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. 2022 Jan 18;66(1):e01736-21. doi: 10.1128/AAC.01736-21

Antimicrobial Resistance in Campylobacter coli and Campylobacter jejuni from Human Campylobacteriosis in Taiwan, 2016 to 2019

Ying-Shu Liao a, Bo-Han Chen a, Ru-Hsiou Teng a, You-Wun Wang a, Jui-Hsien Chang a, Shiu-Yun Liang a, Chi-Sen Tsao a, Yu-Ping Hong a,b, Hui-Yung Sung a, Chien-Shun Chiou a,✉
PMCID: PMC8765299  PMID: 34748382

ABSTRACT

Campylobacter coli and Campylobacter Jejuni are highly resistant to most therapeutic antimicrobials in Taiwan; rapid diagnostics of resistance in bacterial isolates is crucial for the treatment of campylobacteriosis. We characterized 219 (40 C. coli and 179 C. jejuni) isolates recovered from humans from 2016 to 2019 using whole-genome sequencing to investigate the genetic diversity among isolates and the genetic resistance determinants associated with antimicrobial resistance. Susceptibility testing with 8 antimicrobials was conducted to assess the concordance between phenotypic resistance and genetic determinants. The conventional and core genome multilocus sequence typing analysis revealed diverse clonality among the isolates. Mutations in gyrA (T86I, D90N), rpsL (K43R, K88R), and 23S rRNA (A2075G) were found in 91.8%, 3.2%, and 6.4% of the isolates, respectively. The horizontally transferable resistance genes ant(6)-I, aad9, aph(3′)-IIIa, aph(2″), blaOXA, catA/fexA, cfr(C), erm(B), lnu, sat4, and tet were identified in 24.2%, 21.5%, 33.3%, 11.9%, 96.3%, 10.0%, 0.9%, 6.8%, 3.2%, 13.2%, and 96.3%, respectively. High-level resistance to 8 antimicrobials in isolates was 100% predictable by the known resistance determinants, whereas low-level resistance to azithromycin, clindamycin, nalidixic acid, ciprofloxacin, and florfenicol in isolates was associated with sequence variations in CmeA and CmeB of the CmeABC efflux pump. Resistance-enhancing CmeB variants were identified in 62.1% (136/219) of isolates. In conclusion, an extremely high proportion of C. coli (100%) and C. jejuni (88.3%) were multidrug-resistant, and a high proportion (62.5%) of C. coli isolates were resistant to azithromycin, erythromycin, and clindamycin, which would complicate the treatment of invasive campylobacteriosis in this country.

KEYWORDS: Campylobacter, antimicrobial resistance, drug resistance mechanism, whole-genome sequencing, resistance genetic determinant, multidrug resistance efflux pump

INTRODUCTION

Campylobacter species are microaerophilic, mobile, Gram-negative spiral, rod-shaped, or curved bacteria. Many Campylobacter species are zoonotic pathogens, which have been associated with a range of gastrointestinal diseases and, in some cases, life-threatening extragastrointestinal infections, called campylobacteriosis, in humans (1). Among the Campylobacter species, C. jejuni and C. coli are the most prevalent causative agents for gastroenteritis (2). Poultry, domestic animals, and wild animals are the primary reservoirs of Campylobacter species; infections in humans are mostly attributed to the consumption of contaminated meat products, especially fresh and frozen chicken meat, or water (2, 3).

The incidence and prevalence of campylobacteriosis have increased in both developed and developing countries in recent decades and may vary among countries or regions (2, 4). Campylobacteriosis is the most commonly reported gastrointestinal infection in humans in the European Union since 2005 and accounted for 66.8% of the total confirmed human zoonoses in the European Union in 2019 (5). Among the isolates with species information, 83.1% were C. jejuni and 10.8% were C. coli (5). Campylobacteriosis is also the most common foodborne disease in the United States, Australia, and New Zealand (6–8). In Taiwan, the nationwide prevalence of campylobacteriosis has not been well estimated. In a study, Campylobacter was isolated from 6.8% of fecal specimens from children with diarrhea in northern Taiwan around 2004 (9). Of the isolates recovered, 95.1% (58/61) were C. jejuni and 4.9% (3/61) were C. coli. Another study, using a commercial molecular diagnostic kit to detect 15 gastrointestinal pathogens, including bacteria, viruses, and parasites, indicates that Campylobacter was the leading bacterial agent, accounting for 16.7% of the pathogens detected in stool specimens from patients (all ages) who had diarrhea and visited clinics in 2014 (10).

Most Campylobacter infections are self-limited and require only supportive therapy; however, appropriate antimicrobial therapy is beneficial in immunocompromised patients and those with severe and persistent symptoms (11). Some antimicrobials have been employed in the treatment of campylobacteriosis (1, 12, 13). Fluoroquinolones and macrolides are the drugs of the choice for diarrhea due to Campylobacter infection; however, high proportions of ciprofloxacin resistance have been observed in C. coli and C. jejuni from humans and food-producing animals in Europe and many other countries (9, 14, 15). Macrolides such as azithromycin and erythromycin are the alternatives of choice for the treatment of infections caused by fluoroquinolone-resistant Campylobacter (13). However, the emergence of resistance to macrolides has highlighted a great medical concern. Although the prevalence of macrolide resistance in C. jejuni remains low, the resistance has been more common in C. coli (15–17). The trend of resistance development in Campylobacter species has emphasized a need for monitoring resistance to the therapeutic drugs.

