Antimicrobial Susceptibility Patterns and Genetic Diversity of Campylobacter spp. Isolates from Patients with Diarrhea in South Korea

This study aimed to characterize the latest antimicrobial resistance patterns and genetic diversity of Campylobacter spp. isolated from patients with acute diarrhea in Korea. In total, 371 clinical isolates (361 Campylobacter jejuni and 10 Campylobacter coli) were collected from patients with diarrhea in 106 medical institutions of six provinces during the seasonal peak (April–September 2022) in South Korea. We then assessed their antimicrobial susceptibility to eight antimicrobial agents and performed multilocus sequence typing (MLST). This study investigated the antimicrobial resistance (AMR) profiles to tetracycline (32.3%), nalidixic acid (64.9%), and ciprofloxacin (83.3%), confirming high levels of the latter even after its Korean ban in 2010. However, tetracycline resistance displayed a decreasing trend. Alternatively, significantly lower resistance rates to clindamycin (0.8%), azithromycin (0.53%), erythromycin (0.53%), and gentamicin (0.53%) as well as absolute susceptibility to florfenicol (0%) were observed. Four C. jejuni and three C. coli isolates (7/371, 1.88%) were classified as multidrug-resistant (MDR) to at least three antimicrobial classes. MLST identified a high genetic diversity with 21 clonal complexes (CCs) and sixty-six sequence types (STs), including eight novel STs. The high CC frequency of C. jejuni comprised CC21 (37.7%), CC22 (13.8%), and CC206 (9.4%), while C. coli was predominated by CC828 (90%). The high CC21 and CC828 strain prevalence in this study was consistent with their worldwide distribution. This study highlights that quinolone- and tetracycline-resistant Campylobacter circulate in Korea with diverse genotypes, providing important information that could contribute to controlling and preventing increasing antimicrobial resistance in patients.


Introduction
Campylobacter species are a zoonotic pathogen causing foodborne diarrheal diseases worldwide, with Campylobacter jejuni and Campylobacter coli being the most important pathogenic species, responsible for approximately 90% of human infections [1,2].In 2021, the European Center for Disease Prevention and Control reported approximately 129,960 human campylobacteriosis cases [3].The main Campylobacter transmission route to humans is contaminated food handling, preparation, and consumption, especially that of poultry origin [4].Although contamination is the primary cause of retail raw poultry, it can be transmitted to humans through cross-contamination on cutting boards, knives, cucumbers, and hands while handling contaminated poultry at home or in restaurants [5].
Campylobacter identification from patient stool is difficult because of the specific media and culture conditions.Isolation and cultivation of Campylobacter requires difficult culture conditions because of microaerophilic conditions and cultivation in a specific medium, and maintenance culture of the pathogen is difficult compared with other Gram-negative bacteria (pathogenic E. coli).In the case of Campylobacter jejuni NCTC11168, the doubling time in rich media (blood-based agar or MH agar) is 112 min under microaerobic conditions, which is slower than in E. coli (~20 min, aerobic condition).The most problematic aspect is that the positive culture rate of Campylobacter spp.decreases in inverse proportion to the time the sample is exposed to oxygen.Placing specimens in the transport medium is thought to prolong organism survival, but the length of successful storage is poorly defined [6].
Thus, fewer studies are available on clinical isolates than on poultry and food isolates from Korea.Although Campylobacter is reportedly an important food poisoning causative agent along with Escherichia coli and Salmonella, its importance has been evaluated less due to its low isolation rate in Korea [7,8].Most campylobacteriosis cases are usually self-limiting, without any requirement for hospitalization and antimicrobial treatment.However, therapy is required in children with fever and increasing bloody diarrhea, as well as in elderly or immunocompromised patients with severe and prolonged systemic disorders [9,10].In severe cases, macrolides (erythromycin) and fluoroquinolones (ciprofloxacin) are considered first-and second-line antimicrobials, respectively, for human Campylobacter infection treatment [11].
Antimicrobial overuse both in animals and in humans has led to increased antibioticresistant Campylobacter populations [12].Therefore, C. jejuni resistance monitoring is highly relevant to public health.In particular, the widespread and indiscriminate antibiotic supplementation of poultry feed increases resistant bacterial emergence, especially that of multidrug-resistant bacteria, causing serious problems in human treatment and prevention [13].In the European Union, antibiotic use was banned, except for disease treatment, in 2006 [14], starting with Sweden even earlier in 1986 [15].In Korea, antibiotic supplementation of formulated feed was banned in July 2010 [16].Since changes in antibiotic resistance could appear in various ways depending on the antibiotic type or investigation period and region, assessing antibiotic resistance distribution patterns is pivotal for efficient antibiotic use [16].Therefore, developed countries (e.g., Denmark, Japan, and Canada) are continuously investigating antibiotic resistance against pathogenic bacteria at the national level [17][18][19].
To date, multiple studies have addressed Campylobacter antibiotic resistance distribution from domestic poultry-derived isolates [20,21].However, studies on clinical antibiotic resistance distribution and Campylobacter clonal characteristics in patients with acute diarrhea after the ban of antibiotic supplementation of formulated feed remain scarce.
Thus, this study investigated the antibiotic resistance distribution in clinical patients and performed Campylobacter spp.epidemic clonal analysis in patients with acute diarrhea, systematically collecting samples from more than 100 medical institutions in six regions nationwide in South Korea.

