial resistance were determined. Clonality was evaluated with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).
Results: In total, 232 ESBL-EC isolates were obtained from 1,614 non-duplicated samples (14.4% positive rate). The ESBL-EC isolates showed regional and source-related differences. blaCTX-M-55 (N=100), blaCTX-M-14 (N=65), blaCTX-M-15 (N=33), and blaCTX-M-65 (N=23) were common ESBL types. The ESBL-EC isolates showed high resistance rates for various antimicrobial classes; however, all isolates were susceptible to carbapenem. One swine-originating colistin-resistant isolate did not carry any known mcr gene. PFGE was successful for 197 of the 232 isolates, and most PFGE types were heterogeneous, except for some dominant PFGE types (O, R, T, U, and V). MLST of 88 isolates was performed for representative PFGE types; however, no dominant sequence type was observed.
Conclusions: The proportion of ESBL-EC in swine industry-related samples was significant, and the isolates harbored common clinical ESBL gene types. These molecular epidemiologic data could provide important evidence for antimicrobial-resistance control through a one health approach.
Keywords: Extended-spectrum-β-lactamase, Escherichia coli, Antimicrobial resistance, One health, Swine, Mobilized colistin resistance
Antimicrobial resistance (AMR) is a major challenge that requires multi-sectional efforts, such as those relating to human and veterinary medicine, agricultural sciences, epidemiology, and economics . One health is a broad and flexible concept with various facets, such as the environment, community, and nosocomial super-bacteria AMR network . Bacteria from livestock may be carriers of clinically relevant resistance genes for veterinary and human antimicrobials. The exposure to multidrug-resistant bacteria during farming, slaughtering, and distribution may cause transmission to occupationally exposed workers. Therefore, elucidating the current epidemiology and transmission mode of antimicrobial resistant bacteria among humans, animals, and environments is very important to establish an effective strategy for the controlling AMR.
Although a recent domestic study reported the isolation of CTX-M-55 or CTX-M-65-producing
A study was launched in 2017 with support from the Korea Centers for Disease Control and Prevention wherein we evaluated the prevalence of third generation-cephalosporinand colistin-resistant
A total of 1,614 non-duplicated samples were prospectively collected from 19 swine farms, 35 retail stores, and seven slaughterhouses in five administrative districts throughout Korea from August 2017 to July 2018. The samples included swabs from the following: nose, skin (groin region), rectum, and stool of swine; a pigsty fence, floor, and ventilation fan and soil surrounding a pigsty; pooled human stool in a toilet; knives, cutting boards, and slaughterhouse floor. Additionally, 0.5–1 L of livestock wastewater was collected; 10 g of pork meat was sampled in the slaughterhouses. Pork for sale, knives, cutting boards, and showcases in retail stores were also sampled using a swab. We also obtained swab samples from the nose, groin, axillary, antecubital fossa, inter-finger spaces (both hands), and stool of 45 workers after obtaining written informed consent.
The feces of livestock and workers were collected in stool boxes, and the soil was collected in sterilized bags. Swabbed samples were collected with sterilized cotton swabs and put into a transport medium (Copan diagnostics, Murrieta, CA, USA). Analysis was performed at Research Institute of Bacterial Resistance, Yonsei University College of Medicine. Samples were stored at 4°C before analysis and inoculated in culture media within 24-h of sampling. This study was approved by the Institutional Review Board of National Health Insurance Service Ilsan Hospital, Goyang, Korea (NHIMC 2017-07-041).
A total of 25 g of soil surrounding a pigsty was mixed with 225 mL of buffered peptone water (BD Biosciences, San Jose, CA, USA) and incubated at 36 ± 1°C for 18 hours. After the sewage was filtered with a 0.2 μm filter, the content on the surface of the filter was inoculated into Mueller-Hinton (MH) broth and incubated at 36 ± 1°C for 18–24 hours. Other environmental samples were inoculated into 10 mL of MH broth and incubated at 36 ± 1°C for 18–24 hours. In addition, 10 g of pork meat was placed in 10 mL of MH broth and incubated at 36 ± 1°C for 18– 24 hours. After enrichment, 10–100 μL of the liquid culture was used to inoculate ChromID ESBL (BioMérieux, Marcy l’Etoile, France) agar to screen for ESBL-EC. Other swab samples from swine and workers were directly inoculated onto ChromID ESBL agar and incubated at 36 ± 1°C for 18–24 hours.
The disk diffusion method and broth microdilution (BMD) were performed for testing AMR. Briefly, fresh grown
All isolates resistant to cefotaxime or ceftazidime were analyzed by PCR and sequencing for ESBL genes (
Pulsed-field gel electrophoresis (PFGE) was performed as described previously , and the patterns were analyzed using InfoQuest FP software (Bio-Rad) with stored isolates (-70°C in skim milk). The dendrogram was generated based on the unweighted pair group method, with an arithmetic average from Dice’s coefficient with 1% band position tolerance and 0.5% optimization settings. The sequence types (STs) were determined by multilocus sequence typing (MLST) of representative isolates .