The antimicrobial susceptibility of bacterial isolates is primarily determined using the broth microdilution and agar diffusion methods (18). The methods are time-consuming and labor-intensive, and the protocols have to be strictly standardized to obtain results that are comparable among laboratories. Several commercial testing kits have been available for susceptibility testing, but each can only test a limited number of antimicrobials. With the advance of next-generation sequencing techniques and the reduction of the cost, whole-genome sequencing (WGS) of bacterial isolates has been widely employed in characterizing bacterial isolates for evolutionary study, epidemiological investigation, and virulence and resistance gene identification (19–22). Several studies have shown that the resistance determinants identified from whole-genome sequences can accurately predict phenotypic resistance (23–26). In this study, we characterized clinical Campylobacter isolates using WGS to investigate the genetic clonality among isolates and to assess the concordance between resistance genotypes and resistance phenotypes.

RESULTS

Genetic relatedness among isolates.

Of the 219 Campylobacter isolates characterized in this study, 40 (18.3%) were C. coli and 179 (81.7%) were C. jejuni. The conventional 7-locus multilocus sequence type (MLST) analysis revealed that the isolates were relatively diverse, with a total of 99 sequence types (STs) in the 219 isolates, among which 21 were found in the 40 C. coli isolates and 78 in the 179 C. jejuni isolates. Clustering analysis of the ST profiles distinctly separated C. coli isolates from C. jejuni isolates. Most STs found in C. coli belonged to clonal complex 828 (CC828) and CC1150 (Fig. 1). CC257/CC464/CC574, CC21, and CC45 were the major clonal complexes for the C. jejuni isolates. The genetic relationships among the isolates were further established using the core genome MLST (cgMLST) profiles. The cgMLST tree also revealed several distinct genetic lineages; nevertheless, there were 24 clusters, each comprising closely related isolates with a distance of only 10 loci or less (Fig. 2). The isolates in each cluster could likely link to a common epidemiological event.

FIG 1.

FIG 1

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Minimum spanning tree constructed with the conventional 7-locus MLST profiles of 219 Campylobacter isolates. The size of each circle is proportional to the number of isolates, with the sequence type (ST) labeled. The difference of 1, 2, 3, and >3 loci between STs is presented by a solid red, blue, and green line and a dashed gray line, respectively. A clonal complex (CC) is designated for STs among which an ST shares 4 loci or more with the closest one.

FIG 2.

FIG 2

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A maximum parsimony tree constructed with cgMLST profiles of 219 Campylobacter isolates. C. coli isolates are circled within a dotted ellipse. Blue arrows indicate clusters that each comprise genetically close isolates harboring WT-CmeB and RE-CmeB.

Antimicrobial resistance determinants.

Antimicrobial resistance determinants were identified from the WGS data for 219 isolates using the AMRFinderPlus and the ResFinder 4.1 pipelines. Mutations in gyrA (encoding gyrase subunit A), rpsL (encoding 30S ribosomal protein S12), and 23S rRNA, associated with antimicrobial resistance, were found in 91.8%, 3.2%, and 6.4% of the Campylobacter isolates, respectively (Table 1). T86I and D90N mutations in gyrA, which confer resistance to quinolones and fluoroquinolones (27), were identified in 97.5% of C. coli and 90.5% of C. jejuni isolates. K43R and K88R mutations in rpsL, which are associated with streptomycin resistance (28), were found in 17.5% of C. coli but not in C. jejuni isolates. A2075G substitution in 23S rRNA, which confers resistance to macrolides and lincosamides (29), was identified in 32.5% of C. coli but only in 0.6% of C. jejuni isolates.

TABLE 1.

Antimicrobial resistance determinants in Campylobacter coli and C. jejuni isolates

