Abstract
Understanding the antimicrobial resistance of Campylobacter jejuni and Salmonella spp. isolated from patients with enteritis will aid in therapeutic decision-making. This study aimed to characterize C. jejuni and Salmonella spp. isolates from patients with enteritis. For C. jejuni, the resistance rates against ampicillin, tetracycline, and ciprofloxacin were 17.2%, 23.8%, and 46.4%, respectively. All the C. jejuni isolates were susceptible to erythromycin, which is recommended as a first-choice antimicrobial if Campylobacter enteritis is strongly suspected. C. jejuni was classified into 64 sequence types (STs), and the five major STs were ST22, ST354, ST21, ST918, and ST50. The ciprofloxacin-resistance rate of ST22 was 85.7%. For Salmonella, the resistance rates against ampicillin, cefotaxime, streptomycin, kanamycin, tetracycline, and nalidixic acid were 14.7%, 2.0%, 57.8%, 10.8%, 16.7%, and 11.8%, respectively. All the Salmonella spp. isolates were susceptible to ciprofloxacin. Therefore, fluoroquinolones are the recommended antimicrobials against Salmonella enteritis. S. Thompson, S. Enteritidis, and S. Schwarzengrund were the three most prevalent serotypes. The two cefotaxime-resistant isolates were serotyped as S. Typhimurium and were found to harbor blaCMY-2. The results of this study would help select antimicrobials for treating patients with Campylobacter and Salmonella enteritis.
Keywords: antimicrobial resistance, Campylobacter, enteritis, Salmonella
Campylobacter spp. and non-typhoidal Salmonella spp. are bacterial pathogens causing foodborne illnesses globally. The World Health Organization has stated that these two genera are among the four key global causative agents of diarrhea [40, 42]. Although several Campylobacter spp. have been identified as pathogens in human campylobacteriosis, Campylobacter jejuni infections account for more than 80% of human campylobacteriosis [4, 8, 21, 46]. The annual number of food poisoning outbreaks in Japan caused by Campylobacter spp. was higher than 200 between 2014 and 2018 [44]. In Japan, chicken and beef are considered major sources of foodborne campylobacteriosis [4, 8]. Salmonella spp. are serotyped into more than 2,600 serotypes based on three structures, somatic (O), flagellar, and capsular surface antigens. Four O-serogroups (O:4, O:7, O:8, and O:9) are major contributors to human non-typhoidal salmonellosis [12, 19, 20, 27]. Following the Infectious Agents Surveillance Report (https://kansen-levelmap.mhlw.go.jp/Byogentai/Pdf/data81j.pdf), the top seven frequent Salmonella serotypes in patients in 2021 were S. Typhimurium, S. Schwarzengrund, S. Infantis, S. Thompson, S. Enteritidis, S. Braenderup and S. Corvallis. Although the annual number of food poisoning outbreaks in Japan caused by Salmonella spp. was less than 50 since 2011, the average number of patients per outbreak is 38 [44]. Eggs, vegetables, and chicken are considered major sources of foodborne salmonellosis in Japan [18].
Campylobacter and Salmonella infections typically cause acute self-limiting enteritis. Although antimicrobial therapy is not recommended in various cases, it may be lifesaving in patients with severe symptoms and health risk groups such as infants, the older population, and immunocompromised patients [25, 40, 42]. Fluoroquinolones, such as ciprofloxacin and levofloxacin, and third-generation cephalosporins (TGCs), such as cefotaxime and ceftriaxone, have been classified as “critically important antimicrobials for human medicine” by the World Health Organization [41]. These antimicrobials, along with penicillins, tetracyclines, aminoglycosides, sulfonamides, and macrolides, are also used to treat bacterial infections in food-producing animals in Japan [23]. Since numerous Japanese studies have reported multidrug-resistant C. jejuni and Salmonella spp. isolated from broilers, cattle, and pigs [14, 15, 17, 28, 30, 33], humans can get infected with these multidrug-resistant species by consuming various foods derived from these animals. Furthermore, there are Japanese reports on the isolation of fluoroquinolone-resistant Campylobacter spp. and TGC-resistant Salmonella spp. from enteritis patients [26, 27]. Therefore, antimicrobial resistance in the two genera is an important issue in the chemotherapeutic treatment of patients with enteritis. This study aimed to determine the antimicrobial resistance profiles of the two genera isolated from patients with enteritis. Moreover, we characterized these isolates genotypically and serologically. The results of this study would help characterize Campylobacter jejuni and Salmonella spp. isolated from patients with enteritis and select antimicrobials for treating enteritis patients.
