Antimicrobial Resistance of Campylobacter Species Isolated from Broilers in Live Bird Markets in Shanghai,China

Abstract

This study was conducted to determine the prevalence of antimicrobial resistance in Campylobacter spp. isolates from broilers in live bird markets (LBMs). A total of 209 Campylobacter spp. isolates (84 Campylobacter jejuni; 125 Campylobacter coli) were recovered from 364 broiler cecum samples collected from five LBMs in Shanghai, China. Minimum inhibitory concentrations of 13 antimicrobials were determined using agar dilution method. More than 96% of the Campylobacter spp. isolates were resistant to quinolones and tetracyclines. A high prevalence of macrolide resistance (erythromycin, 84.0%; azithromycin, 80.8%) was observed in C. coli, but not in C. jejuni (erythromycin, 6.0%; azithromycin, 2.4%). C. coli also showed significantly higher resistance than C. jejuni to clindamycin, gentamicin, and kanamycin. In contrast, C. coli isolates had lower resistance to florfenicol than the C. jejuni isolates. The majority of the C. jejuni (88.1%) and C. coli (97.6%) isolates exhibited multidrug resistance (MDR) to three or more classes of antimicrobials. All of the 208 ciprofloxacin-resistant Campylobacter spp. isolates were positive for the C257T mutation of the gyrA gene. In addition, the tet(O) gene was identified in all of the 202 doxycycline-resistant Campylobacter spp. isolates. Furthermore, 75.7% and 20.4% of the 103 azithromycin-resistant Campylobacter spp. isolates were positive for the A2075G mutation of the 23S rRNA gene and the presence of the erm(B) gene, respectively. Moreover, the cat gene was found in 14.3% (8/56) and 76.8% (73/95) of the chloramphenicol-resistant C. jejuni and C. coli isolates, respectively. To the best of our knowledge, this is the first report of the prevalence of antimicrobial resistance among Campylobacter spp. isolates originating from LBMs. The high prevalence of MDR Campylobacter spp. isolates in LBMs highlights the need to implement efficient intervention measures to control not only Campylobacter contamination in LBMs but also dissemination of antimicrobial resistance among Campylobacter spp. in poultry production.

Introduction

C ampylobacter spp., especially Campylobacter jejuni and Campylobacter coli, are leading causes of bacterial gastroenteritis globally (Koluman and Dikici, 2013). Although usually self-limiting, severe or long-lasting Campylobacter spp. infections, especially of patients with compromised immune systems, often require antimicrobial therapy (McGill et al., 2009). Campylobacter spp. are prevalent in food-producing animals, and the use of antimicrobials in food animal production has led to an increasing prevalence of antimicrobial-resistant Campylobacter spp. infection transmitted to humans through the food chain, thereby presenting a major threat to public health (Mäesaar et al., 2016).

The handling and consumption of contaminated poultry, particularly broilers, seem to be the most common risk factor (Damjanova et al., 2011). Broilers are regarded as a main reservoir of Campylobacter spp., and the colonization level of Campylobacter spp. in broiler ceca can reach 10⁹ CFU/g of cecal content (Stern et al., 2008). Campylobacter contamination often occurs during the slaughtering process and consumption of meat from a contaminated carcass is a significant source of human campylobacteriosis (Melero et al., 2012).

In China and other Asian countries, live bird markets (LBMs) are among the most important termini of the poultry industry, as people in these countries prefer to purchase live or freshly slaughtered poultry. Birds sold in LBMs are continuingly introduced from different sources and caged at high densities with those collected from other areas. These conditions provide an optimal environment for amplification and persistence of infectious agents. Hence, LBMs are potential reservoirs of poultry-associated pathogens, such as various avian influenza viruses that continue to cause human infection and death (Zhou et al., 2009; Cowling et al., 2013; He et al., 2014). In addition, unlike other consummation termini of the poultry industry, consumers at LBMs are in direct and close contact with live or freshly slaughtered poultry. Together, the conditions of LBMs likely increase the risk for human Campylobacter spp. infection. However, studies concerning Campylobacter spp. from LBMs are very limited (Mansouri-najand et al., 2012). Therefore, in this study, we investigated the prevalence and antimicrobial resistance of C. jejuni and C. coli isolates collected from five LBMs in Shanghai, one of the largest cities in China.

