Antimicrobial Susceptibilities of Campylobacter jejuni and Campylobacter coli Strains Isolated from Two Early Stages of Poultry Production

Abstract

Our aim was to monitor the resistance of Campylobacter isolates from two initial stages of broiler production in 5 grandparent breeder broiler farms (GPBFs) and 12 parent breeder broiler farms (PBFs) in which no antimicrobials were used during the study. Susceptibility tests were carried out for 805 strains (697 Campylobacter jejuni and 108 Campylobacter coli) against nalidixic acid, ciprofloxacin, erythromycin, amoxicillin, amoxicillin plus clavulanic acid, tetracycline, gentamicin, and chloramphenicol using the disk-diffusion method. Quinolone resistance was the most abundant overall (74.9%) and at each stage of production. The second largest resistance was for tetracycline with 48.2%. The resistance against amoxicillin plus clavulanic acid, gentamicin, and chloramphenicol was not found. The percentages of resistance and multidrug-resistant (MDR) isolates were always higher in the PBFs than in the GPBFs. However, pan-susceptible populations (total 10.3%) were isolated in our survey. C. coli isolates were more resistant to tetracycline and erythromycin (96.3% and 23.1%, respectively) than for C. jejuni (40.7% and 0%, respectively) and were more MDR (33.3% vs. 11.9%). In conclusion, as other authors have shown, even in the absence of antibiotic pressure, relatively high rates of quinolone resistance are found in Campylobacter. However, a decrease in quinolone resistance has been observed compared to other studies in Spain [i.e., 99%; Saenz et al. Antimicrob. Agents Chemother. 2000;44(2):267–271]. MDR, fluoroquinolone-, macrolide-, and tetracycline-resistant Campylobacter populations are issues of concern in public health.

Introduction

Thermophilic species of Campylobacter spp. are considered as the main cause for bacterial gastroenteritis worldwide. Most of the campylobacteriosis, ∼95%, are caused by Campylobacter jejuni, followed by Campylobacter coli with 5%.^3,22 Among the different risk factors in the acquisition of the infection, it is well known that the handling and/or the consumption of chicken meat are the main causes.³²

Antimicrobial treatment of campylobacteriosis is recommended only in severe gastroenteritis or immunocompromised patients, and the risk of treatment failure is present when the infection is caused by resistant isolates. Quinolones and macrolides are the drugs of choice in human campylobacteriosis, followed by tetracyclines as an alternative drug.²¹ Therefore, considering the main cause of infection, it is a matter of concern for public health the investigation of the data of antimicrobial resistance among the Campylobacter spp. isolates from poultry and poultry products.

Production of broiler chicks is designed worldwide in several consecutive stages. Three to five pedigree farms maintain the purity of the different broiler breeds. They sell embryonated eggs to the primary breeder companies: great grandparent breeder farms and grandparent breeder farms. Subsequently, they sell hatching eggs to become meat birds and layers at the production farms: parent farms and broiler farms. At each stage, the number of birds increases exponentially.

The prevalence rates of the Campylobacter spp. in poultry farms are highly variable among the European countries.¹⁶ Prevalence depends on climatic, biosecurity, flock production system, and rearing conditions in each country.¹¹

Antibiotics have been widely used in animal production for decades worldwide. However, owing to the emergence of microbes resistant to antibiotics that are used to treat human and animal infections, the European Union has banned the use of antimicrobials in feed as growth promoters from 1 January of 2006, and only certain compounds are allowed for the treatment of animal diseases.

