Campylobacter coli Isolates from Finnish Farrowing Farms Using Aminopenicillins: High Prevalence of bla OXA-61 and β-Lactamase Production,But Low MIC Values

Abstract

Antimicrobial treatment of animals may select resistance in Campylobacter to antimicrobial agents belonging to several classes of compounds. We investigated the effect of widely used aminopenicillin therapy on the minimum inhibitory concentration (MIC) levels in porcine Campylobacter coli isolates and investigated the presence of a β-lactamase gene and β-lactamase production. Epidemiological cut-off values (ECOFFs) were applied to detect decreased susceptibility. Fifty-three isolates were obtained from aminopenicillin-treated (ampicillin or amoxicillin) sows and piglets during and up to 3 weeks post-treatment. All isolates had ampicillin MICs below the ECOFF (≤8 μg/mL). An additional 63 isolates were sampled before treatment or from other untreated sows and piglets. Of these isolates, four had ampicillin MICs above the ECOFF. All ciprofloxacin MICs were below the ECOFF (≤1 μg/mL), except for three isolates from untreated sows and four isolates after aminopenicillin therapy. One isolate originating from an untreated sow had an erythromycin MIC above the ECOFF (>16 μg/mL). None of the isolates had MICs above the ECOFFs for two or three studied antimicrobials simultaneously. Of the 116 C. coli isolates, 90 (77.6%) isolates carried the bla _OXA-61 β-lactamase gene, and 63 (70.0%) of those produced β-lactamase. The isolates producing β-lactamase had higher ampicillin MICs than those without the bla _OXA-61 gene and production of β-lactamase. Proportion of the bla _OXA-61-positive C. coli isolates was similar among untreated animals or during and after the treatment. In conclusion, C. coli isolates did not acquire high ampicillin MICs even though aminopenicillins were administered at therapeutic levels for several days. The bla _OXA-61 gene and production of β-lactamase increased ampicillin MICs in C. coli, but the values remained mainly under the ECOFF. We also demonstrated that aminopenicillin therapy did not select simultaneously resistance to the major antimicrobials used in human therapy against campylobacteriosis (i.e., erythromycin and ciprofloxacin).

Introduction

H umans usually acquire Campylobacter infection via food, and the antimicrobial treatment of food-producing animals contributes to campylobacteriosis with antimicrobial resistant isolates (Aarestrup and Wegener, 1999; Anderson et al., 2003). Campylobacter coli, a commonly detected Campylobacter species in pigs (EFSA and ECDC, 2011), is the most important cause of campylobacteriosis after Campylobacter jejuni (Tam et al., 2003). Antimicrobial treatment of pigs is associated with increasing macrolide and fluoroquinolone resistance in C. coli (Delsol et al. 2004; Juntunen et al., 2010, 2011; Taylor et al., 2009). Antimicrobial agents of these groups (i.e., erythromycin and ciprofloxacin, respectively) are commonly used drugs in the case of severe or prolonged human infection (Blaser and Engberg, 2008). The typical molecular mechanisms detected in porcine C. coli isolates conferring macrolide and fluoroquinolone resistance are mutations in 23S rRNA (A2075G) and gyrA (C257T), respectively (Delsol et al., 2004; Juntunen et al., 2010, 2011; Payot et al., 2004; Qin et al., 2011). A high percentage of Campylobacter isolates produce β-lactamase (Saenz et al., 2000; Tajada et al., 1996), but a combination of amoxicillin and clavulanic acid has been reported to be an effective alternative for human therapy (Griggs et al., 2009; Hakanen et al., 2003; Rodriguez-Avial et al., 2006). Resistance to β-lactams in Campylobacter is known to be mediated by OXA-61 β-lactamase, which is encoded by the bla _OXA-61 gene (Alfredson and Korolik, 2005), but evidence of another β-lactamase exists (Griggs et al., 2009).