MATERIALS AND METHODS
Isolates
This study used 151 clinical isolates of C. jejuni and 102 clinical isolates of Salmonella spp. belonging to O:4 (36 isolates), O:7 (40 isolates), O:8 (7 isolates), and O:9 (19 isolates) serogroups. These isolates were obtained from the stool specimens of 253 different patients with enteritis between December 2019 and April 2022 at BML, Inc., in Saitama, Japan. The stool specimens for C. jejuni and Salmonella spp. were obtained from hospitals in 29 (61.7%) and 24 (51.1%) of the 47 prefectures in Japan, respectively. For the isolation of C. jejuni, each specimen was streaked on a modified charcoal cefoperazone deoxycholate agar plates containing a chromogenic substrate (BDTM mCCDA Clear-HT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) and incubated microaerobically at 42°C for 48 hr using AnaeroPack-Microaero (Mitsubishi Gas Chemicals, Tokyo, Japan). For the isolation of Salmonella spp. each specimen was streaked onto a modified Salmonella–Shigella agar plate (Eiken Chemical Co., Tokyo, Japan) and incubated at 37°C for 24 hr. In this study, no human participants were directly involved. Hence, clearance of human ethics is not required. We used isolates routinely cultured from clinical specimens from hospitals. At the laboratory, these isolates were collected in MicrobankTM vials (Pro-Lab Diagnostics Inc., Round Rock, TX, USA) and stored at −80°C. To characterize these isolates, C. jejuni isolates were grown on brain heart infusion agar containing 5% horse blood (Oxoid Ltd., Hampshire, UK) microaerobically at 42°C using AnaeroPack-Microaero (Mitsubishi Gas Chemicals), while Salmonella spp. isolates were grown on brain heart infusion agar (Oxoid) aerobically at 37°C.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing of C. jejuni and Salmonella spp. isolates was conducted using the broth microdilution method using dried plates (Eiken Chemical). C. jejuni ATCC 33560 and Escherichia coli ATCC 25922 were used as quality control strains for C. jejuni and Salmonella spp., respectively.
Antimicrobial susceptibility testing of C. jejuni isolates was conducted against ampicillin (0.12–256 mg/L), streptomycin (0.12–128 mg/L), tetracycline (0.12–128 mg/L), chloramphenicol (0.12–256 mg/L), nalidixic acid (0.12–128 mg/L), ciprofloxacin (0.03–64 mg/L), erythromycin (0.12–128 mg/L), and gentamicin (0.12–256 mg/L). The breakpoints for ampicillin (32 mg/L), streptomycin (32 mg/L), erythromycin (32 mg/L), tetracycline (16 mg/L), nalidixic acid (32 mg/L), ciprofloxacin (4 mg/L), and chloramphenicol (16 mg/L) were adopted from the Clinical and Laboratory Standards Institute (CLSI) [6] and Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) system [24]. The breakpoint for gentamicin (2 mg/L) was specified by the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme [9].
Antimicrobial susceptibility testing for Salmonella spp. isolates was conducted against ampicillin (1–128 mg/L), cefazolin (1–128 mg/L), cefotaxime (0.5–64 mg/L), streptomycin (1–128 mg/L), kanamycin (1–128 mg/L), tetracycline (0.5–64 mg/L), nalidixic acid (1–128 mg/L), ciprofloxacin (0.03–4 mg/L), colistin (0.12–16 mg/L), chloramphenicol (1–128 mg/L), gentamicin (0.5–64 mg/L), and trimethoprim (0.25–16 mg/L). The breakpoints for ampicillin (32 mg/L), cefazolin (8 mg/L), cefotaxime (4 mg/L), streptomycin (32 mg/L), kanamycin (64 mg/L), tetracycline (16 mg/L), nalidixic acid (32 mg/L), ciprofloxacin (1 mg/L), colistin (4 mg/L), chloramphenicol (32 mg/L), gentamicin (16 mg/L), and trimethoprim (16 mg/L) were adopted from the CLSI [7] and JVARM system [24].
Multilocus sequence typing of C. jejuni isolates
Multilocus sequence typing of C. jejuni isolates was performed following the seven-locus scheme for Campylobacter, employing the primer sets and experimental conditions suggested by the Campylobacter multilocus sequence typing (MLST) database (http://pubmlst.org/campylobacter/).
Sequencing of partial gyrA genes in ciprofloxacin-resistant C. jejuni
In one C. jejuni isolate per sequence type determined by MLST, partial gyrA genes of the isolates were amplified using polymerase chain reaction (PCR) [47], and the PCR products were directly sequenced.
Serotyping of Salmonella spp. isolates
Somatic antigens of Salmonella spp. isolates were confirmed by slide agglutination using O antisera (Denka Co., Tokyo, Japan). Salmonella isolates were further tested for flagella antigens via tube agglutination using H antisera (Denka). Serovars were determined based on the combinations of O and H group antigens following the Kauffmann–White scheme [13]. Isolates agglutinated with anti-O:4 and anti-H:i serum but not anti-H:1 or anti-H:2 serum were confirmed as monophasic variants of S. Typhimurium using a previously reported PCR method [11].
Determination of antimicrobial resistance genes and sequence types based on MLST in cefotaxime-resistant Salmonella spp. isolates using whole-genome sequence analysis
DNA was extracted from cefotaxime-resistant strains using the DNeasy® UltraClean® Microbial Kit (Qiagen GmbH, Hilden, Germany). Whole-genome sequence analysis was performed as previously described [34]. Sequencing libraries for each isolate were prepared using the QIAseq FX Library Kit (Qiagen) to obtain paired-end sequences (300 bp × 2) using the Illumina MiSeq platform. The draft genome sequence was assembled using A5-miseq with only Illumina short-read data. Gene annotation was performed using DFAST version 1.2.3 with the following databases: DFAST default database [39], ResFinder database [45], and Bacterial Antimicrobial Resistance Reference Gene database (PRJNA313047). MLST was performed using the “mlst” program version 2.16.2 (https://github.com/tseemann/mlst) with the PubMLST database (https://pubmlst.org/).