Bacterial Isolate Collection
In total, 352 and 19 Campylobacter spp.isolates from fecal samples and rectal swabs, respectively, were collected from patients with diarrhea and campylobacteriosis from 106 medical institutions in six provinces (Seoul, Gyeonggi, Chungcheong [Chungbuk and Chungnam], Gyeongsang [Gyeongbuk and Gyeongnam], Jeolla [Jeonbuk and Jeonnam], and Gangwon/Jeju) in South Korea between April and September 2022.Of the 33,511 PCRtested cases for the causative agent of acute diarrhea, 2492 Campylobacter spp.-positive specimens (positive rate: 7.4%), 361 and 10 strains being identified as C. jejuni and C. coli, respectively, were confirmed (371 isolates/2492 positive cases, isolation rate 14.9%).In order to collect unbiased strains nationwide, the minimum collected strain number of 15 or more from each of the six regions was set (Seoul, Gyeonggi, Jeolla, Chungcheong, Gyeongsang, Gangwon, and Jeju) (Table 1).Thus, strains nationwide based on isolation from single patients were collected.

Identification and Growth Conditions
The strains were cultured on blood-based agar with 5% defibrinated sheep blood and incubated at 42 • C for 48 h under microaerobic conditions (85% nitrogen, 10% carbon dioxide, and 5% oxygen) [22].Then, the colonies were subcultured on the antibiotic supplement (cefoperazone 16 mg and amphotericin 5 mg; Oxoid, Basingstoke, UK)-containing modified charcoal cefoperazone desoxycholate agar (mCCDA, Oxoid) and incubated at 42 • C for 48 h under microaerophilic conditions [23].We stored the Campylobacter isolates as frozen stocks at −80 • C in Tryptic Soy Broth containing 20% glycerol supplemented with 5% defibrinated sheep blood.All presumptive Campylobacter spp.isolates at the species level were identified (i.e., C. jejuni and C. coli) using the Matrix-Assisted Laser Desorption/Ionization biotyper (Bruker) and VITEK 2 (Biomerieux) [24].The genomic DNA were extracted using the MagNA Pure 96 System (Roche Diagnostics, Pleasanton, CA, USA), fully automated, for high throughput nucleic acid purification according to the manufacturer's instructions.The eluted DNA was stored at −20 • C for further MLST screening.

Clonal Population
All C. jejuni and C. coli isolates were characterized by multilocus sequence typing (MLST) based on primers for seven housekeeping genes of each isolate, including aspA, glnA, gltA, glyA, pgm, tkt, and uncA [25].Allele numbers for each housekeeping gene, sequence types (STs), and clonal complexes (CCs) were assigned by submitting the DNA sequences to the Campylobacter MLST database (https://pubmlst.org/campylobacter/,accessed on 31 October 2023) at the University of Oxford.The PubMLST algorithm was used to assign CCs that were conditional on having at least five or more identical alleles regarding the founder allelic profile.Therefore, Phyloviz 2.0 software was used to analyze the genetic relationships between isolates constructed from MLST profiles [26].

Statistical Analysis
Statistical analysis was performed using GraphPad Software (version 8.3.0;GraphPad Software, San Diego, CA, USA).Fisher's exact test was used to compare differences in the ratios of infection between patient gender and in the ratios of resistance between the two Campylobacter species.
In this study, our MLST identified eight and two STs for C. jejuni and C. coli, respectively.Our MLST data-based minimum spanning trees for the C. jejuni and C. coli isolates cluster analysis indicates that our human campylobacteriosis-related Campylobacter isolates belong to diverse clones (Figure 2).