For categorical variables (source, province, ESBL type), we used count and percentages of the group from which they were derived. AMR rate was calculated as the percentage of isolates that showed resistance to certain antimicrobials. The relative ratio of ESBL genotypes was defined as the percentage of the total. The prevalence or positive rate of ESBL-EC isolates was derived by comparing the number of samples with ESBL-EC with the total number of samples studied and was expressed as a percentage. Multiple samples from the same swine were calculated as one sample, and the swine was defined to be ESBLpositive even if only one sample from it was positive (nose, skin at groin region, rectum, and stool). Chi-square test was used for the comparative analysis of categorical variables using IBM SPSS Statistics for Windows software version 23.0 (IBM Corp., Armonk, NY, USA). Statistical significance of the results was defined at
The overall ESBL-EC positive rate was 14.4% (232/1,614), with the rates being 8.9% in pork, 18.4% in swine, 9.0% in workers, and 20.6% in the environment. ESBL-EC positive rate varied according to sample source and geographic location (Table 1). The ESBL-EC positive rate of pork samples from Seoul/Gyeonggi was significantly higher than that of samples from Chungcheong, Gyeongsang, and Jeolla (
The ESBL-EC isolates showed AMR to gentamicin (41.1%), amikacin (51.1%), ciprofloxacin (32.5%), and trimethoprimsulfamethoxazole (57.6%), in addition to β-lactams. All isolates were susceptible to imipenem, meropenem, or ertapenem. The AMR rates differed according to sample source. Isolates from swine showed higher resistance rates for β-lactams, amikacin, gentamicin, and ciprofloxacin than isolates from pork samples (Fig. 1A).
All ESBL-EC isolates contained a single CTX-M type ESBL gene. Common genotypes included
PFGE was successful in 197 of the 232 isolates, and most of PFGE types were heterogeneous (total 105 PFGE types) with some dominant PFGE types (O, R, T, U, and V), which were detected with a cut-off level of 5% (if more than 10 isolates had the same PFGE type). Thirty-five isolates failed to grow in repeated subculture of stored isolates (-70°C in skim milk) or could not be analyzed by repeated PFGE.
MLST of 88 isolates was performed with representative PFGE types (Fig. 2A and 2B), and they showed heterogeneous STs, with ST101 (N = 8), ST457 (N = 7), ST10 (N = 5), ST117 (N = 3), ST206 (N = 3), ST410 (N = 3), and ST744 (N = 3). No dominant ST was detected by MLST with representative PFGE types (Fig. 2C). ST131 was not detected in this study.
The isolation of ESBL-EC with
The community prevalence of ESBL increased gradually in the mid-2000s, owing to the wide spread of ST131 with
This study also included the monitoring of carbapenemaseproducing and colistin-resistant
The extensive use of antimicrobials has resulted in the generation of antimicrobial concentration gradients in humans and livestock, thus accelerating the emergence and spread of antimicrobial-resistant bacteria among humans and animals [10, 11]. Environmental contamination and livestock production systems have been implicated as likely reservoirs of AMR and promote AMR transmission to humans via the colonization of commensal bacteria, such as
In summary, the current nationwide molecular epidemiology of major antimicrobial-resistant organisms was characterized in swine-related industries. The proportion of ESBL-EC in swine industry-related samples was high, and a number of dominant PFGE types and clinically common ESBL genes were observed in these samples. The spread of resistant bacteria to humans and animals via foodstuffs needs to be decreased, and the concentrations of antimicrobials and antimicrobial-resistant bacteria introduced into the environment need to be minimized. In this regard, our epidemiological data could be useful for developing evidence-based policies for the control of antimicrobial-resistant bacteria in livestock to improve animal and human health in line with the “one health” concept.
The limitation of this study is that evidences of the spread of ESBL-EC among workers, livestock, the environment, and slaughterhouses were not documented and the prevalence of ESBLEC in livestock was not evaluated in a longitudinal study. Further studies are needed on samples from swine at various breeding stages.
I thank So Ra Yoon, PhD for the statistics from research Institute of national health insurance Ilsan hospital.
Kim YA and Lee K conceived the experiments. Seo YH and Park GE conducted the experiments. Kim H and Lee H analyzed the results. Kim YA and Kim H wrote the manuscript. All authors reviewed the manuscript.
No potential conflicts of interest relevant to this paper were reported.
This study was supported by research funds from the Korea Centers for Disease Control and Prevention (Project No. 2017NER54060 and 2020ER540500).