There are a variety of studies about the resistance rates against antimicrobials of the avian Campylobacter spp. isolates in different countries. Most of them reflect the diversity of the resistance rates among countries and even within a single country.¹² There are some studies in Spain about the rates of C. jejuni and C. coli resistance in isolates recovered from poultry, and most of them cover a limited number of isolates.^7,20,23,29 Most of them emphasize the high resistance rates of fluoroquinolones and tetracyclines. The resistance rates against several compounds, such as fluoroquinolones, tetracyclines, and beta-lactams, are highly variable worldwide and range between total susceptibility and 100% of resistance.^{4,6,14,15,27,35} On the other hand, the resistance rates against macrolides are generally low: in most cases, between 0.3% and 20% in most of the countries.¹² Further, the macrolide resistance is usually linked to C. coli.^12,18 Resistance to aminoglycosides and phenicols is, with few exceptions,^4,30 low, ranging from <0.1% to 36%.^2,35

The aim of our study was to monitor the resistance against eight antimicrobials of the Campylobacter strains isolated during 4 years (2002–2005) in the early stages of the broiler production (5 grandparent breeder broiler farms and 12 parent breeder broiler farms) in Spain.

Materials and Methods

Characteristics of the farms

During the 4 years of the study period (2002–2005), five grandparent breeder broiler farms (GPBFs) were sampled and investigated for the presence of Campylobacter spp. These five farms are located in a central province of Spain, within an area of ∼30 km². Birds are moved to the GPBFs at about 20 weeks of age from two rearing farms (RFs) where birds are raised from the hatcheries. During the study, 18 different flocks inhabited the GPBFs. The next stage, parent breeders are produced from fertile eggs of the grandparent flocks, and are reared, housed, and managed in the parent breeder broiler farms (PBFs) after their arrival from the GPBF hatcheries at 1-day old. In any case, a traceability of PBF birds was possible for each flock.

Thirteen PBFs were investigated, and one flock of each was sampled. The PBFs are located in 13 different Spanish provinces that are different to the GPBF's province.

While GPBFs are facilities with high biosecurity measures, PBFs are more heterogeneous in their biosecurity characteristics, but all the farms chosen for the study are Salmonella- and pleuropneumonia-like organism- (PPLO, such as Mycoplasma gallisepticum) free.

Some of the birds at these stages of production enter the food chain. For example, there are several selections of males during the productive life of the grandparent breeders, at least one selection at around 6 weeks of age in which the unselected males are destined for the slaughterhouse. Further, at the end of the productive life of birds (around 60 weeks old), all of them are processed as meat derivatives or meat broth.

As per the recommendations of the EU (see the European Parliament resolution of 27 October 2011 on the public health threat of antimicrobial resistance, accessible through www.europarl.europa.eu/sides/getDoc.do?pubRef=-//EP//TEXT+TA+P7-TA-2011-0473+0+DOC+XML+V0//EN&language=EN last accession 29/3/2012), hygiene measures and good husbandry minimize the need for antibiotics, and they are only used when needed for the actual treatment of the disease with the correct dosage, dose intervals, and duration. As stated by the producers, no disease affected the animals during the study, and therefore the animals in the farms of study received no antimicrobial treatment.

Sampling scheme: Campylobacter spp. isolation and identification

As an initial step in our study, the RFs and three GPBFs were sampled with the objective of confirming the presence or absence of Campylobacter spp. in birds without taking into account the age of the flocks. We confirmed the high prevalence of the Campylobacter spp. in the sampled GPBF birds (flocks D [43 weeks], G [57 weeks], and O [57 weeks]; see Table 1) and the absence of Campylobacter in the RFs.

Table 1.