Materials and Methods

Sample collection

From November 2012 to April 2013, a total of 364 cecum samples from broilers were randomly collected from five LBMs located in five geographically separated districts (Minhang, Fengxian, Pudong, Songjiang, and Yangpu) of Shanghai (Table 1). For each LBM, 71–77 samples were collected at three to four time points between November 2012 and April 2013. Each cecum sample was collected from a different chicken during evisceration and put into a stomacher bag, and then rapidly transported on ice to our laboratory within 2 h.

Table 1.

The Prevalence of Campylobacter Species in Cecum Samples Collected from LBMs in Shanghai, China

District	Nos. of cecum samples	No. of Campylobacter jejuni^a	No. of Campylobacter coli^a	No. of total isolates from each district ^a
Minhang	71	20 (28.2)	47 (66.2)	67 (94.4)
Fengxian	77	16 (20.8)	22 (28.6)	38 (49.4)
Songjiang	73	12 (16.4)	34 (46.6)	46 (63)
Putuo	71	20 (28.2)	8 (11.3)	28 (39.4)
Yangpu	72	16 (22.2)	14 (19.4)	30 (41.7)
Total (%)	364	84 (23.1)	125 (34.3)	209 (57.4)

Numbers in parentheses indicate percentages.

LBM, live bird market.

Isolation and identification of Campylobacter spp.

A loopful of fecal content from each sample was directly streaked onto a Campylobacter spp. Selective Agar (Base) plate (Sigma, St. Louis, MO) containing 5% fresh sterile defibrinated sheep blood and Campylobacter supplement III (Sigma) (Ma et al., 2014). The plates were incubated under microaerobic conditions (CO₂, 10%; O₂, 5%; N₂, 85%) at 42°C for 48 h. One presumptive colony from each plate was subcultured and identified by PCR strategies (Keramas et al., 2003) and API-Campy kits (BioMérieux, Marcy l'Etoile, France). All the identified isolates were stored at −80°C in brain-heart infusion broth with 20% (v/v) glycerol.

Antibiotic susceptibility testing

The standard agar dilution method, as described by the Clinical Laboratory Standards Institute (CLSI, 2010), was employed to determine the susceptibility of Campylobacter spp. isolates to 13 antibiotic agents: nalidixic acid, ciprofloxacin, enrofloxacin, levofloxacin, erythromycin, tetracycline, doxycycline azithromycin, clindamycin, chloramphenicol, florfenicol, gentamicin, and kanamycin. These antimicrobials, with the exception of nalidixic acid (Sigma), were all obtained from the China Institute of Veterinary Drug Control (Beijing, China). Breakpoints for each antimicrobial agent and minimum inhibitory concentration (MIC) ranges for quality control strains C. jejuni ATCC 33560 and Escherichia coli ATCC25922 are shown in Table 2. A Campylobacter spp. isolate simultaneously resistant to three or more classes of antimicrobials was defined as multidrug resistant.

Table 2.

Antimicrobial Susceptibility of 84 C. jejuni and 125 C. coli Strains Isolated from LBMs in Shanghai, China