Resistance determinant Total (n = 219) C. coli (n = 40) C. jejuni (n = 179) Antimicrobials Reference(s)
gyrA 91.8 97.5 90.5 Quinolones, fluoroquinolones 27
 T86I 91.3 97.5 89.9
 T86I, D90N 0.5 0.0 0.6
rpsL (S12) 3.2 17.5 0.0 Streptomycin 28
 K43R 1.4 7.5 0.0
 K88R 1.8 10.0 0.0
23S rRNA (A2075G) 6.4 32.5 0.6 Macrolides, lincosamides 29
ant(6)-I 24.2 70.0 14.0 Streptomycin 30, 31
 aadE 21.0 52.5 14.0
 aadE-Cc 1.8 10.0 0.0
 ant(6)-Ia 9.1 37.5 2.8
aad9 21.5 70.0 10.6 Spectinomycin 34
aph(3′)-IIIa 33.3 75.0 24.0 Kanamycin, neomycin, lividomycin, paromomycin, livostamycin, butirosin, amikacin, isepamicin 30
aph(2″) 11.9 40.0 5.6 Gentamicin 32, 33
 aac(6′)-Ie/aph(2″)-Ia 5.0 25.0 0.6
 aph(2″)_1f 3.7 12.5 1.7
 aph(2″)_1h 3.7 5.0 3.4
blaOXA 96.3 95.0 96.6 Amoxicillin, ampicillin, ticarcillin 35
 blaOXA-61 family 74.0 95.0 69.3
 blaOXA-184 family 22.4 0.0 27.4
catA/fexA 10.0 12.5 9.5
 catA 10.0 12.5 9.5 Chloramphenicol 36, 37
 fexA 0.9 0.0 1.1 Chloramphenicol, florfenicol
cfr(C) 0.9 5.0 0.0 Phenicols, lincosamides, oxazolidinones, pleuromutilins, streptogramin A 38
erm(B) 6.8 30.0 1.7 Macrolides, lincosamides, streptogramin 39
lnu 3.2 7.5 2.2 Lincosamides 40, 41
 lnu(C) 1.8 7.5 0.6
 lnu(P) 1.4 0.0 1.7
sat4 13.2 42.5 6.7 Streptothricin 42
tet 96.3 97.5 96.1 Tetracycline 43, 44
 tet(L) 11.4 0.0 14.0
 tet(O) 79.0 67.5 81.6
 tet(O/M/O) 17.4 42.5 11.7
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Horizontally transferable resistance genes, including ant(6)-I [aadE, aadE-Cc, ant(6)-Ia], aad9, aph(3′)-IIIa, aph(2″) [aac(6′)-Ie/aph(2″)-Ia, aph(2″)-If, aph(2″)-Ih], blaOXA (blaOXA-61 family and blaOXA-184 family), catA/fexA, cfr(C), erm(B), lnu [lnu(C), lnu(P)], sat4, and tet [tet(L), tet(O), tet(O/M/O)] were identified in 24.2%, 21.5%, 33.3%, 11.9%, 96.3%, 10.0%, 0.9%, 6.8%, 3.2%, 13.2%, and 96.3% of the isolates, respectively (Table 1). The aminoglycoside resistance genes ant(6)-I, aad9, aph(3′)-IIIa, and aph(2″), which confer resistance to streptomycin, spectinomycin, kanamycin, and gentamicin (30–34), respectively, existed in a higher proportion of C. coli than C. jejuni isolates. Clustering analysis of blaOXA amino acid sequences revealed that they belonged to the blaOXA-61 and blaOXA-184 families, which confer resistance to ampicillin, amoxicillin, and ticarcillin (35). C. coli isolates harbored only genes belonging to the blaOXA-61 family; whereas C. jejuni carried genes belonging to both the blaOXA-61 and blaOXA-184 families. catA, a chloramphenicol resistance gene (36), was carried by 12.5% of C. coli and 9.5% of C. jejuni isolates; fexA, which confers resistance to chloramphenicol and florfenicol (37), was found in only 2 C. jejuni isolates. cfr(C), which encodes an rRNA methyltransferase (38), was found in two C. coli isolates, one of which had a nonsense mutation. erm(B), which encodes a 23S rRNA methyltransferase and confers resistance to macrolides, lincosamides, and streptogramin B (39), was detected in 30% of C. coli isolates and 1.7% of C. jejuni isolates. lnu(C) and lnu(P), which encode lincosamide nucleotidyltransferase (40, 41), were found in 3 C. coli and 5 C. jejuni isolates. Lnu(C) confers resistance to lincomycin but not to clindamycin in Streptococcus agalactiae; however, the enzyme expressed in Escherichia coli can inactivate both lincomycin and clindamycin (40). In this study, the lnu(C)- and lnu(P)-carrying Campylobacter isolates were not resistant to clindamycin. The streptothricin resistance gene, sat4 (42), was detected in 42.5% of C. coli isolates but a lower proportion (6.7%) of C. jejuni isolates. The tetracycline resistance genes, tet(O) and tet(O/M/O) (43, 44), were found in 97.5% of C. coli isolates; tet(L), tet(O), and tet(O/M/O) were present in 96.1% of C. jejuni isolates. C. coli was more resistant than C. jejuni. According to the resistance determinants identified, all (40/40) C. coli isolates and 88.3% (158/179) of C. jejuni isolates were expected to be multidrug-resistant, as the isolates displayed resistance to ≥3 classes of antimicrobials.

Resistance phenotypes.

Susceptibility testing results revealed that the phenotypic resistance to 3 antimicrobials (erythromycin, gentamicin, and tetracycline) in the isolates was completely concordant with the presence of the genetic resistance determinants identified using the AMRFinderPlus and ResFinder 4.1 pipelines (Table 2). All but one isolate resistant to azithromycin, nalidixic acid, and ciprofloxacin carried the identified resistance determinants, erm(B) and A2075G substitution in 23S rRNA, and T86I and T86I and D90N substitutions in gyrA. Isolates that harbored the genetic determinants exhibited high-level resistance to the 3 antimicrobials. One C. jejuni isolate that did not harbor any known resistance determinant identified by the two pipelines displayed low-level resistance to azithromycin, nalidixic acid, and ciprofloxacin. Phenotypic resistance to clindamycin was found in 20.5% of the isolates, but only 13.2% of the isolates carried the resistance determinants erm(B), cfr(C), and A2075G substitution in 23S rRNA. Similarly, a much higher proportion of isolates displayed phenotypic resistance than genotypic resistance to florfenicol, as 10.5% of isolates were resistant to florfenicol, but only 1.4% of isolates carried the resistance determinants fexA and cfr(C). Clindamycin- and florfenicol-resistant isolates that did not carry any known resistance determinant had a low MIC range. The phenotypic and genotypic resistance data together indicated that the Campylobacter isolates would be extremely resistant to nalidixic acid, ciprofloxacin, tetracycline, and ampicillin, with a resistance rate between 92.2% and 96.3% (Table 2). C. coli was more resistant than C. jejuni. Of the C. coli isolates, 62.5% were resistant to azithromycin, erythromycin, and clindamycin, 60.0% were resistant to both ciprofloxacin and erythromycin, and 70% to 80% were resistant to the aminoglycosides, including streptomycin, spectinomycin, and kanamycin. In contrast, C. jejuni isolates had a much lower resistance rate (2.2% to 24.0%) to the macrolides, lincosamides, and aminoglycosides.