Data of Isolates Tested by Farm and Flock

Poultry production stage	Farm	Flock	Sampling year	No. of birds/farm	Age of selected birds (weeks)	Campylobacter positive swabs/total cloacal swabs	Prevalence (%)	No. of tested isolates/total isolates
GPBF	1	A	2002–2003	14,000	28	79/80	98.7	74/79
		B	2003	14,000	25	26/30	86.7	16/26
		C	2004	14,000	26	18/40	45	12/18
	2	D	2002	14,000	43	65/80	81.2	60/65
		E	2002–2003	14,000	21	20/40	50	15/20
		F	2003–2004	14,000	37	15/15	100	8/15
	3	G	2002	16,000	57	39/40	97.5	38/39
		H	2002–2003	16,000	23	15/15	100	13/15
		I	2003–2004	16,000	27	20/20	100	15/20
		J	2005	16,000	30	37/80	46.2	27/37
	4	L	2002–2003	16,000	23	67/80	83.7	63/67
		M^a	2003	16,000	18	73/80	91.2	60/73
		N	2004	16,000	23	16/40	40	12/16
	5	O	2002	16,000	57	70/80	87.5	69/70
		P	2002–2003	16,000	23	78/80	97.5	74/78
		Q^a	2003	16,000	13	27/60	45	27/27
		R	2004	16,000	20	30/60	50	24/30
PBF	P1	P-A	2003	8,300	16	9/10	90	9/9
	P2	P-B	2003	11,600	16	20/40	50	18/20
	P3	P-C	2003	16,500	17	37/40	92.5	28/37
	P4	P-D	2003	8,800	11	6/20	30	6/6
	P5	P-E	2003	16,000	14	19/30	63.3	19/19
	P6	P-F	2003	16,000	25	35/40	87.5	19/35
	P7	P-G	2003	12,000	10	10/15	66.7	10/10
	P8	P-H	2003	12,000	5	25/30	83.3	25/25
	P9	P-I	2003	20,000	26	23/25	92	23/23
	P10	P-J	2003	20,000	12	22/30	73.3	18/22
	P12	P-L	2004	8,000	9	16/30	53.3	12/16
	P13	P-M	2004	16,000	18	15/30	50	11/15

Characteristics such as the number of birds and their age at the time of sampling are shown. The number of Campylobacter spp.-positive swabs, the prevalence and the number of isolates in which the disk-diffusion method was performed, are also reflected.

Samples obtained in the rearing farms (RFs) before the placement of the birds to their production farm (GPBF). The RFs are not a stage in poultry production by themselves; they are considered as a part of the GPBFs where the birds are raised from hatcheries to 20 weeks old.

PBFs, parent breeder broiler farms; GPBFs, grandparent breeder broiler farms.

After ascertaining the high prevalence of Campylobacter spp. in the sampled GPBF birds, several consecutive flocks were sampled in each of the GPBFs. We chose one sampling period by flock in their first weeks of production, and in all the cases, the prevalence of the Campylobacter spp. was at least 30%. Routinely, we continued sampling RFs, and they largely remained negative for the Campylobacter spp. with two exceptions: flocks M and Q (see Table 1).

For the next production stage at the PBFs, we investigated one flock by each farm. Previous data in these farms confirmed that the prevalence was independent of the age of the birds; therefore, flocks from 5 to 26 weeks were studied. Detailed information on the number of samples investigated in each flock and farm is shown in Table 1.

In all the cases, the cloacal samples were taken from individual birds using sterile swabs (Amies medium; Pronadisa), and within a period of <6 hr, the swabs were streaked onto two selective media: Karmali (CM0935B; Oxoid) and Abeyta-Hunt-Bark Agar (AHBm) (AHBm prepared following the FDA recommendations: http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/UCM061209 last accession to the webpage: 13/10/2012) and cultured in a microaerobic atmosphere at 42°C for 48 hr. Presumptive colonies were propagated in Columbia agar supplemented with 5% of sheep blood (Becton & Dickinson). Only one colony per sampled poultry was investigated. After 24 hr, at 42°C, the species were identified by means of the hippurate test, as previously described.³³ Doubtful identifications were confirmed using a previously described multiplex PCR.¹ The isolates were stored at −80°C in a brain–heart infusion broth (BHI broth; Oxoid) supplemented with 15% of glycerol.

Susceptibility tests

After a freeze storage period, the isolates were propagated in Columbia agar supplemented with 5% of sheep blood (Becton & Dickinson). In those viable isolates (see Table 1), the disk-diffusion method (DDM) was used for the susceptibility test, according to the CLSI guidelines and recommendations: that is, the density of inocula, time of incubation, and quality-control strain C. jejuni ATCC 33560 at each batch of tests, among others.⁵ The method was carried out with the following antimicrobials purchased from Oxoid: nalidixic acid (NAL; 30 μg, CT0031B), ciprofloxacin (CIP; 5 μg, CT0425B), erythromycin (ERY; 15 μg, CT020B), gentamicin (GEN; 10 μg, CT0794B), amoxicillin (AMX; 25 μg, CT0061B), amoxicillin plus clavulanic acid (AMC; 30 μg, CT0223B), chloramphenicol (CHL; 30 μg, CT0013B), and tetracycline (TCY; 30 μg, CT0054B). Each antimicrobial has been codified following the WHONET recommendations.³¹

The Mueller–Hinton agar supplemented with 5% of defibrinated sheep blood (Becton & Dickinson) was used as the culture medium in all the susceptibility tests.