	MIC QC range (μg/mL) ^a		MIC breakpoint (μg/mL) ^b			C. jejuni		C. coli
Antimicrobial agent	ATCC33560	ATCC25922	S	I	R	MIC₅₀/MIC₉₀ (μg/mL)	Resistant isolates (%) ^c	MIC₅₀/MIC₉₀ (μg/mL)	Resistant isolates (%) ^c
Nalidixic acid	4–16	—	≤16	32	≥64	256/256	84 (100)	256/256	125 (100)
Ciprofloxacin	0.06–0.5	—	≤1	2	≥4	128/256	83 (98.8)	32/128	125 (100)
Enrofloxacin	—	0.008–0.03	≤0.5	1–2	≥4	16/128	83 (98.8)	8/16	122 (97.6)
Levofloxacin	0.03–0.25	—	≤2	4	≥8	32/256	83 (98.8)	16/32	122 (97.6)
Erythromycin	1–4	—	≤8	16	≥32	2/8	5 (6.0)	256/256	105 (84.0)
Azithromycin	0.03–0.12	—	≤2	4	≥8	0.125/1	2 (2.4)	256/256	101 (80.8)
Clindamycin	0.12–0.5	—	≤2	4	≥8	2/8	14 (16.7)	64/256	106 (84.8)
Chloramphenicol	1–4	—	≤8	16	≥32	32/64	56 (66.7)	32/64	95 (76.0)
Florfenicol	0.5–2	—	≤4	8	≥16	32/64	67 (79.8)	4/8	8 (6.4)
Tetracycline	0.25–1	—	≤4	8	≥16	256/256	83 (98.8)	256/256	122 (97.6)
Doxycycline	0.25–2	—	≤2	4	≥8	64/128	81 (96.4)	64/128	121 (96.8)
Gentamicin	0.5–4	—	≤2	4	≥8	0.25/256	15 (17.9)	256/256	117 (93.6)
Kanamycin	—	1–4	≤16	32	≥64	8/256	23 (27.4)	256/256	116 (92.8)

The QC ranges of C. jejuni ATCC 33560 were directly adopted from CLSI (2010). Due to the lack of QC ranges of C. jejuni ATCC 33560 for enrofloxacin and kanamycin, we used E. coli ATCC 25922 as QC strain for these two antimicrobial agents (CLSI, 2010).

MIC breakpoints for nalidixic acid, ciprofloxacin, erythromycin, azithromycin, clindamycin, florfenicol, tetracycline, doxycycline, and gentamicin are those recommended by the CLSI (2010). Since standardized MIC breakpoints for enrofloxacin, levofloxacin, chloramphenicol, and kanamycin are not available for Campylobacter spp., we used the breakpoints of Enterobacteriaceae for these four antimicrobial agents as recommended by CLSI (2010).

Numbers in parentheses indicate percentages.

I, intermediate; QC, quality control; R, resistant; S, susceptible.

Detection of resistance determinants

The presence of genes involved in macrolide, tetracycline, and phenicol resistance was detected by PCR. Genomic DNA of Campylobacter spp. isolates was extracted using the TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China), according to the manufacturer's directions, and used as templates for subsequent PCR reactions. The detection of the tet(O) (Qin et al., 2011), cat (Wang and Taylor, 1990), cfr (Kehrenberg and Schwarz, 2004), cmlA & floR (Dai et al., 2008), and erm(B) (Spiliopoulou et al., 2004) genes was conducted by PCR analysis, as previously described. In addition, the mismatch amplification mutation assay (MAMA) PCR was used to detect the C257T mutation in the quinolone resistance-determining region (QRDR), which confers high-level quinolone resistance (Zirnstein et al., 2000), and the mutations A2074C and A2075G in the 23S rRNA gene, which are associated with macrolide resistance (Alonso et al., 2005). Moreover, the macrolide-resistant isolates were also screened for mutations in the L4 and L22 ribosomal protein genes (Cagliero et al., 2006).

Statistical analysis

The resistance frequencies for each class of antimicrobial agents and multidrug resistance (MDR) rates between C. jejuni and C. coli isolates from LBMs were compared with the chi-square test using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL). Differences were considered significant at p < 0.05.

Results and Discussion

Incidence of Campylobacter spp.