TABLE 2.

Genotypic and phenotypic resistance in Campylobacter coli and C. jejuni

Antimicrobial Genotype
 Phenotypea
 Resistance determinants
Total (n = 219) C. coli (n = 40) C. jejuni (n = 179) Total (n = 219) C. coli (n = 40) C. jejuni (n = 179)
Azithromycin 13.2 62.5 2.2 13.7 62.5 2.8 erm(B), A2075G in 23S rRNA
Erythromycin 13.2 62.5 2.2 13.2 62.5 2.2 erm(B), A2075G in 23S rRNA
Clindamycin 13.2 62.5 2.2 20.5 62.5 11.2 erm(B), A2075G in 23S rRNA, cfr(C)
Nalidixic acid 91.8 97.5 90.5 92.2 97.5 91.1 T86I, D90N in gyrA
Ciprofloxacin 91.8 97.5 90.5 92.2 97.5 91.1 T86I, D90N in gyrA
Ciprofloxacin and erythromycin 12.3 60.0 1.7 12.3 60.0 1.7 erm(B), T86I & D90N in gyrA; A2075G in 23S rRNA
Gentamicin 11.9 40.0 5.6 11.9 40.0 5.6 aac(6′)-Ie/aph(2″)-Ia, aph(2″)-If, aph(2″)-Ih
Tetracycline 96.3 97.5 96.1 96.3 97.5 96.1 tet(L), tet(O), tet(O/M/O)
Florfenicol 1.4 2.5 1.1 10.5 7.5 11.2 fex, cfr(C)
Chloramphenicol 10.0 12.5 9.5 ND ND ND catA13, fexA, cfr(C)
Ampicillin etc.b 96.3 95.0 96.6 ND ND ND bla OXA
Lincomycin 3.7 10.0 2.2 ND ND ND lnu(C), lnu(P), cfr(C)
Kanamycin etc.c 33.3 75.0 24.0 ND ND ND aph(3′)-IIIa
Spectinomycin 21.5 70.0 10.6 ND ND ND aad9
Streptomycin 26.0 80.0 14.0 ND ND ND aadE, aadE-Cc, ant(6)-Ia, K43R and K88R in rpsL
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a

ND, not determined.

b

Ampicillin, amoxicillin, ticarcillin.

c

Kanamycin, neomycin, lividomycin, paromomycin, livostamycin, butirosin, amikacin, isepamicin.

CmeABC sequences.

CmeABC is a well-studied tripartite multidrug efflux pump in Campylobacter, consisting of a periplasmic fusion protein, CmeA, an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily, CmeB, and an outer membrane protein, CmeC; sequence variations in CmeB have been reported to be associated with an enhanced efflux function (45). The CmeB sequences from the 219 isolates studied comprised 1,038, 1,039, or 1,040 amino acid residues. Clustering analysis of CmeB sequences using the maximum parsimony algorithm grouped the sequences into two distinct clusters (Fig. 3). Cluster A and cluster B shared only 87.84% amino acid sequence identity. The sequences in each cluster were considerably divergent; sequences in cluster A shared ≥95.62% identity, while those in B shared ≥98.92% identity. Cluster A comprised CmeB sequences from both C. coli and C. jejuni isolates. Most sequences in cluster B were from C. jejuni isolates; only 3 were from C. coli isolates. Among the sequences in cluster B, one was shared by two species (2 C. coli and 27 C. jejuni isolates). Cluster A was grouped with a CmeB sequence from C. jejuni NCTC11168 that carries a wild-type CmeB (WT-CmeB), while cluster B was grouped with a sequence from C. jejuni NT161 that harbors a resistance-enhancing CmeB (RE-CmeB) (45) (Fig. 3). CmeB variants (RE-CmeB), which were grouped in cluster B, were found in 136 (62.1%) isolates, 3 (7.5%) C. coli and 133 (74.3%) C. jejuni isolates.

FIG 3.

FIG 3

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Maximum parsimony tree constructed with CmeB sequences. A wild-type CmeB from C. jejuni NCTC11168 (GenBank accession number NC_002163.1) and a resistance-enhancing CmeB from C. jejuni NT161 (GenBank accession number KT778507.1) are included as references.

Since 81.7% (179) of isolates were C. jejuni, they were used to compare the difference in the MIC distributions of antimicrobials between isolates carrying WT-CmeB and RE-CmeB. The difference in the MIC distribution between WT-CmeB and RE-CmeB-carrying isolates was statistically significant for clindamycin, florfenicol, azithromycin, and erythromycin (see Fig. S1 in the supplemental material). However, no resistance enhancement was observed for gentamicin (Fig. S1). Compared to the WT-CmeB-carrying isolates, RE-CmeB-carrying C. jejuni isolates could have 2- to 8-fold higher MICs for the 4 antimicrobials. Among the isolates that harbored an RE-CmeB but no other known resistance determinant, 16 and 18 C. jejuni isolates, respectively, had a MIC greater than the epidemiological cutoff values for clindamycin and florfenicol (Fig. S1). One RE-CmeB-carrying isolate (R18.1545) did not harbor any known resistance determinant but had MICs greater than the epidemiological cutoff values for azithromycin, nalidixic acid, and ciprofloxacin. Nonetheless, all other isolates carrying only RE-CmeB but no known resistant determinant displayed only low-level resistance to the antimicrobials.