The diameters of the inhibition zones were measured with a digital caliper (Mitutoyo Corp.) in millimeters and transferred to a database. To discriminate between resistant and susceptible isolates, we have followed the cutoff values provided by the Comité de l'Antibiogramme de la Societé Française de Microbiologie (available at http://sfm-microbiologie.org/UserFiles/file/CASFM/casfm_2010.pdf Last accession 1/19/2012). The diameters of the inhibition zones of our isolates were compared with the selected antimicrobial cutoff values for the inhibition zones. Diameters in millimeters lesser than those of the cutoff values were considered as resistant. To assure the reproducibility of the results, a selection of isolates by flock were rechecked by the DDM (data not shown).

Following the previously published criteria,¹⁷ we have considered an isolate as multidrug resistant (MDR) when it is resistant to three or more groups of the antimicrobials.

Statistical analysis of results

The prevalence of each antimicrobial resistance in the isolates was compared statistically with the Campylobacter species using χ² analyses. Statistical significance was defined at the p≤0.05 or p≤0.001 levels when applicable. Statgraphics plus software (Statistical Graphics Corporation) was used for all statistical analyses.

Results

Total resistance data among poultry isolates

After the sampling periods, we obtained 932 poultry isolates during the 4 years of the study. The susceptibility of the viable isolates (805) was investigated. From the 805 isolates analyzed, 697 were identified as C. jejuni and 108 as C. coli.

As seen in Table 2, the NAL and CIP resistances were the most prevalent in our study, followed by TCY, AMX, and ERY resistances; besides, 14.4% of the isolates could be considered as MDR. Two different MDR resistotypes were identified: NAL^R CIP^R AMX^R TCY^R and NAL^R CIP^R ERY^R TCY^R. The pan-susceptible isolates were found to be at 10.3% of the total. In any case, no resistance against AMC, GEN, or CHL was detected in any of the isolates from the study.

Table 2.

Global Resistance Percentages and Comparison of Resistance Between Production Stages

	Grandparents		Parents		Whole data		Statistics
Antimicrobial	No. of strains/total	Percentage (%)	No. of strains/total	Percentage (%)	No. of strains/total	Global percentage (%)	p-Value^a
NAL^R	432/607	71.2	171/198	86.4	603/805	74.9	<0.001
CIP^R	430/607	70.8	165/198	83.3	595/805	73.9	<0.001
TCY^R	242/607	39.9	146/198	73.7	388/805	48.2	<0.001
AMX^R	79/607	13.0	80/198	40.4	159/805	19.8	<0.001
ERY^R	—	—	25/198	12.6	25/805	3.1	<0.001
MDR	40/607	6.6	76/198	38.4	116/805	14.4	<0.001
Pansusceptible	65/607	10.7	18/198	9.1	83/805	10.3	>0.5

p-Value obtained in the statistical comparison between the two stages of production.

MDR and pan-susceptible percentages have also been represented. Each antimicrobial has been codified following the WHONET recommendations: nalidixic acid (NAL), ciprofloxacin (CIP), erythromycin (ERY), gentamicin (GEN), amoxicillin (AMX), amoxicillin plus clavulanic acid (AMC), chloramphenicol (CHL), and tetracycline (TCY).

MDR, multidrug resistant.

Comparison of resistance by the production-rearing step

About 607 and 198 out of the 805 isolates belonged to the GPBFs and PBFs, respectively. The comparison of the resistance percentages between the two production steps showed statistically significant differences (p<0.001) in the susceptibilities to all the antimicrobials tested. In all cases, as shown in Table 2, the percentages of resistance are clearly higher in the PBFs than in the GPBFs.