Discussion
This clinical-focused study aimed to compensate for the lack of campylobacteriosisrelated research in domestic clinical patients.Therefore, isolates from patients with acute diarrhea treated at 106 medical institutions in six regions nationwide in South Korea were systematically collected and identified, and their antimicrobial resistance patterns and epidemic clones were investigated.Laboratory monitoring of acute diarrhea in Korea confirmed that Campylobacter is the third most common pathogen, following pathogenic Escherichia coli and Salmonella [27].However, Campylobacter pathogenicity is evaluated as less important because of its difficult isolation and culture due to complex culture conditions and the low isolation rate [28].
The PCR-positive rate was 7.4% among the total requests (2492/33,511), the pathogen isolation rate from the PCR-positive patients was 14.9% (371/2492), and the pathogen isolation rate from the total requests was 1.1% (371/33,511) (Figure 1).This result was similar to other domestic data of Ryoo [8], demonstrating that the proportion of Campylobacter spp.among domestic acute gastroenteritis cases increased steadily between 2007 and 2019, accounting for 0.3-1.5%.Campylobacter spp. is reportedly the most common causative agent among patients with gastroenteritis [8].However, different from European or North American patterns, it is the third most common causative agent in Korea, the reasons for which are unclear.Infections occurred most frequently in children (of 1-9 years) to young adults (of 15-29 years) (Supplementary Figure S1), indicating a pattern similar to the results of studies in Denmark and others [29,30].
In this study, the Gangwon/Jeju region had the highest PCR positivity rate (10.2%) and Campylobacter spp.isolation rate (27%), and the Seoul region had the lowest PCR positivity rate (4.0%) but a high isolation rate (26.5%).It is not believed that poultry consumption is particularly high in the Gangwon/Jeju region, nor that sanitary conditions are particularly poor.However, in the Gangwon/Jeju region, the hospitals where samples were collected account for a larger proportion of samples collected through tertiary hospitals than the other regions, resulting in more severe symptoms than patients who visited primary and secondary hospitals.This is because PCR and pathogen isolation were performed on samples that were present.In the case of Seoul, it is located in the same region as the laboratory and was conducted within 12 h from sample collection to the start of the experiment; therefore, the samples were exposed to oxygen for a shorter time than in other regions.Therefore, the isolation rate of pathogens is high.According to a report by Cho et al., the incidence rate of Campylobacter spp. was reported to be 1.23 times higher in rural regions than in urban regions [31].By contrast, rates of 2.46% of Campylobacter spp.isolated in city regions and 0.58% in rural regions have been reported [27].However, herein, rather than these regional characteristics, differences such as the severity of patient symptoms or delivery time to the laboratory after specimen collection are considered as the most important factors.
Campylobacteriosis is usually self-limiting and rarely requires antibiotic treatment, except for severe or prolonged cases [1,2].Fluoroquinolones are broad-spectrum antimicrobials for treating a multitude of infections, including undiagnosed diarrhea cases, as priority therapy in Korea [32].Therefore, increased ciprofloxacin resistance is considered a major public health concern.Such high antibiotic resistance might be closely related to antibiotic consumption on commercial farms.It was banned in 2010 because of the indiscriminate misuse of antibiotics, problems with antibiotic residues, and an increase in antibiotic multidrug resistant strains, and the use of commercial laying hens was banned in July 2017 [33].However, the fact that consumption is not decreasing is because of customary use in farms and anxiety about a decrease in income due to non-use.The annual antibiotic consumption in Korea is 3 and 13.6 times higher than that in the United States and the European Union, respectively [13].
In Korea, antibiotic use in commercial animals was banned in 2010 in an effort to suppress the increase in fluoroquinolone resistance [23].Nevertheless, from 2009 to 2013, the nalidixic acid resistance rate was 100% for human isolates and 95.2% for animal isolates, and the ciprofloxacin resistance rate was 96.8% for human isolates and 92.9% for animal isolates [34].The fluoroquinolone resistance of C. jejuni was ciprofloxacin (89.4 to 100%) and nalidixic acid (90.9 to 100%), and there was no decreasing trend of antibiotic resistance [35,36].Even our study result could not confirm a significant antibiotic resistance reduction against ciprofloxacin (83.3%) and nalidixic acid (64.9%), and maintenance of a high-level antibiotic resistance was described (Table 2).C. jejuni isolates from poultry meat yielded similar results.In the case of ciprofloxacin, chicken and duck meat-derived samples displayed 83.3-85.6% and 85.6-87.8%resistance, respectively.For nalidixic acid, chicken and duck meat-derived samples exhibited 92.9-97.8% and 92.7-97.8%resistance, respectively.So, the impact of banning antibiotics appears to be insignificant [21,37].These results could be obtained potentially because mass fluoroquinolone use is still allowed for poultry in Korea.In particular, enrofloxacin displays the highest proportion as a poultry treatment [38].Enrofloxacin is well absorbed when orally administered to poultry, and compared to first-generation nalidixic acid, it has excellent bioavailability in animals and has a low plasma protein binding rate, making it advantageous in terms of pharmacokinetics [39].Both enrofloxacin and ciprofloxacin are second-generation fluoroquinolones