RESULTS
For C. jejuni, the resistance rates against ampicillin, streptomycin, tetracycline, nalidixic acid, and ciprofloxacin were 17.2%, 2.6%, 23.8%, 47.0%, and 46.4%, respectively (Table 1). All the C. jejuni isolates were susceptible to erythromycin, gentamicin, and chloramphenicol. In addition, C. jejuni was classified into 64 sequence types (STs) using MLST (Table 2). The five major STs were ST22 (14 isolates), ST354 (12 isolates), ST21 (9 isolates), ST918 (9 isolates), and ST50 (8 isolates). The ciprofloxacin resistance rates of ST22, ST354, ST21, ST918, and ST50 were 85.7% (12/14), 25.0% (3/12), 100.0% (9/9), 33.3% (3/9), and 0.0% (0/8), respectively. Ciprofloxacin resistance was observed in 32 (50.0%) of these 64 STs. Among the 32 STs, the Thr86Ile substitution (mediated by the C257T mutation in the gyrA genes) was detected in 31 STs. The remaining one ST (ST11491) had the Thr86Lys substitution (mediated by the C257A mutation). Moreover. ST8071 and ST10424 had the Asp90Asn substitution (mediated by the G268A mutation) and the Val149Ile substitution (mediated by the G508A mutation), respectively, in addition to the Thr86Ile substitution.
Table 1. Antimicrobial resistance rates of Campylobacter jejuni and Salmonella spp. isolates.
| Antimicrobial | No. of resistant isolates (%) | |
|---|---|---|
| Campylobacter jejuni | Salmoella spp. | |
| Ampicillin | 26 (17.2) | 15 (14.7) |
| Cefazolin | NT | 2 (2.0) |
| Cefotaxime | NT | 2 (2.0) |
| Streptomycin | 4 (2.6) | 59 (57.8) |
| Erythromycin | 0 (0.0) | NT |
| Gentamicin | 0 (0.0) | 1 (1.0) |
| Kanamycin | NT | 11 (10.8) |
| Tetracycline | 36 (23.8) | 17 (16.7) |
| Nalidixic acid | 71 (47.0) | 12 (11.8) |
| Ciprofloxacin | 70 (46.4) | 0 (0.0) |
| Colistin | NT | 8 (7.8) |
| Chloramphenicol | 0 (0.0) | 3 (2.9) |
| Trimethoprim | NT | 10 (9.8) |
NT: not tested.
Table 2. Antimicrobial resistane profiles of 151 Campylobacter jejuni isolates.
| CC (No.) | ST | ARP | No. | CC (No.) | ST | ARP | No. |
|---|---|---|---|---|---|---|---|
| 21 (35) | 19 | NA+CPFX | 5 | 354 (17) | 354 | ABPC+NA+CPFX | 1 |
| 21 | TC+NA+CPFX | 6 | TC+NA+CPFX | 1 | |||
| SM+NA+CPFX | 1 | NA+CPFX | 1 | ||||
| NA+CPFX | 2 | ABPC | 1 | ||||
| 50 | ABPC | 2 | Susceptible | 8 | |||
| TC | 2 | 1723 | Susceptible | 1 | |||
| Susceptible | 4 | 4091 | Susceptible | 1 | |||
| 53 | Susceptible | 2 | 5721 | Susceptible | 1 | ||
| 806 | TC+NA+CPFX | 1 | 10010 | Susceptible | 1 | ||
| TC | 1 | 10432 | Susceptible | 1 | |||
| 883 | Susceptible | 2 | 443 (6) | 51 | ABPC+NA+CPFX | 1 | |
| 4253 | Susceptible | 3 | 440 | NA+CPFX | 1 | ||
| 4526 | TC+NA+CPFX | 2 | NA | 1 | |||
| 5649 | NA+CPFX | 1 | Susceptible | 2 | |||
| 9776 | NA+CPFX | 1 | 1904 | ABPC | 1 | ||
| 22 (14) | 22 | ABPC+NA+CPFX | 2 | 464 (7) | 4106 | TC | 1 |
| TC+NA+CPFX | 2 | 4389 | ABPC | 1 | |||
| NA+CPFX | 8 | 5731 | Susceptible | 1 | |||
| ABPC | 1 | 6704 | ABPC | 3 | |||
| Susceptible | 1 | 10424 | NA+CPFX | 1 | |||
| 42 (5) | 42 | TC+NA+CPFX | 1 | 508 (1) | 508 | Susceptible | 1 |
| Susceptible | 1 | 574 (1) | 9996 | Susceptible | 1 | ||
| 447 | NA+CPFX | 2 | 607 (5) | 607 | NA+CPFX | 3 | |
| 459 | TC+NA+CPFX | 1 | 4600 | ABPC | 1 | ||
| 45 (7) | 11 | Susceptible | 1 | 10431 | Susceptible | 1 | |
| 45 | ABPC+NA+CPFX | 2 | 658 (4) | 1044 | Susceptible | 3 | |
| TC+NA+CPFX | 2 | 10443 | Susceptible | 1 | |||
| ABPC | 1 | Unassigned (20) | 468 | Susceptible | 1 | ||
| Susceptible | 1 | 922 | ABPC+TC+NA+CPFX | 2 | |||
| 48 (9) | 918 | NA+CPFX | 3 | NA+CPFX | 1 | ||
| Susceptible | 6 | 2274 | TC+NA+CPFX | 1 | |||
| 52 (3) | 52 | NA+CPFX | 2 | NA+CPFX | 1 | ||
| 10440 | Susceptible | 1 | 2535 | ABPC+NA+CPFX | 1 | ||
| 61 (5) | 61 | Susceptible | 2 | 4325 | NA+CPFX | 1 | |
| 628 | Susceptible | 1 | ABPC | 2 | |||
| 1244 | TC | 1 | 4622 | ABPC+TC | 1 | ||
| 11491 | TC+NA+CPFX | 1 | 6609 | TC+SM+NA+CPFX | 1 | ||
| 257 (4) | 257 | Susceptible | 2 | 8071 | TC+NA+CPFX | 1 | |
| 824 | Susceptible | 1 | NA+CPFX | 2 | |||
| 4022 | ABPC+SM+TC | 1 | 10006 | NA+CPFX | 1 | ||
| 283 (3) | 4063 | Susceptible | 2 | 10437 | SM+TC+NA+CPFX | 1 | |
| 10486 | TC+NA+CPFX | 1 | 11069 | ABPC+TC+NA+CPFX | 1 | ||
| 353 (5) | 400 | NA+CPFX | 1 | 11080 | NA+CPFX | 1 | |
| 10425 | TC | 4 | 11081 | ABPC+NA+CPFX | 1 |
CC: clonal complex, ST: sequence type, ARP: antimicrobial resistance profile, ABPC: ampicillin, SM: streptomycin, TC: tetracycline, NA: nalidixic acid, CPFX: ciprofloxacin.