The percentage of TCY resistance (73.7%) among the PBF isolates is almost twice that in the GPBF isolates (39.9%). It is remarkable that none of the GPBF isolates were resistant to ERY as opposed to 12.6% of the PBF isolates.

Regarding the MDR populations, the prevalence of MDR at the PBFs was almost six times compared to the GPBFs (38.4% in PBFs vs. 6.6% in GPBFs). Moreover, two different MDR phenotypes were found at the PBFs (NAL^R CIP^R AMX^R TCY^R and NAL^R CIP^R ERY^R TCY^R) compared to the one in the GPBFs (NAL^R CIP^R AMX^R TCY^R).

Total comparison of resistance between C. jejuni and C. coli

As shown in Table 3, statistically significant differences were found (p<0.001) for all the tested antimicrobials with the exception of AMX.

Table 3.

Total Comparison of Resistance Between Campylobacter jejuni and Campylobacter coli

Antimicrobial	No. of strains/total C. jejuni	C. jejuni (%)	No. of strains/total C. coli	C. coli (%)	Statisticsp-Value^a
NAL^R	538/697	77.2	65/108	60.2	<0.001
CIP^R	530/697	76.0	65/108	60.2	<0.001
TCY^R	284/697	40.7	104/108	96.3	<0.001
AMX^R	144/697	20.7	15/108	13.9	>0.05
ERY^R	—	0.0	25/108	23.1	<0.001
MDR	80/697	11.5	36/108	33.3	<0.001
Pan-susceptible	83/697	11.9	—	0.0	<0.001

p-Value obtained in the statistical comparison between the two species.

MDR and pan-susceptible percentages have also been represented. Each antimicrobial has been codified following the WHONET recommendations: NAL, CIP, ERY, GEN, AMX, AMC, CHL, and TCY.

Resistances against NAL and ciprofloxacin were higher in C. jejuni than in C. coli (for example, 77.2% vs. 60.2%, p<0.001, for NAL). Among the C. coli isolates, the TCY resistance was the main resistance, and it also was significantly higher than in the C. jejuni isolates (96.3% vs. 40.7%, p<0.001). The ERY resistance was restricted to a single population of C. coli isolates (farm P8, belonging to PBFs, as seen in Table 1).

It is remarkable that none of the C. coli isolates in our study could be considered as pan-susceptible as opposed to the 11.9% of the C. jejuni isolates. In general, the C. coli isolates tended to be more MDR than C. jejuni (33.3% vs. 11.5%, p<0.001).

Comparison of the resistance between C. jejuni and C. coli at each production stage

Among the 607 isolates from the GPBFs, 573 were identified as C. jejuni, whereas 34 were C. coli. As shown in Table 4, all the C. coli isolates from this step of poultry production were resistant only to TCY, with the susceptibility for the rest of the antimicrobials. These C. coli isolates, from flocks M and Q, were genetically related, and this was proved with by means of molecular markers, as shown in a previous work.²⁵

Table 4.

Comparison of Resistance Between Campylobacter jejuni and Campylobacter coli in Each Production Stage

	GPBF					PBF
					Statistics					Statistics
Antimicrobial	No. of strains/total C. jejuni	C. jejuni (%)	No. of strains/total C. coli	C. coli (%)	p-Value	No. of strains/total C. jejuni	C. jejuni (%)	No. strains/total C. coli	C. coli (%)	p-Value^a
NAL^R	432/573	75.4	—	0.0	<0.001					>0.05
CIP^R	430/573	75.0	—	0.0	<0.001	100/124	80.6	65/74	87.8	>0.1
TCY^R	208/573	36.3	34/34	100.0	<0.001	76/124	61.3	70/74	94.6	<0.001
AMX^R	79/573	13.8	—	0.0	<0.05	65/124	52.4	15/74	20.3	<0.001
ERY^R	—	0.0	—	0.0	—	—	0.0	25/74	33.8	<0.001
MDR	40/573	7.0	—	0.0	>0.05	40/124	32.3	36/74	48.6	<0.05
Pan-susceptible	65/573	11.3	—	0.0	<0.05	18/124	14.5	—	0.0	<0.001

p-Value obtained in the statistical comparison between the two species at each production stage.