We have previously detected the co-selection of resistance to several classes of antimicrobial agents in C. coli when pigs are treated with a macrolide or a fluoroquinolone (Juntunen et al., 2010, 2011). Griggs et al. (2005) determined the proportion of ciprofloxacin-resistant Campylobacter during fluoroquinolone treatment of poultry flocks and reported a higher percentage of ampicillin resistance in ciprofloxacin-resistant Campylobacter isolates (57.9%, 168/290) than in ciprofloxacin-susceptible (38.5%, 69/179) Campylobacter isolates.

Aminopenicillins are widely used in pig production, but there is a lack of knowledge as to whether aminopenicillin therapy selects ampicillin resistance in porcine C. coli isolates or whether it could also select isolates resistant to other groups of antimicrobial agents (e.g., ciprofloxacin and erythromycin). Therefore, we investigated whether the therapeutic usage of aminopenicillins selects antimicrobial resistance in porcine C. coli isolates. We sampled sows and piglets at seven farrowing farms at which aminopenicillins are used, and we determined the antimicrobial susceptibilities in C. coli before, during, and after the therapy.

Methods

Antimicrobial therapies at the farms

Seven Finnish farrowing farms that treat piglets or sows with injectable aminopenicillins (ampicillin [Duphacillin vet 150 mg/L; Scanvet Eläinlääkkeet Oy, Parola, Finland], amoxicillin [Amovet vet 150 mg/L; Orion Pharma Animal Health, Turku, Finland, or Betamox vet 150 mg/L; Vet Medic Pharmaceuticals Oy, Parola, Finland]) were selected for this study. Aminopenicillins were administered for the treatment of diarrhea or MMA (mastitis, metritis, agalactia) syndrome at 15 mg/kg intramuscularly for 3–5 days. Other antimicrobials (enrofloxacin, danofloxacin, procaine benzylpenicillin, sulfadiazine, trimethoprim, tetracycline, or tylosin) were occasionally used at some of the farms for the treatment of other infections according to the instructions of a local veterinarian, but none of the sampled animals were known to be treated with these drugs before or during the sample collection.

Sampling, isolation, confirmation, and antimicrobial susceptibilities of C. coli

Sixty-three C. coli were isolated from untreated animals or immediately before aminopenicillin therapy. One isolate per untreated sow (n=53) or piglet (n=10) was included in this study. Twenty-six animals (seven sows and 19 piglets) received aminopenicillin therapy, and 14 and 39 isolates were obtained during and after the therapy, respectively. Isolates collected after treatment were clustered into two groups: those collected 2–7 days after the last day of treatment and those collected 9–22 days after the last day of treatment. An individual pig or sow was sampled once at most at each sampling (before; during; 2–7 days after; and 9–22 days after treatment), and one isolate per sampling was included for minimum inhibitory concentration (MIC) determinations. Samples were collected between July 2007 and November 2009, and C. coli were isolated and confirmed by PCR as previously described (Juntunen et al., 2010). Susceptibilities to ampicillin, ciprofloxacin, and erythromycin were determined either with the VetMIC (National Veterinary Institute, Uppsala, Sweden; www.sva.se/en/service-and-products/VetMIC) or the agar dilution method (CLSI, 2002). The epidemiological cut-off values (ECOFFs), which divide bacterial population into wild-type and non-wild-type populations, were applied to detect decreased susceptibility. Development of increased MIC values can be observed earlier using ECOFF values, because ECOFF values are usually lower than clinical breakpoints, which refer instead to expected clinical outcome. The ECOFFs determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2011) for C. coli were ≤8, ≤1, and ≤16 μg/mL for ampicillin, ciprofloxacin, and erythromycin, respectively. C. jejuni ATCC 33560 was used as a control strain in all MIC determinations, and E. coli ATCC 25922 was included for controlling ampicillin MIC determinations.