For Salmonella spp., the resistance rates against ampicillin, cefazolin, cefotaxime, streptomycin, gentamicin, kanamycin, tetracycline, nalidixic acid, colistin, chloramphenicol, and trimethoprim were 14.7%, 2.0%, 2.0%, 57.8%, 1.0%, 10.8%, 16.7%, 11.8%, 7.8%, 2.9%, and 9.8%, respectively. All the isolates were susceptible to ciprofloxacin. In the O:4 serogroup (36 isolates), S. Schwarzengrund was the most prevalent serovar (13 isolates), and nine (69.2%) isolates were resistant to kanamycin (Table 3). The S. Typhimurium monophasic variant was the second most prevalent serovar (nine isolates), and five (55.6%) isolates were resistant to ampicillin and streptomycin. The two cefotaxime-resistant isolates obtained from two different prefectures were serotyped as S. Typhimurium, and they harbored the AmpC-type β-lactamase gene of blaCMY-2 (Table 4). These two cefotaxime-resistant isolates also had seven genes encoding resistance to aminoglycoside (aac(6’)-Iaa, ant(3”)-Ib, and aph(6)-Id), phenicol (floR), quinolone (qnrB19), sulfonamide (sul2), and tetracycline (tet(A)). In the O:7 serogroup (40 isolates), S. Thompson was the most prevalent serovar (22 isolates). Among these 40 isolates, 39 (97.5%) did not show multidrug resistance. In the O:8 serogroup (seven isolates), S. Manhattan was the most prevalent serovar, and two isolates were resistant to streptomycin and tetracycline. In the O:9 serogroup (19 isolates), S. Enteritidis was the most prevalent serovar (15 isolates), and seven (46.7%) isolates were resistant to ampicillin, streptomycin, and nalidixic acid.
Table 3. Antimicrobial resisntace profiles of 102 Salmonella isolates.
| O serogroup (No.) | Serovar (No.) | ARP | No. |
|---|---|---|---|
| O:4 (36) | Schwarzengrund (13) | SM+KM+TC+NA+TMP | 1 |
| SM+KM+TC+TMP | 2 | ||
| SM+KM+TMP | 2 | ||
| SM+KM+TC | 1 | ||
| SM+TC+TMP | 1 | ||
| KM+TMP | 3 | ||
| Susceptible | 3 | ||
| Typhimurium (5) | ABPC+CEZ+CTX+SM+TC+NA+CP | 2 | |
| SM+GM+KM+TC+TMP | 1 | ||
| SM | 1 | ||
| Susceptible | 1 | ||
| Typhimurium monophasic variant (9) | ABPC+SM+KM+TC+CL+CP | 1 | |
| ABPC+SM+TC | 3 | ||
| ABPC+SM | 1 | ||
| SM+TC | 2 | ||
| SM | 1 | ||
| TC | 1 | ||
| Agona (3) | SM | 3 | |
| Saintpoul (4) | SM | 1 | |
| Susceptible | 3 | ||
| Paratyphi B (1) | Susceptible | 1 | |
| Brandenburg (1) | Susceptible | 1 | |
| O:7 (40) | Thompson (22) | SM+NA | 1 |
| SM | 10 | ||
| Susceptible | 11 | ||
| Infantis (7) | SM | 4 | |
| Susceptible | 3 | ||
| Braenderup (7) | SM | 5 | |
| Susceptible | 2 | ||
| Virchow (2) | SM | 2 | |
| Oranienburg (1) | SM | 1 | |
| Montevideo (1) | Susceptible | 1 | |
| O:8 (7) | Manhattan (3) | SM+TC | 2 |
| Susceptible | 1 | ||
| Corvallis (2) | Susceptible | 2 | |
| Bovismorbificans (1) | SM | 1 | |
| Untypable (1) | Susceptible | 1 | |
| O:9 (19) | Enteritidis (15) | ABPC+SM+NA+CL | 5 |
| ABPC+SM+NA | 2 | ||
| ABPC+NA | 1 | ||
| CL | 2 | ||
| Susceptible | 5 | ||
| Panama (3) | SM | 2 | |
| Susceptible | 1 | ||
| Durban (1) | SM | 1 | |
ARP: antimicrobial resistance profile, ABPC: ampicillin, SM: streptomycin, TC: tetracycline, CL: colistin, CP: chloramphenicol, NA: nalidixic acid, TMP: trimetoprim.