MDR and pan-susceptible percentages have also been represented. Each antimicrobial has been codified following the WHONET recommendations: NAL, CIP, ERY, GEN, AMX, AMC, CHL, and TCY.

Regarding the 198 isolates from the 12 parent breeder broiler farms (PBFs), 124 were identified as C. jejuni and 74 as C. coli. As shown in Table 4, most of the C. coli isolates were TCY resistant (94.6%). It is noteworthy that the AMX resistance was more than twice higher in C. jejuni than in C. coli (52.4% vs. 20.3%). The pan-susceptible isolates were detected only among the C. jejuni isolates (14.5%). On the other hand, almost half (48.6%) of the C. coli isolates from this step of production were of the MDR phenotypes: NAL^R CIP^R AMX^R TCY^R and NAL^R CIP^R ERY^R TCY^R.

Discussion

Campylobacteriosis caused by resistant strains limit the treatment or could cause treatment failure of the disease. Risk studies point to poultry handling or consumption as the main risk factor in acquiring the disease. Therefore, monitoring and control of the resistances of the Campylobacter spp. in poultry farms are major issues.

For resistance detection, the DDM is a well-known established method described in several articles¹⁷ as suitable for the screening of large number of isolates. Although it does not provide a direct MIC (minimal inhibitory concentration) value, it is commonly accepted that the results of DDM (in terms of resistant or susceptible) are comparable to the agar-dilution or E-test results.¹⁷ Further, it is accepted by the CLSI and other organizations (i.e., EUCAST) for the discrimination between resistant and susceptible strains.

Our study could be considered as sufficiently representative of the general data in Spain, because (1) GPBFs together with PBFs cover almost all of the Spanish territory, and (2) GPBF production provides almost 50% of the parent broiler production in Spain.

As seen in Table 2, the resistance percentages were always higher in the PBFs than in the GPBFs. There were also more MDR and less pan-susceptible strains in the PBFs than in the GPBFs. A possible explanation might be that the lesser biosecurity measures in the PBFs allowed different antimicrobial-resistant bacteria from the outside environment to reach the birds and colonize them. Another explanation might be the higher prevalence of C. coli in the PBFs than in the GPBFs in our study, because it is recognized that C. coli are more resistant to antimicrobials than C. jejuni.^6,8

All the sampling periods in the study reached a Campylobacter prevalence ≥30%, which gave a total number of isolates (805) enough to ensure the robustness of the study and the observation of a true trend of the susceptibility to antimicrobials in the two stages of poultry production. With the future objective of investigating the resistance mechanisms of the isolates, a selection of resistant and susceptible isolates were selected and rechecked by the DDM, and their MIC was confirmed by the E-test (BioMerieux) with no variation of results (data not shown).

Analyzing the data from our study, the highest percentages of resistance were against quinolones and fluoroquinolones with 74.9% and 73.9% for NAL and CIP, respectively. There were significant differences between the two species, C. jejuni being more fluoroquinolone resistant than C. coli, although most of the articles show higher rates of fluoroquinolone resistance among the C. coli populations of poultry origin.⁸ The quinolone and fluoroquinolone resistance rates vary worldwide between susceptibility²⁷ and extremely high rates.^4,29 In our study, the CIP total resistance percentage, even though high at 73.9%, is lesser than in other studies conducted in Spain (99%²⁹ and 95.45%,²⁰ in both cases at slaughterhouses). Further, our results are comparable with those for other countries, for example, in Italy, 80% (C. jejuni) and 82.6% (C. coli) resistances to NAL.³⁰ In those studies, the same screening method was performed (DDM). It has been shown that the fluoroquinolone resistance did not decrease several years after the ban of the use of quinolones as growth promoters in the United States.^28,34 One possible reason for this is that the fluoroquinolone resistance seems to provide a certain advantage in the colonization of birds without causing a loss to their biological fitness.¹⁹ However, 25% reduction in average resistance to quinolone was observed compared to a study conducted 10 years ago,²⁹ and it is clearly lesser than in a recent study.²⁰ Good biosecurity measures could allow the producers to reduce the use of antimicrobials in animal therapeutics at the farm level and limit the presence of the resistant populations of bacteria. The poultry industry must continue with their good practices in the use and dosage of antimicrobials in the production of broilers.