A cgMLST tree was constructed to show the phylogenetic relationships among the RE-CmeB-carrying isolates. The cgMLST tree revealed that the 3 RE-CmeB-carrying C. coli isolates were located in a common lineage but widely distributed (Fig. 2). RE-CmeB-carrying C. jejuni isolates were distributed in multiple lineages. As indicated in Fig. 2, there were 5 clusters, each comprising closely related WT-CmeB-carrying and RE-CmeB-carrying isolates. The observation suggested that cmeB genes could be horizontally exchanged frequently among bacterial strains.

To investigate the origin of the RE-CmeB, CmeB sequences of C. coli and C. jejuni isolates were compared with those from other Campylobacter species. As shown in Fig. 4, the WT-CmeB sequences from C. coli and C. jejuni (cluster A) were more closely related to those from C. lari, C. fetus, C. hyointestinalis, C. upsaliensis, and C. hepaticus, whereas 1 RE-CmeB sequence in cluster B, which was found in 16 C. jejuni isolates from Taiwan, was identical to one carried by a C. fetus strain. Distantly related CmeB sequences were found not only in C. coli and C. jejuni but also in C. lari, C. fetus, C. hyointestinalis, and C. upsaliensis (Fig. 4).

FIG 4.

FIG 4

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Maximum parsimony tree constructed with CmeB sequences from Campylobacter species.

The CmeA sequences from the 219 isolates studied comprised 355 or 367 amino acid residues. Clustering analysis revealed two distinct clusters for the CmeA sequences from the 219 isolates; all isolates in cluster A were C. jejuni and all in cluster B were C. coli (Fig. S2). However, cluster A had no significant difference from cluster B in the MIC distribution of florfenicol (Fig. S2). Reanalysis of the sequences from 179 C. jejuni isolates only revealed two distinct clusters that had a significant difference in the MIC distribution of florfenicol (Fig. S3). The profiles of MIC distribution for clusters A and B of CmeA were concordant with those for clusters A and B of CmeB (Fig. S3). Except for one isolate (R17.0993), the isolates in clusters A and B of CmeB were, respectively, grouped in clusters A and B of CmeA. Isolate R17.0993 harbored a CmeB that was grouped in cluster B, but its CmeA was located in cluster A of CmeA. In addition, the CmeA from C. jejuni NT161, which carries a resistance-enhancing CmeABC, was also grouped in cluster A.

The CmeC sequences from the 219 isolates comprised 491 or 492 amino acid residues. The two CmeC clusters for the 179 C. jejuni isolates had no significant difference in the florfenicol MIC distribution (Fig. S3).

DISCUSSION

One primary purpose of this study is to predict the antimicrobial resistance in Campylobacter isolates recovered in Taiwan using WGS data. Several studies have applied WGS to assess the concordance between phenotypic resistance and genotypic resistance in Campylobacter and obtained promising results (21, 23, 24, 26, 46). Our data show that the phenotypic resistance to 6 antimicrobials, azithromycin, erythromycin, ciprofloxacin, nalidixic resistance acid, gentamicin, and tetracycline, are predicted accurately by the genetic resistance determinants identified using the AMRFinderPlus and ResFinder 4.1 pipelines (47, 48). However, the resistance rates for clindamycin and florfenicol determined by susceptibility testing are much higher than those predicted by the resistance determinants. Our data indicate that clindamycin- and florfenicol-resistant isolates carrying the known resistance genes, erm(B), fexA, cfr(C), and A2075G substitution in 23S rRNA, exhibit a higher level of resistance to the two antimicrobials. In contrast, resistant isolates that do not carry any of the relevant resistance determinants identified by the AMRFinderPlus and ResFinder 4.1 pipelines exhibit only low-level resistance to clindamycin and florfenicol.

Our analysis reveals that the low-level resistance to azithromycin, nalidixic acid, ciprofloxacin, clindamycin, and florfenicol is associated with sequence variations in CmeB. Further analysis indicates that the resistance enhancement could also be contributed by CmeA but not CmeC. CmeB is an inner membrane efflux transporter of the tripartite multidrug efflux pump CmeABC, belonging to the resistance-nodulation-division superfamily (49). CmeABC can expel structurally diverse molecules and heavy metals. Studies have shown that an impaired CmeB could result in a significant decrease in resistance to multiple classes of antimicrobials, heavy metals, and bile salts (50, 51). Structural modeling conducted by Yao and colleagues suggests that sequence variations in the drug-binding pocket of CmeB likely contribute to the enhanced efflux function (45). A Campylobacter strain harboring a resistance-enhancing CmeABC could have 8-, 4-, 4-, 2-, and 2-fold higher MICs for florfenicol, chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, respectively, than one carrying a WT-CmeABC (45). Our study also shows that isolates harboring an RE-CmeB could exhibit 2- to 8-fold higher MICs for azithromycin, erythromycin, clindamycin, and florfenicol than isolates carrying a WT-CmeB. However, the effect of RE-CmeB on resistance enhancement is not obvious for nalidixic acid, ciprofloxacin, and tetracycline, as most (91.8% to 96.3%) isolates have carried resistance determinants that confer high levels of resistance to the antimicrobials.