Overall, Campylobacter spp. were isolated in 209 (57.4%) of the 364 broiler cecum samples. Campylobacter spp. isolation rates varied from 39.4% to 94.4% across the five surveyed LBMs (Table 1). Among these samples, the prevalence of C. jejuni and C. coli was 23.1% and 34.3%, respectively, which corresponds well with previous reports on the greater proportion of C. coli than C. jejuni in broilers (Henry et al., 2011; Ma et al., 2014). However, the observation that C. coli was the predominant Campylobacter spp. in broilers was contrary to other reports (Chen et al., 2010; Anderson et al., 2012). It was consistent with the speculation that C. coli, instead of C. jejuni, is becoming the predominant Campylobacter spp. in chickens in China, which might be attributed to the fact that C. coli is intrinsically more adaptable to antimicrobial treatment, and the extensive use of antimicrobials in broiler production may have favorably selected C. coli over C. jejuni (Ma et al., 2014; Wang et al., 2016).

Quinolone resistance

High prevalence of quinolone resistance was observed among the 209 Campylobacter spp. isolates (nalidixic acid, 100%; ciprofloxacin, 99.5%; enrofloxacin, 98.1%; levofloxacin, 98.1%) (Table 2). These results are similar to previous observations in China that almost 100% of Campylobacter spp. strains isolated from chickens were resistant to quinolones (Chen et al., 2010; Ma et al., 2014), which might be caused by the extended use of these antimicrobials in chicken production. In contrast, a very low incidence of quinolone-resistant Campylobacter spp. has been reported in Canada (Agunos et al., 2013), the United States (Ge et al., 2013), and France (Guyard-Nicodème et al., 2015), which may be benefited from prudent use policy of antimicrobials in these countries.

Quinolone resistance in Campylobacter spp. is usually conferred by point mutations in the QRDR of DNA gyrase A (GyrA), and the C257T mutation, leading to the Thr-86-Ile substitution in the gyrase, is the most frequently detected (Hormeño et al., 2016). All of the 208 Campylobacter spp. (C. jejuni = 83; C. coli = 125) isolates resistant to ciprofloxacin were positive for the C257T mutation in the QRDR of gyrA. However, some Campylobacter spp. isolates examined in this study showed a high level of fluoroquinolone resistance. For example, 21 of 84 C. jejuni strains exhibited ciprofloxacin MICs of ≥256 μg/mL. However, the C257T mutation is not sufficient to confer such a high level of fluoroquinolone resistance in Campylobacter spp.; thus other mechanisms, such as additional mutations the QRDR of gyrA and enhanced activity of the MDR efflux pump CmeABC, may be involved (Guo et al., 2010; Dai et al., 2015).

Tetracycline resistance

High tetracycline (tetracycline, 98.1%; doxycycline, 96.7%) resistance rate was also present in the Campylobacter spp. isolates of this study, which was consistent with other investigations performed in China (Chen et al., 2010; Ma et al., 2014). Tetracycline resistance in Campylobacter spp. is mainly conferred by the ribosomal protection protein gene tet(O), which is located either on the chromosome or a transmissible plasmid (Gibreel et al., 2004; Abdi-Hachesoo et al., 2014). In this study, all the 202 doxycycline-resistant Campylobacter spp. strains (81 C. jejuni and 121 C. coli) were positive for the tet(O) gene. Interestingly, three doxycycline-sensitive and tet(O)-negative Campylobacter spp. isolates (2 C. jejuni and 1 C. coli) exhibited high-level resistance to tetracycline (MIC, ≥256 μg/mL), indicating the possible existence of an unknown tetracycline resistance mechanism in these trains.

Macrolide and lincosamide resistance

The prevalence of macrolide and lincosamide resistance in C. coli was significantly higher than in C. jejuni (erythromycin, 84.0% vs. 6.0%; azithromycin, 80.8% vs. 2.4%; clindamycin, 84.8% vs. 16.7%, respectively) (Table 2) (p < 0.01). Resistance rates of C. coli (84.0%) to erythromycin were lower than that reported in previous studies performed in China (Chen et al., 2010; Ma et al., 2014), but higher than in Australia (Obeng et al., 2012) and Poland (Wieczorek et al., 2013). Furthermore, the incidence of erythromycin-resistant C. jejuni (6.0%) was comparable to some other findings (Gblossi Bernadette et al., 2012; Torralbo et al., 2015), but higher than that reported in most previous studies (Obeng et al., 2012; Shin et al., 2012).