antibiotics with similar structures and antimicrobial activities.Enrofloxacin is reportedly converted into ciprofloxacin in certain animals (e.g., pigs and cattle) through metabolism [40].Moreover, enrofloxacin use in poultry production increases the fluoroquinolone resistance of C. jejuni, leading to unsuccessful treatment of infected humans through food [41,42].In addition, as a result of administering 50 ppm of enrofloxacin to chicken, fluoroquinolone resistance rapidly increased and gyrA mutation was confirmed in multiple individuals [43].According to the domestic nationwide antibiotic usage and resistance monitoring data, among quinolone drugs, ciprofloxacin was not sold between 2010 and 2022, but enrofloxacin was the most consumed, i.e., 27,138-58,003 kg per year.However, due to the ban on enrofloxacin use in poultry from 31 October 2021, the sales decreased to approximately 27 tons in 2022, compared to 58 tons in 2021 [44].Due to the same ban, the resistance correlation to the fluoroquinolone ciprofloxacin should be continuously analyzed and the resistance correlation between poultry and patient isolates should also be monitored.
Tetracycline has been a widely used antibiotic globally due to its low cost and high convenience.The resistance rate, which was expected to decrease due to the ban on tetracycline use for growth promotion in the EU in 2006, increased from less than 30% in 2001 to 47% in 2013 in Salmonella Typhimurium isolated from pigs, potentially due to the increased therapeutic use [45].However, our results demonstrated 32.3% of tetracycline resistance in human patients (Table 2).According to another domestic report, the tetracycline resistance rate between 2009 and 2013 was 74.6% for human isolates and 81.0% for animal isolates [35], and the tetracycline resistance rate among human isolates in Gyeonggi-do, Korea, in 2010 was 69% [46].Additionally, according to a domestic report on poultry, the tetracycline resistance rate of poultry isolates in 2013 was 52% in chickens and 97% in ducks [38].Poultry isolates from 2016 to 2017 showed a resistance rate of 27.8% in chickens and a resistance rate of 57.8% in ducks [21], suggesting that tetracycline resistance in humans and poultry is decreasing in Korea.This could be potentially due to the lack of tetracycline sales for livestock use between 2011 and 2020 and the 55% sales volume reduction of oxytetracycline, a tetracycline family member, during the same period [30].As a result, consistent with the data from One Health antimicrobial-resistant organisms, the tetracycline resistance of C. jejuni isolated from poultry decreased rapidly from 76.7% in 2012 to 37.5% in 2021 [47].
The most prevalent clonal complexes (CCs) (CC21, CC22, and CC206) accounted for 61% of the isolates and the most prevalent STs were ST4253, ST22, and ST572, with 66, 50, and 28 isolates, respectively.The most dominant CC in all human isolates was CC21, comprising 12 STs, of which the most common was ST4253 (66/136, 48.5%), also reported from a human clinical C. jejuni population in Japan [48].Furthermore, CCs such as CC22, CC45, CC48, CC354, and CC443 were frequently reported from poultry meat isolates in Korea [20,49].This study also identified 50, 22, 11, 17, and 4 isolates of the aforementioned complexes, respectively (Table 3).In Korea, ST21 of CC21 appeared to be dominant in each region in the human patient-derived samples [49].Our study demonstrated the dominance of ST4253 of CC21 in patient-derived samples.
Between 2012-2017, as a result of C. jejuni genotype analysis for domestic patients with diarrhea in the acute diarrhea laboratory surveillance project, the main CCs were CC21 (89/194, 45.9%), CC443 (17/194, 8.8%), and CC22 (8/194, 4.1%).CC206 was analyzed in one sample out of 194 (1/194, 0.5%) [50].This study analyzed CC21 (136/371, 37.7%), CC22 (50/371, 13.8%), and CC206 (34/371, 9.4%) in order (Table 4).Therefore, CC21 prevalence was still predominant in patients with diarrhea for 10 years.In particular, the increase in CC22 and CC206 indicates changes in the domestic epidemic.CC22 has been reported in domestic chicken [21].However, CC206 is a clone found only in human isolates that have not been previously reported in poultry.Therefore, the gradually emerging CC206 clone in Korea would require clinical monitoring in the future.Previous studies have also reported that CC21 is the most commonly isolated C. jejuni CC in China, including human and chicken isolates [11,51].Moreover, CC21 has also been isolated and reported in ducks in Korea [20], thereby providing evidence that poultry is a C. jejuni reservoir and a main transmission route for human infection.Therefore, C. jejuni MLST profiles in Asian patients with diarrhea revealed that CC21 is the most commonly isolated in China [11,51], Japan [48], and South Korea.Concerning the C. coli strains, our MLST analysis revealed that 90% (9/10) belonged to CC828, while 10% (1/10) represented a novel and previously unreported CC in the MLST database (Table 3).The CC828 complex, comprising eight STs (ST830, ST854, ST860, ST872, ST9201, ST9867, ST12600, and ST12613) yielded the highest diversity.The high prevalence of the CC828 strains in this study was consistent with its worldwide distribution [52].
In Korea, several studies have reported Campylobacter spp.prevalence in poultry [20,21,23,49].However, clinical Campylobacter characterization remains limited.In this study, we described the characteristics of antimicrobial susceptibility profiles and molecular typing of clinical Campylobacter isolates from patients with acute diarrhea in South Korea.Furthermore, we underpinned the high genetic diversity of Campylobacter isolates circulating in Korea.Systematic Campylobacter surveillance and molecular characterization could provide important information to prevent Campylobacter infections in patients with diarrhea.