We also investigated the association between the MIC distributions of antimicrobials and CmeA sequence variations. CmeA sequences from the 179 C. jejuni isolates are grouped into two clusters with a short distance (Fig. S3). As shown in Fig. S3, the two clusters of CmeA have a significant difference in the MIC distribution of florfenicol (Fig. S3). The profiles of MIC distribution for the two clusters of CmeA are concordant with those for the clusters of CmeB. However, the CmeA sequence of C. jejuni NT161 that carries a resistance-enhancing CmeABC (45) is grouped in cluster A of CmeA in which isolates have a low range of florfenicol MICs (Fig. S3). Thus, further study is necessary to investigate the key variations in CmeA associated with resistance enhancement.

cgMLST analysis is applied to investigate the genetic relatedness among the Campylobacter isolates. The cgMLST tree reveals that some RE-CmeB-carrying C. jejuni isolates are very closely related to WT-CmeB-carrying isolates (Fig. 2), suggesting that a cmeB gene could be exchanged easily among C. jejuni strains. Our study further indicates that a cmeB gene could also be exchanged frequently across different Campylobacter species. For example, one RE-CmeB is found to be shared by 2 C. coli and 27 C. jejuni isolates (Fig. 3), while another RE-CmeB is found in 13 C. jejuni isolates from Taiwan and a C. fetus strain from another country (Fig. 4). Several studies have indicated that Campylobacter species can take up foreign DNA in high efficiency via the natural transformation mechanism (52). The mechanism should have played a critical role in the horizontal transfer of cmeB as well as other resistance determinants among Campylobacter strains and species.

As predicted by the identified resistance determinants, the Campylobacter isolates from human campylobacteriosis in Taiwan are highly resistant. All C. coli isolates and 88.3% of C. jejuni isolates characterized are expected to be multidrug-resistant. Both C. coli and C. jejuni displayed extremely high resistance prevalence to ciprofloxacin, nalidixic acid, tetracycline, and ampicillin, with a resistance rate ranging from 91.1% to 97.5% (Table 2). In total, the Campylobacter isolates have a moderate resistance prevalence (10.0% to 20.5%) to the macrolides (azithromycin, erythromycin), clindamycin, gentamicin, and chloramphenicol and a high resistance prevalence (21.5% to 33.3%) to aminoglycosides (kanamycin, streptomycin, and spectinomycin). However, C. coli is more resistant than C. jejuni. There are only 2.8% and 2.2% of C. jejuni isolates resistant to azithromycin and erythromycin, but 62.5% of C. coli isolates are resistant to the two macrolides. The two macrolides have been the drugs of choice for the treatment of human campylobacteriosis, as a large proportion of infections worldwide have been caused by fluoroquinolone-resistant strains (15, 53). The high prevalence of resistance to the two macrolides in C. coli is jeopardizing the medical treatment of campylobacteriosis, as nearly all Campylobacter isolates from Taiwan have been fluoroquinolone-resistant.

Macrolide resistance in Campylobacter species can be attributed to the target modification via nucleotide substitution and methylation in the 23S rRNA and the alteration of the 50S ribosomal subunit proteins L4 and L22, enzymatic inactivation of macrolides, and expelling of macrolides via efflux pump (54). In this study, the high-level resistance to macrolides (azithromycin and erythromycin) and lincosamide (clindamycin) in the Campylobacter isolates is attributed to erm(B) and A2057G substitution in 23S rRNA. Macrolide resistance due to A2057G mutation in Campylobacter species has been frequently reported (29); however, the resistance associated with erm(B) has not been commonly identified outside China (55). erm(B) encodes an rRNA methyltransferase that confers resistance to macrolide-lincosamide-streptogramin B (56). Macrolide resistance mediated by erm(B) has been detected in many Gram-positive bacterial species (57) and was first found in Campylobacter in 2014 (39). In Campylobacter, erm(B) is carried on chromosomes and plasmids (55). In this study, erm(B) is found in 6.8% of isolates (12 C. coli and 3 C. jejuni isolates). These erm(B)-carrying isolates are all multidrug-resistant, and each harbors at least 6 resistance determinants conferring resistance to multiple classes of antimicrobials. To determine the location of erm(B), we further determined the complete genome sequences of 2 genetically distant erm(B)-carrying C. coli isolates, R18.1828 (ST5506; sequences submitted under GenBank accession number NZ_CP076509) and R19.1157 (ST1145; GenBank accession number NZ_CP076513). Both erm(B)-carrying isolates concurrently harbor aac(6′)-Ie/aph(2″)-Ia, aad9, aadE, ant(6)-Ia, aph(3′)-IIIa, blaOXA-193, sat4, and tet(O/M/O). The two isolates have no plasmid; erm(B) is located in different chromosomal multidrug resistance genomic islands. Another analysis also indicates that the 15 erm(B)-carrying isolates are distributed in multiple distinct lineages. Thus, the isolates should have acquired erm(B) from multiple source attributions. Campylobacter species can acquire DNA horizontally via natural transformation (52); the capability would facilitate the acquisition and spread of resistance determinants among bacterial strains. The emergency and spread of erm(B) among Campylobacter species have sounded an alarm to the antimicrobial treatment of severe campylobacteriosis in Taiwan.

An intact cfr(C) gene is carried by a C. coli isolate, which is multidrug-resistant to high levels of florfenicol, clindamycin, azithromycin, erythromycin, nalidixic acid, ciprofloxacin, and tetracycline. The cfr(C) gene was first identified in a conjugative plasmid in a C. coli isolate and detected in 10% of C. coli but not in C. jejuni isolates from different cattle farms in the United States (38). cfr(C) encodes an rRNA methyltransferase; it belongs to cfr multiresistance gene family and confers resistance to at least 5 antimicrobial classes, phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (38, 58). The study conducted by LaMarre et al. (59) indicates that a cfr-carrying Staphylococcus aureus strain could have a low fitness cost. Thus, cfr(C)-carrying C. coli could also fit well and could have the potential to spread rapidly.