In Campylobacter spp., macrolide resistance is often mediated by point mutations in the 23S rRNA gene, especially A2074C and A2075G (Lehtopolku et al., 2011). In this study, no A2074C mutation was detected among the 103 azithromycin-resistant Campylobacter spp. isolates (2 C. jejuni and 101 C. coli), while 75.7% (78/103) of these strains, except 25 C. coli, harbored the A2075G point mutation as determined by MAMA PCR. Screening of the erm(B) gene and mutations in the L4 and L22 genes in the 25 C. coli isolates without the 23 s rRNA mutations revealed that 21 harbored the erm(B) gene, but no L4 or L22 mutations were found. The erm(B)-positive rate in LBM-origin Campylobacter spp. isolates (10.0%, 21/209) was significantly higher than that in Campylobacter spp. strains from other sources (3.7%, 58/1554) (Wang et al., 2014). The high density and continued introduction of broilers in LBMs may provide optimal conditions for the spread and persistence of erm(B)-carrying Campylobacter spp. isolates, finally resulting in the high incidence of the erm(B) gene. Notably, there were four C. coli isolates demonstrating high-level macrolide resistance that did not contain the 23 s rRNA mutations or the erm(B) gene. This may be attributed to the enhanced activity of the CmeABC efflux pump and/or other unknown mechanisms, thus further studies are warranted (Guo et al., 2010; Qin et al., 2014).

Phenicol resistance

Interestingly, the florfenicol resistance rate of C. jejuni was significantly higher compared with C. coli (79.8% vs. 6.4%, respectively) (Table 2) (p < 0.01), but the Campylobacter spp. with the higher chloramphenicol resistance rate was C. coli, rather than C. jejuni (76.0% vs. 66.7%, respectively). 29.2% Campylobacter spp. strains (C. jejuni, 54; C. coli, 7) were resistant to both phenicols. Low chloramphenicol resistance rates in Campylobacter spp. isolates were found in investigations conducted in Spain (Perez-Boto et al., 2013), Japan (Ozawa et al., 2012), and Iran (Chakeri et al., 2012). However, it is worth noting that the incidence of chloramphenicol resistance among Campylobacter spp. isolates was very high in China (Chen et al., 2010) and Brazil (de Moura et al., 2013).

In this study, of the 151 (C. jejuni, 56; C. coli, 95) chloramphenicol-resistant Campylobacter spp. isolates, 81 (C. jejuni, 8; C. coli, 73) were positive for the cat gene, but not cmlA. In addition, the floR and cfr genes, which confer florfenicol resistance, were not detected in any of the 75 (C. jejuni, 67; C. coli, 8) florfenicol-resistant Campylobacter spp. isolates.

Aminoglycoside resistance

A very high prevalence of gentamicin (93.6%) and kanamycin (92.8%) resistance was observed among the C. coli isolates, but the corresponding resistance rates of C. jejuni were only 17.9% and 27.4%, respectively (Table 2). In addition, as mentioned above, the resistance rates of the C. coli isolates to azithromycin and clindamycin were also significantly higher than those of the C. jejuni isolates. Some previous studies also reported that resistance to some antimicrobial classes (i.e., aminoglycosides, macrolides, and lincosamides) was much higher in C. coli than C. jejuni (Ma et al., 2014; Torralbo et al., 2015). Further research is needed to better elucidate the molecular mechanism leading to the differences in antimicrobial resistance between C. jejuni and C. coli.