Table 4. Antimicrobial resistance genes in cefotaxime-resistant isolates.
| Serovar | Isolate | ST | Antimicrobial resistance profile | Antimicrobial resistance genes |
|---|---|---|---|---|
| Typhimurium | 202203 | 19 | ABPC+CEZ+CTX+SM+TC+NA+CP | aac(6’)-Iaa, ant(3”)-Ib, blaCMY-2, aph(6)-Id, floR, qnrB19, sul2, tet(A) |
| Typhimurium | 202204 | 19 | ABPC+CEZ+CTX+SM+TC+NA+CP | aac(6’)-Iaa, ant(3”)-Ib, blaCMY-2, aph(6)-Id, floR, qnrB19, sul2, tet(A) |
ST: sequence type, ABPC: ampicillin, CEZ: cefazolin, CTX: cefotaxime, SM: streptomycin, TC: tetracycline, NA: nalidixic acid, CP: chloramphenicol.
DISCUSSION
In Japan, fluoroquinolones are recommended as first-choice antimicrobials for empiric therapy of patients with diarrhea and Salmonella enteritis [25]. Moreover, macrolides are recommended as first-choice antimicrobials if Campylobacter enteritis is strongly suspected or patients have been exposed to regions where quinolone-resistant Campylobacter spp. is prevalent [25]. Ohishi et al. [26] reported that the ciprofloxacin-resistance rate in C. jejuni isolated from patients between 2007 and 2014 is 44.3%. Moreover, Yamada et al. [43] reported that the ciprofloxacin-resistance rate in C. jejuni isolated from patients between 2009 and 2017 was 41.9%, and the Thr86Ile substitution in GyrA was observed in 93.9% of ciprofloxacin-resistant C. jejuni isolates. The Thr86Ile point mutation in GyrA has been identified as the predominant mutation and is associated with increased quinolone resistance [16]. In the present study, the ciprofloxacin-resistance rate in C. jejuni was 46.4%, and the Thr86Ile substitution in GyrA was observed in 96.9% of ciprofloxacin-resistant C. jejuni isolates, suggesting that the fluoroquinolone-resistance rate has consistently been more than 40% in the last decade, and macrolides must ideally be the first-choice antimicrobials if Campylobacter enteritis is strongly suspected, given that erythromycin resistance was not observed in C. jejuni isolates. The top five STs were ST22, ST354, ST21, ST918, and ST50 in the present study. Among these, ST21 is one of the predominant STs in C. jejuni isolated from cattle [33], and the remaining 4 STs are less abundant in C. jejuni isolated from cattle and poultry [2, 26, 33]. We reported that seven (77.8%) of the nine ST21 isolates from cattle are resistant to tetracycline, nalidixic acid, and ciprofloxacin [33]. Of the nine ST21 isolates obtained in the present study, six (66.7%) were also resistant to these three antimicrobials. Moreover, we reported that the two most abundant STs in C. jejuni isolated from Japanese layer flocks are ST4389 (eight isolates) and ST6704 (seven isolates), and 93.3% (14/15) of them are ampicillin-resistant [32]. In that study, two ST354 and two ST918 isolates were obtained from layer flocks, of which 75.0% (3/4) were susceptible to all the tested antimicrobials. These results suggest that some of the STs adapted to cattle and poultry are likely to be pathogenic to humans, and the causative foods of human C. jejuni infection can be identified using a combination of MLST and antimicrobial resistance profiles of C. jejuni isolates. Asakura et al. [3] reported ST22 as the most prevalent ST in C. jejuni isolated from human campylobacteriosis cases in Osaka Prefecture, Japan, between 2010 and 2011. In contrast, ST22 C. jejuni is less abundant in cattle and poultry [2, 30, 32, 33]. Thus, humans might exhibit enteritis easily when infected with ST22, compared to that with other STs. This may be the reason ST22 is the predominant ST isolated from stool specimens of campylobacteriosis patients. Moreover, Takahashi et al. [38] reported that serotype HS:19 isolates in Japan accounted for 67 out of 102 (65.7%) C. jejuni isolates obtained from patients with Guillain-Barré syndrome (GBS), a severe post-infection autoimmune disease. Akase et al. [1] reported 98.9% (87/88) of serotype HS:19 isolated from patients with GBS, sporadic diarrheal patients, and poultry meat samples to be ST22. To understand the relationship between ST22 isolates from humans and livestock, further characterization of these ST22 isolates is needed.
To the best of our knowledge, there are no reports about the isolation of fluoroquinolone-resistant Salmonella spp. from enteritis patients in Japan this decade. In this study, ciprofloxacin resistance was not observed in Salmonella isolates. Therefore, fluoroquinolones are the ideal first-choice antimicrobials against Salmonella enteritis. In addition, ceftriaxone is recommended as a second-choice antimicrobial for Salmonella enteritis [25]. Although the prevalence of TGC-resistant Salmonella spp. in chicken products and broilers has increased since 2005 [5, 10, 24, 28], the prevalence has decreased after withdrawing the use of TGC in broiler production in 2012 [5, 35]. We recently reported that 1.3% (4/309) of Salmonella isolates (three S. Infantis and one S. Manhattan strains) isolated from chicken products sampled between January 2018 and October 2021 were resistant to cefotaxime and harbored blaCMY-2 or blaTEM-52B [31]. In the present study, cefotaxime resistance was very low (2.0%, 2/102), and both the TGC-resistant Salmonella isolates were S. Typhimurium harboring blaCMY-2. Although TGC-resistant S. Typhimurium has never been isolated from broilers, layers, or pigs in Japan [5, 28, 29, 35, 36], it has been isolated from cattle, and all of them have been found to harbor blaCMY-2 [17]. Shimojima et al. [37] investigated the presence of Salmonella in 993 imported meat products (281 chicken, 393 pork, and 319 beef products) between 2009 and 2017, but no TGC-resistant S. Typhimurium was isolated.