WGS data are useful for the prediction of antimicrobial resistance and are also frequently used in the epidemiological study of bacterial organisms for disease surveillance and outbreak detection (20, 60). In this study, the conventional 7-locus MLST profiles and the core genome MLST profiles are retrieved from the WGS data to investigate genetic relatedness among the isolates. As expected, the 7-locus MLST data show that the Campylobacter isolates from human campylobacteriosis in Taiwan belong to diverse clones (Fig. 1). The cgMLST analysis, based on 2,242 core genes, provides a much higher resolution in discriminating among clonal isolates (Fig. 2). The cgMLST analysis identifies 24 clusters of closely related isolates; isolates in each cluster differ by 10 loci or less. Among the 24 clusters, only 5 contain isolates that are epidemiologically related; 19 clusters consist of epidemiologically unrelated isolates recovered in various years and locations (counties or cities). Because the isolates characterized in this study represent only a very small proportion of infections in Taiwan, the isolates in each of the clusters with no epidemiological relevance may still likely link to a prolonged ongoing outbreak during that period. Campylobacteriosis is not a notifiable disease in Taiwan; the prevalence of this disease is still unknown. Thus, a national campylobacteriosis surveillance program is needed in this country for monitoring the trend of antimicrobial resistance and real-time detection of ongoing disease clusters.

In conclusion, Campylobacter isolates recovered from humans in Taiwan from 2016 to 2019 are highly resistant; more than 90% of isolates are resistant to quinolones/fluoroquinolones (nalidixic acid/ciprofloxacin), β-lactams (ampicillin etc.), and aminoglycosides (kanamycin etc.). C. coli is more resistant than C. jejuni. All C. coli and 88.3% of C. jejuni isolates are multidrug-resistant. Although C. jejuni has a low resistance prevalence for azithromycin, erythromycin, and clindamycin, the primary alternative drugs for the treatment of fluoroquinolone-resistant Campylobacter infections, a large proportion (62.5%) of C. coli isolates have been resistant to these 3 antimicrobials. This study indicates that resistance determinants identified from WGS data using the AMRFinderPlus and ResFinder 4.1 pipelines can accurately predict high-level resistance in isolates to the corresponding antimicrobials. However, sequence variations in the CmeA and CmeB components of the CmeABC efflux pump can contribute to low-level resistance of many antimicrobials, in this study, including azithromycin, nalidixic acid, ciprofloxacin, clindamycin, and florfenicol. Alarmingly, a large proportion of C. coli isolates have been found to be resistant to azithromycin, erythromycin, and clindamycin, which would complicate the treatment of invasive campylobacteriosis in Taiwan.

MATERIALS AND METHODS

Bacterial isolates.

The 219 Campylobacter sp. isolates included in this study were randomly selected from a collection of 1,234 isolates obtained from 21 collaborative hospitals in Taiwan between 2016 and 2019. All isolates were recovered from humans with campylobacteriosis and were submitted, under the request of a foodborne disease surveillance project, to the Taiwan Centers for Disease Control (Taiwan CDC) for molecular typing using an international standardized pulsed-field gel electrophoresis protocol. The collaborative hospitals were recruited roughly based on the population in the four regions—northern Taiwan (10 hospitals), central Taiwan (4 hospitals), southern Taiwan (6 hospitals), and eastern Taiwan (1 hospital). Each hospital submitted 1 to 436 isolates to Taiwan CDC over the 4 years. There were 83.6% of the isolates recovered from patients in northern Taiwan, where around half of the national population resides. The Campylobacter species of the isolates were identified using the Bruker matrix-assisted laser desorption ionization (MALDI) biotyper (Bruker Daltonics).

WGS and sequence analysis.

WGS of bacterial isolates was conducted in the Central Region laboratory of Taiwan CDC using the Illumina MiSeq sequencing platform. DNA of isolates was extracted using the Qiagen DNeasy blood and tissue kit (Qiagen Co., Germany); library construction was performed using the Illumina DNA Prep, (M) Tagmentation system (Illumina Co., USA), and sequencing was run with the MiSeq reagent kit version 3 (600 cycles). All the procedures were manipulated by following the manufacturer’s instructions. Illumina sequence reads were assembled using SPAdes assembler version 3.12.0 (http://cab.spbu.ru/software/spades/) (61). The assembled sequences were subjected to identification of bacterial species using FastANI (https://github.com/ParBLiSS/FastANI) (62), antimicrobial resistance genes using AMRFinderPlus (https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/) (48), point mutations in genes that are associated with antimicrobial resistance using AMRFinderPlus and the ResFinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/) (47, 48), and sequence types (STs) using the tool and database of PubMLST (https://pubmlst.org/organisms/campylobacter-jejunicoli) (63). Genetic relationships among isolates constructed with the conventional 7-locus MLST profiles were performed using the tools provided in the BioNumerics software version7.6 (Applied Maths; bioMérieux).

cgMLST analysis.

cgMLST profiles of isolates were generated from assembled WGS reads using the Campylobacter cgMLST scheme and the profiling tool provided on the cgMLST@Taiwan website (http://rdvd.cdc.gov.tw/cgMLST/). The cgMLST scheme, comprising 2,242 core genes identified from 250 C. coli and 250 C. jejuni genomes, was developed in-house for fine typing of C. coli and C. jejuni. Genetic relationships among isolates constructed with cgMLST profiles were performed using the tools provided in the BioNumerics software version 7.6.

Antimicrobial susceptibility testing.