TCY resistance was the second largest in our study, with a total percentage of 48.2%. The prevalence of TCY resistance in the Campylobacter spp. varies among countries between susceptibility²⁷ and high rates of close to 100%.^4,14 Our data are comparable to those from other countries, for example, the United States, in slaughterhouses at 46%.³⁵ It is important to note the high percentage of TCY resistance among the C. coli isolates, at 96.3% of the total samples (Table 3). This result is similar to that of the study of de Jong and collaborators on the avian C. coli isolates in Spain⁷ (93.1%). The resistance to TCY has been linked to C. coli in several studies⁸ as caused by the regular use of TCYs in a swine husbandry.²⁴ Even though swine is the main animal host of C. coli, it also appears in poultry at a relatively high frequency.¹³

The percentage of the AMX resistance may be considered as relatively low (17.3%) and is lesser than in a previous study conducted in Spain,²⁹ where the beta-lactam tested (ampicillin) gave 55% of resistance in Campylobacter of broiler origin. Although the beta-lactam resistance values are variable among the countries and even within countries, for example, France 13%–35%,²¹ our data are similar to those from other countries.¹² Since no beta-lactams are used in antibiotic treatment of human campylobacteriosis, the European Food Safety Authority (EFSA) considers their surveillance as of low priority.⁹

Macrolides are considered as the treatment of choice for severe campylobacteriosis, and the resistance rates are generally low among avian isolates (0.3%–20%).¹² In our study, the total resistance (C. jejuni plus C. coli) was relatively low (2.3%) and lesser than that found by us (3.8%) in a previous study in Spain with human isolates.²⁶ Besides, they were similar to those found in other European countries such as UK (2%).¹² It is noteworthy that all the ERY-resistant isolates from our study belonged to a single C. coli population isolated from a PBF (P8, see Tables 1 and 3). Similar to TCY, macrolide resistance has been usually linked with C. coli, and it is believed that the cause is because of the use of macrolide derivatives (tylosin) as the growth promoter for swine, years ago.¹⁰ The rate of the ERY resistance among our C. coli isolates (23.1%) is very similar to that found in a recent study conducted at European level at 24.1%.⁷

No CHL or GEN resistance was found during the study. With the exception of a recent study with cloacal isolates from China⁴ and data from Italy,³⁰ in general, the CHL and GEN resistances are documented as of low occurrence among the Campylobacter strains.¹²

In our study, pan-susceptible isolates constituted 10.3% despite the fact that they shared the same ecological niche with the rest of the resistant isolates. In most cases, those pan-susceptible populations did not persist between consecutive flocks in the GPBFs (not shown in this study).

In our study, the MDR percentage was significantly higher in C. coli than in C. jejuni (33.3% vs. 11.5%, respectively, p<0.001, Table 3), which supports the previous studies showing a high frequency of MDR phenotypes in C. coli from turkeys and broilers.^6,8 Further, none of the C. coli isolates in our study could be considered as pan-susceptible.

In conclusion, our study gives new data on the antimicrobial resistance prevalence among the zoonotic pathogens C. jejuni and C. coli. Differences in the prevalence and patterns of resistances were found between the two species and at different stages of broiler production. The resistance percentages are clearly lesser than in previous studies conducted in Spain several years ago and are comparable to those in other European countries. Surveillance of the Campylobacter MDR isolates and, specifically, certain C. coli-resistant populations should be the issues of interest in public health.

Footnotes

Acknowledgments

This work was supported by the Instituto de Salud Carlos III of the Spanish Ministry of Science and Innovation (PI05/00042). We gratefully thank Juana Bustos for her assistance in the analysis of data and Cristina Acebal for her critical review of the manuscript.

Disclosure Statement

No competing financial interests exist.

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