Salmonella Thompson, S. Schwarzengrund, S. Infantis, and S. Braenderup were the prevalent serotypes in this study. Most of the S. Schwarzengrund serotypes were multidrug resistant; however, S. Thompson, S. Infantis, and S. Braenderup were not. S. Schwarzengrund and S. Infantis are the two most prevalent serotypes in chicken meat in Japan, and more than 65% of them are multidrug-resistant [22, 31, 37]. S. Thompson, S. Infantis, and S. Braenderup are the prevalent serotypes in layer breeding chains in Japan, and most of them are not multidrug-resistant [29, 36]. Meanwhile, in beef and pork, S. Typhimurium and its monophasic variant are the two most prevalent serotypes, while S. Thompson, S. Schwarzengrund, S. Infantis, and S. Braenderup are barely isolated [37].
In conclusion, the characteristics of human C. jejuni and Salmonella spp. isolates could represent the characteristics of these two bacterial isolates originating from contaminated food. Monitoring the antimicrobial resistance of C. jejuni and Salmonella spp. isolated from food-producing animals and food would thus aid in the selection of antimicrobials for treating Campylobacter and Salmonella enteritis patients.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Acknowledgments
This study was supported by grants from the Japan Agency for Medical Research and the Development Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108103). The authors wish to acknowledge BML, Inc., for providing the clinical isolates for this study.
REFERENCES
- 1.Akase S, Yokoyama K, Obata H, Monma C, Konishi N, Hatakeyama K, Saiki D, Maeda M, Asayama C, Suzuki J, Sadamasu K. 2022. Multi-locus sequence typing and lipopligosaccharide class analysis of Campylobacter jejuni HS:19 isolated in Japan. Jpn J Infect Dis 75: 199–201. doi: 10.7883/yoken.JJID.2021.341 [DOI] [PubMed] [Google Scholar]
- 2.Asakura H, Brüggemann H, Sheppard SK, Ekawa T, Meyer TF, Yamamoto S, Igimi S. 2012. Molecular evidence for the thriving of Campylobacter jejuni ST-4526 in Japan. PLoS One 7: e48394. doi: 10.1371/journal.pone.0048394 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Asakura H, Taguchi M, Ekawa T, Yamamoto S, Igimi S. 2013. Continued widespread dissemination and increased poultry host fitness of Campylobacter jejuni ST-4526 and ST-4253 in Japan. J Appl Microbiol 114: 1529–1538. doi: 10.1111/jam.12147 [DOI] [PubMed] [Google Scholar]
- 4.Asakura H, Sakata J, Nakamura H, Yamamoto S, Murakami S. 2019. Phylogenetic diversity and antimicrobial resistance of Campylobacter coli from humans and animals in Japan. Microbes Environ 34: 146–154. doi: 10.1264/jsme2.ME18115 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Chuma T, Miyasako D, Dahshan H, Takayama T, Nakamoto Y, Shahada F, Akiba M, Okamoto K. 2013. Chronological change of resistance to β-lactams in Salmonella enterica serovar Infantis isolated from broilers in Japan. Front Microbiol 4: 113. doi: 10.3389/fmicb.2013.00113 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Clinical and Laboratory Standards Institute.2013. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard, 4th ed. CLSI document VET01-A4. Clinical and Laboratory Standards Institute, Wayne. [Google Scholar]
- 7.Clinical and Laboratory Standards Institute.2016. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria, 3rd ed., CLSI guideline M45. Clinical and Laboratory Standards Institute, Wayne. [DOI] [PubMed] [Google Scholar]
- 8.Cody AJ, McCarthy NM, Wimalarathna HL, Colles FM, Clark L, Bowler ICJW, Maiden MCJ, Dingle KE. 2012. A longitudinal 6-year study of the molecular epidemiology of clinical campylobacter isolates in Oxfordshire, United kingdom. J Clin Microbiol 50: 3193–3201. doi: 10.1128/JCM.01086-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.DANMAP2020. Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark. https://www.danmap.org/reports/2020 [accessed on February 21, 2022].