Multidrug resistance

As is shown in Table 3, all, but one, of the Campylobacter spp. isolates were resistant to two or more classes of antimicrobials. Overall, 196 (93.8%) of the 209 Campylobacter spp. isolates exhibited MDR. In addition, the MDR rate was significantly higher (p < 0.05) among C. coli isolates than C. jejuni (97.6%, 122/125 vs. 88.1%, 74/84, respectively). The most common antimicrobial resistance pattern in C. jejuni (48.8%, 41/84) was the combination of quinolones, phenicols, and tetracyclines. In C. coli, 87 of 125 (69.6%) isolates exhibited resistance to all of the antimicrobial classes tested. The difference in dominant MDR patterns between the Campylobacter spp. might be due to the intrinsic ability of C. coli to develop resistance to these antimicrobials (Chen et al., 2010). Interestingly, a similar high MDR prevalence was also observed in the surveillance studies concerning human-origin Campylobacter spp. in China (Zhang et al., 2014; Zhou et al., 2016). The extremely high rates of antimicrobial resistance in Campylobacter spp. isolates from human and LBM origin suggested that some of the antimicrobial classes, such as quinolones and tetracyclines, may no longer be suitable for the treatment of human Campylobacter infection in China.

Table 3.

Antimicrobial Resistance Patterns of Campylobacter Isolates from Five LBMs in Shanghai, China

	No. (%) of Campylobacter strains
Antimicrobial resistance patterns ^a	C. jejuni (n = 84) ^b	C. coli (n = 125) ^b
Q	1 (1.2)	0
Q-A	0	1 (0.8)
Q-T	9 (10.7)	2 (1.6)
Q-M-T	0	1 (0.8)
Q-P-A	0	1 (0.8)
Q-P-T	41 (48.8) ^c	1 (0.8)
Q-T-A	4 (4.8)	6 (4.8)
Q-L-P-T	3 (3.6)	0
Q-L-T-A	1 (1.2)	3 (2.4)
Q-M-L-A	0	1 (0.8)
Q-M-P-T	1 (1.2)	0
Q-M-T-A	0	1 (0.8)
Q-P-T-A	12 (14.3)	6 (4.8)
Q-L-P-T-A	8 (9.5)	0
Q-M-L-P-T	1 (1.2)	1 (0.8)
Q-M-L-T-A	0	14 (11.2)
Q-M-P-T-A	2 (2.4)	0
Q-M-L-P-T-A	1 (1.2)	87 (69.6) ^c
MDR (%)^d	74 (88.1)	122 (97.6)

Abbreviations of antimicrobial agents: Q, quinolones (nalidixic acid, ciprofloxacin, enrofloxacin, and levofloxacin); P, phenicols (florfenicol and chloramphenicol); M, macrolides (erythromycin and azithromycin); T, tetracyclines (tetracycline and doxycycline); L, clindamycin; A, aminoglycosides (gentamicin and kanamycin).

n, the number of Campylobacter isolates.

The bold text indicates the major resistance patterns.

The number (%) of multidrug-resistant Campylobacter.

Conclusions

This is the first report addressing antimicrobial resistance of Campylobacter spp. isolated from LBMs in China. An extremely high rate of antimicrobial resistance was observed in the Campylobacter spp. isolates, which was likely due to the extensive use of various antimicrobials in poultry production. Furthermore, given the fact that consumers purchasing poultry from LBMs have direct contact with live or freshly slaughtered birds, the high prevalence of MDR Campylobacter spp. isolates in LBMs may pose a high risk factor for human Campylobacter spp. infection and treatment. The findings of this study highlight the need for efficient measures to control Campylobacter spp. contamination in LBMs, while the extensive antimicrobial resistance among Campylobacter spp. isolates examined in this study underlines the need for prudent use of antimicrobials in poultry production to minimize the emergence and spread of antibiotic-resistant Campylobacter spp. strains.

Footnotes

Acknowledgment

This work was funded by the National Natural Science Foundation of China (grant nos. 31302145 and 31672606).

Disclosure Statement

No competing financial interests exist.

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