Detection of bla_OXA-61 and production of β-lactamase

C. coli isolates were tested for the presence of bla _OXA-61 by polymerase chain reaction (PCR). The DNA was extracted as previously described (Juntunen et al., 2010), and the primers PJBLF (5′-GGGCTATGGAGACTTTCTCT-3′) and PJBLR (5′-AAAACCTACAATCCAAGCAA-3′) were designed with Primer3Plus (www.primer3plus.com) to produce an amplicon of 488 bp. The sequence of Cj0299, a putative periplasmic beta-lactamase in the C. jejuni NCTC 11168 genome (GenBank accession no. NC002163.1), was used in the primer design. DNA amplifications were performed as previously described (Juntunen et al., 2010), with the exception of an annealing temperature of 56°C. Isolates were additionally tested with another primer pair (469 and 470) to amplify a fragment (281 bp) of the bla _OXA-61 gene (Elviss et al., 2009). The primer pairs used in this study were purchased from Oligomer Oy, Helsinki, Finland. Production of β-lactamase was detected with nitrocefin disks (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) according to the instructions of the manufacturer after growing isolates microaerobically (5% O₂, 10% CO₂, and 85% N₂) on nutrient blood agar (Oxoid Ltd., Bakingstoke, UK) for 48 h. C. jejuni strain NCTC 11168 was used as a positive control.

Detection of gyrA and 23S rRNA gene mutations

A fragment of gyrA and 23S rRNA genes of ciprofloxacin and erythromycin-resistant isolates, respectively, were amplified and sequenced at the Institute of Biotechnology, University of Helsinki (www.biocenter.helsinki.fi/bi/dnagen/index.htm) using primers described previously (Delsol et al., 2004; Juntunen et al., 2010). The sequences were analyzed with FinchTV (www.geospiza.com/Products/finchtv.shtml) and aligned with ClustalW (www.ebi.ac.uk/tools/msa/clustalw2).

Statistical analysis

Fisher's exact test was applied to identify differences in the percentage of resistance between the isolates from untreated and treated animals using SPSS for Windows, release 15.0.1. (SPSS Inc., Chicago, IL). A p-value of<0.05 was considered statistically significant.

Results

Antimicrobial susceptibility

The distribution of the MIC values for ampicillin, ciprofloxacin, and erythromycin are listed in Table 1. Of all 116 isolates, four (3.4%) had an ampicillin MIC=16 μg/mL, which was above the ECOFF for ampicillin. All four isolates were collected from untreated sows. Those isolates cultured during or after aminopenicillin therapy had ampicillin MICs below the ECOFF. The MICs for ciprofloxacin were below the ECOFF (MIC≤1 μg/mL), except for those of three isolates from untreated sows and those of four isolates collected after aminopenicillin therapy. One of 116 isolates had an MIC above the erythromycin ECOFF (MIC>16 μg/mL); this isolate was collected from an untreated sow. None of the isolates had non-wild-type MICs for two or three studied antimicrobials simultaneously. No statistically significant differences between untreated and treated animals were observed with respect to the proportion of isolates with MICs above the ECOFF values for ampicillin, ciprofloxacin, or erythromycin (p>0.05).

Table 1.

Distribution of the Minimum Inhibitory Concentrations (MICs) of Campylobacter coli (n=116) Isolated from the Feces of Untreated and Aminopenicillin-Treated Swine