- 10.Duc VM, Kakiuchi R, Muneyasu H, Toyofuku H, Obi T, Chuma T. 2022. Decreasing trend of β-lactam resistance in Salmonella isolates from broiler chickens due to the cessation of ceftiofur in ovo administration. Vet Anim Sci 16: 100248. doi: 10.1016/j.vas.2022.100248 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Echeita MA, Herrera S, Usera MA. 2001. Atypical, fljB-negative Salmonella enterica subsp. enterica strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium. J Clin Microbiol 39: 2981–2983. doi: 10.1128/JCM.39.8.2981-2983.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Frasson I, Bettanello S, De Canale E, Richter SN, Palù G. 2016. Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy. Gut Pathog 8: 26. doi: 10.1186/s13099-016-0110-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Grimont PAD, Weil F. 2007. Antigenic Formulas of the Salmonella Serovars, 9th ed., WHO Collaborating Centre for Reference and Research on Salmonella: Paris, France, Institute Pasteur, Paris. [Google Scholar]
- 14.Haruna M, Sasaki Y, Murakami M, Ikeda A, Kusukawa M, Tsujiyama Y, Ito K, Asai T, Yamada Y. 2012. Prevalence and antimicrobial susceptibility of Campylobacter in broiler flocks in Japan. Zoonoses Public Health 59: 241–245. doi: 10.1111/j.1863-2378.2011.01441.x [DOI] [PubMed] [Google Scholar]
- 15.Haruna M, Sasaki Y, Murakami M, Mori T, Asai T, Ito K, Yamada Y. 2013. Prevalence and antimicrobial resistance of Campylobacter isolates from beef cattle and pigs in Japan. J Vet Med Sci 75: 625–628. doi: 10.1292/jvms.12-0432 [DOI] [PubMed] [Google Scholar]
- 16.Kijima M, Shirakawa T, Uchiyama M, Kawanishi M, Ozawa M, Koike R. 2019. Trends in the serovar and antimicrobial resistance in clinical isolates of Salmonella enterica from cattle and pigs between 2002 and 2016 in Japan. J Appl Microbiol 127: 1869–1875. doi: 10.1111/jam.14431 [DOI] [PubMed] [Google Scholar]
- 17.Kumagai Y, Pires SM, Kubota K, Asakura H. 2020. Attributing human foodborne diseases to food sources and water in Japan using analysis of outbreak surveillance data. J Food Prot 83: 2087–2094. doi: 10.4315/JFP-20-151 [DOI] [PubMed] [Google Scholar]
- 18.Li X, Singh N, Beshearse E, Blanton JL, DeMent J, Havelaar AH. 2021. Spatial epidemiology of salmonellosis in Florida, 2009–2018. Front Public Health 8: 603005. doi: 10.3389/fpubh.2020.603005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Iovine NM. 2013. Resistance mechanisms in Campylobacter jejuni. Virulence 4: 230–240. doi: 10.4161/viru.23753 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Mascaro V, Pileggi C, Crinò M, Proroga YTR, Carullo MR, Graziani C, Arigoni F, Turno P, Pavia M. 2017. Non-typhoidal Salmonella in Calabria, Italy: a laboratory and patient-based survey. BMJ Open 7: e017037. doi: 10.1136/bmjopen-2017-017037 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Metreveli M, Bulia S, Shalamberidze I, Tevzadze L, Tsanava S, Goenaga JC, Stingl K, Imnadze P. 2022. Campylobacteriosis, shigellosis and salmonellosis in hospitalized children with acute inflammatory diarrhea in Georgia. Pathogens 11: 232. doi: 10.3390/pathogens11020232 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Mori T, Okamura N, Kishino K, Wada S, Zou B, Nanba T, Ito T. 2018. Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry meat in Japan. Food Saf (Tokyo) 6: 126–129. doi: 10.14252/foodsafetyfscj.2017019 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.National Veterinary Assay Laboratory of Japan.2020. Report on the Japanese Veterinary Antimicrobial Resistance Monitoring System 2016–2017; National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo. [Google Scholar]
- 24.Noda T, Murakami K, Etoh Y, Okamoto F, Yatsuyanagi J, Sera N, Furuta M, Onozuka D, Oda T, Asai T, Fujimoto S. 2015. Increase in resistance to extended-spectrum cephalosporins in Salmonella isolated from retail chicken products in Japan. PLoS One 10: e0116927. doi: 10.1371/journal.pone.0116927 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ohnishi K, Ainoda Y, Imamura A, Iwabuchi S, Okuda M, Nakano T. Japanese Association for Infectious Disease/Japanese Society of ChemotherapyJAID/JSC Guide to Clinical Management of Infectious Disease/Guideline-preparing CommitteeIntestinal Infections Working Group (WG).2018. JAID/JSC guidelines for infection treatment 2015 −Intestinal infections. J Infect Chemother 24: 1–17. doi: 10.1016/j.jiac.2017.09.002 [DOI] [PubMed] [Google Scholar]
- 26.Ohishi T, Aoki K, Ishii Y, Usui M, Tamura Y, Kawanishi M, Ohnishi K, Tateda K. 2017. Molecular epidemiological analysis of human- and chicken-derived isolates of Campylobacterjejuni in Japan using next-generation sequencing. J Infect Chemother 23: 165–172. doi: 10.1016/j.jiac.2016.11.011 [DOI] [PubMed] [Google Scholar]
- 27.Saito S, Koori Y, Ohsaki Y, Osaka S, Oana K, Nagano Y, Arakawa Y, Nagano N. 2017. Third-generation cephalosporin-resistant non-typhoidal Salmonella isolated from human feces in Japan. Jpn J Infect Dis 70: 301–304. doi: 10.7883/yoken.JJID.2016.166 [DOI] [PubMed] [Google Scholar]
- 28.Sasaki Y, Ikeda A, Ishikawa K, Murakami M, Kusukawa M, Asai T, Yamada Y. 2012. Prevalence and antimicrobial susceptibility of Salmonella in Japanese broiler flocks. Epidemiol Infect 140: 2074–2081. doi: 10.1017/S0950268812000039 [DOI] [PubMed] [Google Scholar]