The MIC of antimicrobials for isolates was determined in the laboratory of Taiwan CDC using commercial Sensititre Campylobacter CAMPY AST panel plates (Thermo Fisher Scientific Co.) following the manufacturer’s instructions. All Campylobacter isolates were grown for 24 h at 37°C in microaerophilic conditions before susceptibility tests were conducted. C. jejuni ATCC 33560 was used for quality control. The antimicrobials in the panel include azithromycin, erythromycin, clindamycin, nalidixic acid, ciprofloxacin, florfenicol, gentamicin, and tetracycline. The susceptibility results were interpreted using the epidemiological cutoff values set by the U.S. National Antimicrobial Resistance Monitoring System for Campylobacter isolates (https://www.cdc.gov/narms/antibiotics-tested.html).

CmeABC sequences analysis.

CmeABC is a tripartite multidrug efflux pump in Campylobacter (49); amino acid sequence variations in the three protein components CmeA, CmeB, and CmeC likely affect the evicting function of the efflux pump for certain antimicrobials. To compare the amino acid sequences of CmeA, CmeB, and CmeC, we retrieved the sequences from the WGS data of Campylobacter isolates characterized in this study. CmeA, CmeB, and CmeC sequences from C. jejuni NCTC11168 (GenBank accession number NC_002163.1), which carries a wild-type CmeABC, and C. jejuni NT161 (GenBank accession number KT778507.1), which carries a resistance-enhancing CmeABC, were included as references. CmeB sequences for various Campylobacter species were obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). The phylogenetic analysis of CmeA, CmeB, and CmeC sequences was performed using the tools provided in BioNumerics software version 7.6.

Data availability.

The WGS reads for the 219 Campylobacter isolates used in this study have been submitted to the Sequence Read Archive (SRA) database of the NCBI under accession numbers SRR11942617 to SRR11942651, SRR11942653 to SRR11942672, SRR11942674 to SRR11942679, SRR11942681 to SRR11942705, and SRR11942707 to SRR11942841. CmeB sequences carried by various Campylobacter species were obtained from the NCBI database, including C. canadensis (GenBank accession number WP_172233987), C. concisus (MBS5828900), C. corcagiensis (QKF63970), C. cuniculorum (WP_027305763), C. curvus (WP_018137400), C. fetus (EAK5660056, EAJ6189929, EAJ0321195), C. gracilis (MBS6153299), C. helveticus (WP_139455163), C. hepaticus (WP_124134124), C. hominis (WP_012108439), C. hyointestinalis (EAI7421010, WP_059432821), C. insulaenigrae (WP_147500214), C. lari (EAI9310238, EAK9946776), C. mucosalis (WP_169763585), C. peloridis (WP_044599442), C. rectus (QCD47857), C. showae (WP_122871822), C. sputorum (ASM37806), C. subantarcticus (WP_039664716), C. upsaliensis (EAK1340833, EAL3986340), C. ureolyticus (WP_016646469), and C. volucris (WP_195684951).

ACKNOWLEDGMENTS

This study was funded by the Ministry of Health and Welfare, Taiwan (grant number MOHW109-CDC-C-315-144406).

We sincerely thank the hospitals for providing Campylobacter isolates for study. The hospitals include Hualien Tzu Chi Hospital, Taipei Tzu Chi Hospital, Changhua Christian Hospital, Chiayi Christian Hospital, Pingtung Christian Hospital, Chiayi Chang Gung Memorial Hospital, Kaohsiung Chang Gung Memorial Hospital, Linkou Chang Gung Memorial Hospital, China Medical University Hospital, Chung Shan Medical University Hospital, Far Eastern Memorial Hospital, Kaohsiung Veterans General Hospital, Taipei Veterans General Hospital, Lotung Pohai Hospital, MacKay Memorial Hospital, Mackay Memorial Hospital Hsinchu Branch, National Cheng Kung University Hospital, National Taiwan University Hospital, National Taiwan University Hospital Yun-Lin Branch, Shuangho Hospital of Ministry of Health and Welfare, and Tri-Service General Hospital.

Footnotes

Supplemental material is available online only.

Supplemental file 1
supplemental material. Download AAC.01736-21-s0001.pdf, PDF file, 0.8 MB (810.4KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

supplemental material. Download AAC.01736-21-s0001.pdf, PDF file, 0.8 MB (810.4KB, pdf)

Data Availability Statement

The WGS reads for the 219 Campylobacter isolates used in this study have been submitted to the Sequence Read Archive (SRA) database of the NCBI under accession numbers SRR11942617 to SRR11942651, SRR11942653 to SRR11942672, SRR11942674 to SRR11942679, SRR11942681 to SRR11942705, and SRR11942707 to SRR11942841. CmeB sequences carried by various Campylobacter species were obtained from the NCBI database, including C. canadensis (GenBank accession number WP_172233987), C. concisus (MBS5828900), C. corcagiensis (QKF63970), C. cuniculorum (WP_027305763), C. curvus (WP_018137400), C. fetus (EAK5660056, EAJ6189929, EAJ0321195), C. gracilis (MBS6153299), C. helveticus (WP_139455163), C. hepaticus (WP_124134124), C. hominis (WP_012108439), C. hyointestinalis (EAI7421010, WP_059432821), C. insulaenigrae (WP_147500214), C. lari (EAI9310238, EAK9946776), C. mucosalis (WP_169763585), C. peloridis (WP_044599442), C. rectus (QCD47857), C. showae (WP_122871822), C. sputorum (ASM37806), C. subantarcticus (WP_039664716), C. upsaliensis (EAK1340833, EAL3986340), C. ureolyticus (WP_016646469), and C. volucris (WP_195684951).

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