Presence of bla_OXA-61 and production of β-lactamase

Similar proportion of β-lactamase production was detected among the bla _OXA-61-positive C. coli isolates from untreated animals (35/50, 70.0%), and during and after the aminopenicillin treatment (28/40, 70.0%; Table 1). The number of bla _OXA-61-positive isolates was 50/63 (79.4%) among the isolates from the untreated animals and 40/53 (75.5%) among the isolates collected during or after the treatment (p=0.66). Table 2 represents the results for the presence of bla _OXA-61 and the production of β-lactamase. Of the 116 C. coli isolates, 90 (77.6%) carried the bla _OXA-61 β-lactamase gene, and 63 of those (70.0%) produced β-lactamase. The median ampicillin MICs for the bla _OXA-61-negative isolates, bla _OXA-61-positive isolates without β-lactamase production, and bla _OXA-61-positive isolates with β-lactamase production were 1, 4, and 8 μg/mL, respectively. All isolates with an ampicillin MIC of 8–16 μg/mL (n=46) yielded an amplicon for the bla _OXA-61 gene, and 82.6% (38/46) of these isolates produced β-lactamase. The isolates with an ampicillin MIC of <1 μg/mL (n=7) were negative for both the bla _OXA-61 gene and the production of β-lactamase. Of the 63 isolates with an MIC of 1, 2, and 4 μg/mL, 33.3% (6/18), 68.8% (11/16), and 93.1% (27/29) produced a PCR amplicon for bla _OXA-61, respectively. The sensitivity of both primer pairs that were used was similar: each primer pair failed to amplify bla _OXA-61 from only one isolate for which the other pair produced an amplicon for the target gene. The primers designed for this study (PJBLF and PJBLR) amplified a fragment of 488 bp, and the other primer pair (469 and 470) produced an amplicon of 281 bp. The production of β-lactamase was detected in 33.3% (2/6), 27.3% (3/11), and 74.1% (20/27) of the bla _OXA-61-positive isolates with ampicillin MICs of 1, 2, and 4 μg/mL, respectively. None of the isolates negative for bla _OXA-61 produced β-lactamase.

Table 2.

Presence of the bla _OXA-61 Gene, the Production of β-Lactamase, and the Susceptibility to Ampicillin of 116 Campylobacter coli Isolates

	No. of Campylobacter coli isolates with minimum inhibitory concentrations (MICs) of (μg/mL)
	<1		1		2		4		8		16
	bla_OXA-61		bla_OXA-61		bla_OXA-61		bla_OXA-61		bla_OXA-61		bla_OXA-61
β-lactamase production	Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Total
Negative	7	0	12	4	5	8	2	7	0	8	0	0	53
Positive	0	0	0	2	0	3	0	20	0	34	0	4	63
Total	7	0	12	6	5	11	2	27	0	42	0	4	116

Mutations in gyrA and 23S rRNA genes

Six out of seven ciprofloxacin-resistant isolates had the C257T mutation, and no other resistance-related mutations were detected in the gyrA fragment. One erythromycin-resistant isolate had the A2075G (C. coli RM2228 no. A2122G) point mutation in the 23S rRNA gene.

Discussion

The total percentage of ampicillin MICs above the ECOFF was low (3.4%) in Finnish porcine C. coli isolates in our study. The selection of isolates with ampicillin MICs higher than the ECOFF was not detected among the isolates collected during or after the therapy, nor did the ciprofloxacin or erythromycin MICs increase. In fact, all C. coli isolates with an ampicillin MIC above the ECOFF were collected from untreated sows. However, higher percentages (17–52%) for ampicillin resistance have been reported in porcine C. coli isolates in other countries (Aarestrup et al., 1997; Payot et al., 2004; Qin et al., 2011; Shin and Lee, 2007; Varela et al., 2007). In contrast, in a Japanese study (Ishihara et al., 2006), only 1.5% of porcine C. coli was ampicillin-resistant. It should be noted that these previous studies used a higher breakpoint (MIC of ≥32 μg/mL) for the determination of resistance and none of the isolates in our study reached that level.

Elviss et al. (2009) studied the selection of amoxicillin-resistant Campylobacter when poultry flocks were treated with amoxicillin. They concluded that previously susceptible isolates remained susceptible but that pre-existing resistant isolates with bla _OXA-61 in their genome proliferated during therapy. In our collection of isolates, the MIC values for ampicillin for the most part remained at the same level, indicating that the aminopenicillin therapy did not select isolates with high ampicillin MICs from among the pre-existing porcine C. coli population with low MICs.