Conclusions
Our study highlights the antibiotic resistance patterns and genetic diversity of 371 clinical isolates from patients with acute diarrhea treated at more than 100 medical institutions in South Korea.The investigated Campylobacter clinical isolates exhibited drug resistance mainly to fluoroquinolone and tetracycline, and they were highly susceptible to macrolides.In particular, fluoroquinolones-resistant Campylobacter strains are emerging worldwide, representing a significant problem in clinical treatment.Even after the ban on ciprofloxacin use in 2010 in South Korea, no rapid decline could be observed and high-level ciprofloxacin resistance was still identified.However, tetracycline resistance displays a decreasing trend.Macrolide and gentamicin need to be appropriately used to prevent increasing resistance against them in Campylobacter spp.The results did not meet our expectations that the ciprofloxacin resistance of Campylobacter isolated from patients and poultry would decrease after the ban on fluoroquinolone as a growth promoter in poultry in Korea.In addition, as enrofloxacin was banned by the Korean Government since 31 October 2021, we plan to conduct additional research to determine whether this ban has an impact on changes in fluoroquinolone resistance rates through patient-poultry genetic correlation analysis from a One Health perspective.
Moreover, we revealed the high genetic diversity of Campylobacter isolates circulating in South Korea.Our MLST analysis demonstrated that CC21 (ST4253), CC22 (ST22), and CC206 (ST572) were high-frequency clones in C. jejuni, CC828 was a dominant clone in C. coli, and identified eight novel STs.Therefore, it is important to continuously monitor the spread of Campylobacter resistance through antimicrobial susceptibility and molecular analysis of patient isolates, which have been lacking in studies.In conclusion, this study contributes to the establishment of basic data necessary to identify the correlation between clinical isolates and to prevent Campylobacter spread in the potential future event of a large-scale patient outbreak.

Figure 2 .
Figure 2. Minimum spanning tree constructed with the conventional 7-locus MLST profiles of 371 Campylobacter isolates.Each circle size is proportional to the number of isolates, with the sequence type (ST) labeled.Differences of 1, 2, 3, and >3 loci between STs are represented by solid red, black, green line, and gray lines, respectively.A clonal complex (CC) is designated for STs among which an ST shares five loci or more with the closest one.

Table 1 .
Results of PCR and culture tests for bacteria causing acute diarrhea requested from six regions from April to September 2022.

Table 2 .
Distribution of the MICs for C. jejuni (n = 361) and C. coli (n = 10) isolates from human patients.