- 29.Sasaki Y, Yonemitsu K, Uema M, Igimi S, Asakura H. 2019. Salmonella prevalence in laying hen farms and estimation of effective tools for controlling Salmonella infections. J Jpn Soc Poult Dis 55: 159–163 (in Japanese, with an English summary). [Google Scholar]
- 30.Sasaki Y, Iwata T, Uema M, Asakura H. 2020. Prevalence and characterization of Campylobacter in bile from bovine gallbladders. Shokuhin Eiseigaku Zasshi 61: 126–131 (in Japanese with an English summary). doi: 10.3358/shokueishi.61.126 [DOI] [PubMed] [Google Scholar]
- 31.Sasaki Y, Kakizawa H, Baba Y, Ito T, Haremaki Y, Yonemichi M, Ikeda T, Kuroda M, Ohya K, Hara-Kudo Y, Asai T, Asakura H. 2021. Antimicrobial resistance in Salmonella isolated from food workers and chicken products in Japan. Antibiotics (Basel) 10: 1541. doi: 10.3390/antibiotics10121541 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Sasaki Y, Iwata T, Uema M, Yonemitsu K, Igimi S, Asakura H. 2022. Campylobacter spp. prevalence and fluoroquinolone resistance in chicken layer farms. J Vet Med Sci 84: 743–746. doi: 10.1292/jvms.22-0047 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Sasaki Y, Asakura H, Asai T. 2022. Prevalence and fluoroquinolone resistance of Campylobacter spp. isolated from beef cattle in Japan. Anim Dis 2: 15. doi: 10.1186/s44149-022-00048-6 [DOI] [Google Scholar]
- 34.Sekizuka T, Yatsu K, Inamine Y, Segawa T, Nishio M, Kishi N, Kuroda M. 2018. Complete genome sequence of a blaKPC-2-positive Klebsiella pneumoniae strain isolated from the effluent of an urban sewage treatment plant in Japan. MSphere 3: e00314–e00318. doi: 10.1128/mSphere.00314-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Shigemura H, Matsui M, Sekizuka T, Onozuka D, Noda T, Yamashita A, Kuroda M, Suzuki S, Kimura H, Fujimoto S, Oishi K, Sera N, Inoshima Y, Murakami K. 2018. Decrease in the prevalence of extended-spectrum cephalosporin-resistant Salmonella following cessation of ceftiofur use by the Japanese poultry industry. Int J Food Microbiol 274: 45–51. doi: 10.1016/j.ijfoodmicro.2018.03.011 [DOI] [PubMed] [Google Scholar]
- 36.Shigemura H, Maeda T, Nakayama S, Ohishi A, Carle Y, Ookuma E, Etoh Y, Hirai S, Matsui M, Kimura H, Sekizuka T, Kuroda M, Sera N, Inoshima Y, Murakami K. 2021. Transmission of extended-spectrum cephalosporin-resistant Salmonella harboring a blaCMY-2-carrying IncA/C2 plasmid chromosomally integrated by ISEcp1 or IS26 in layer breeding chains in Japan. J Vet Med Sci 83: 1345–1355. doi: 10.1292/jvms.21-0085 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Shimojima Y, Nishino Y, Fukui R, Kuroda S, Suzuki J, Sadamasu K. 2020. Salmonella serovars isolated from retail meats in Tokyo, Japan and their antimicrobial susceptibility. Shokuhin Eiseigaku Zasshi 61: 211–217 (in Japanese, with an English summary). doi: 10.3358/shokueishi.61.211 [DOI] [PubMed] [Google Scholar]
- 38.Takahashi M, Koga M, Yokoyama K, Yuki N. 2005. Epidemiology of Campylobacter jejuni isolated from patients with Guillain-Barré and Fisher syndromes in Japan. J Clin Microbiol 43: 335–339. doi: 10.1128/JCM.43.1.335-339.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Tanizawa Y, Fujisawa T, Nakamura Y. 2018. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 34: 1037–1039. doi: 10.1093/bioinformatics/btx713 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.World Health Organization2018. Factsheet, Salmonella (non-typhoidal). https://www.who.int/news-room/fact-sheets/detail/salmonella-(non-typhoidal) [accessed on January 6, 2023].
- 41.World Health Organization2019. Critically Important Antimicrobials for Human Medicine, 6th Revision, World Health Organization, Geneva. [Google Scholar]
- 42.World Health Organization2020. Factsheet, Campylobacter. (https://www.who.int/news-room/fact-sheets/detail/campylobacter) [accessed on January 6, 2023].
- 43.Yamada K, Saito R, Muto S, Sasaki M, Murakami H, Aoki K, Ishii Y, Tateda K. 2019. Long-term observation of antimicrobial susceptibility and molecular characterisation of Campylobacter jejuni isolated in a Japanese general hospital 2000-2017. J Glob Antimicrob Resist 18: 59–63. doi: 10.1016/j.jgar.2019.02.001 [DOI] [PubMed] [Google Scholar]
- 44.Yoshikura H. 2020. Declining Vibrio parahaemolyticus and Salmonella, increasing Campylobacter and persisting Norovirus food poisonings: Inference derived from food poisoning statistics of Japan. Jpn J Infect Dis 73: 102–110. doi: 10.7883/yoken.JJID.2019.247 [DOI] [PubMed] [Google Scholar]
- 45.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67: 2640–2644. doi: 10.1093/jac/dks261 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Zhang P, Zhang X, Liu Y, Jiang J, Shen Z, Chen Q, Ma X. 2020. Multilocus sequence types and antimicrobial resistance of Campylobacter jejuni and C. coli isolates of human patients from Beijing, China, 2017–2018. Front Microbiol 11: 554784. doi: 10.3389/fmicb.2020.554784 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Zirnstein G, Li Y, Swaminathan B, Angulo F. 1999. Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 37: 3276–3280. doi: 10.1128/JCM.37.10.3276-3280.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]