The proportion of the bla _OXA-61-positive C. coli isolates was similar among untreated animals or during and after the treatment. In addition, the proportion of the β-lactamase-positive isolates from untreated animals was not different from those isolated during and after the treatment, further indicating that the therapy did not select either bla _OXA-61 or β-lactamase production. Most of our isolates were bla _OXA-61-positive and produced β-lactamase, even though the majority of the MICs were below the ECOFF for ampicillin. However, the lower the ampicillin MICs were, the lower the percentage of bla _OXA-61-positive isolates that produced β-lactamase was. Furthermore, the isolates carrying the bla _OXA-61 gene and producing β-lactamase had higher median MICs for ampicillin than the isolates without bla _OXA-61 gene and the production of β-lactamase (8 vs. 1 μg/mL). Similar to our results, previous studies found that more than 80% of Campylobacter isolates produced β-lactamase (Saenz et al., 2000; Tajada et al., 1996), and the production of β-lactamase was reported in a number of ampicillin-susceptible C. jejuni and C. coli isolates (Saenz et al., 2000).

Griggs et al. (2009) reported the existence of a type of β-lactamase other than bla _OXA-61 in some poultry Campylobacter isolates producing β-lactamase, but in our set of isolates, all C. coli that produced β-lactamase had the bla _OXA-61 gene in their genome. In two cases, one primer pair failed to amplify the fragment of the bla _OXA-61 gene, but the other primer pair yielded an amplicon. Our results as well as the previous conclusions (Li et al., 2007) emphasize the role of bla _OXA-61 in the production of β-lactamase in Campylobacter. Even if the bla _OXA-61 gene was prevalent in our set of C. coli isolates, ampicillin MICs remained under the ECOFF value, suggesting that the β-lactamase production is not necessarily sufficient to lead to high ampicillin MIC values in Campylobacter. As concluded by Tajada et al. (1996), changes in the structure of penicillin-binding proteins (PBPs) preventing attachment of β-lactams and inadequate transport through the porins of the outer membrane may be responsible for increased MICs in C. coli and C. jejuni in addition to β-lactamase production. In Helicobacter pylori (a related epsilon-proteobacterial species), point mutations in pbp gene have shown to increase amoxicillin MICs from 0.125 to 0.5–1.0 μg/mL (Gerrits et al., 2006). More studies on PBPs on β-lactam resistance mechanisms in Campylobacter are warranted.

Using a similar sampling protocol, we have demonstrated that macrolide- and fluoroquinolone-resistant C. coli isolates were present soon after the start of the therapy with the antimicrobial of the same class (Juntunen et al., 2010, 2011). Furthermore, use of tylosin co-selected resistance to ciprofloxacin, nalidixic acid, and streptomycin. Danofloxacin therapy also selected erythromycin-resistant C. coli isolates. In contrast, aminopenicillins do not appear to select resistance to these other clinically important antimicrobials. All except one ciprofloxacin-resistant isolate harbored the typical C257T mutation in their gyrA gene and one isolate resistant to erythromycin had the A2075G point mutation in the 23S rRNA gene. These isolates were not simultaneously resistant to ampicillin.

Conclusion

Here, we found that C. coli isolates retained low ampicillin MICs when aminopenicillins were administered at therapeutic levels for several days, even though the bla _OXA-61 gene was common among the isolates. We also demonstrated that aminopenicillin therapy did not select resistance to the major antimicrobials used in human therapy against campylobacteriosis (i.e., erythromycin and ciprofloxacin). The bla _OXA-61 gene and production of β-lactamase increased ampicillin MICs in C. coli, but the values remained mainly under the ECOFF.

Footnotes

Acknowledgments

Olli Peltoniemi, Claudio Oliviero, and Camilla Munsterhjelm are acknowledged for their cooperation in contacting the farms and collecting samples for this study. The authors gratefully acknowledge our laboratory's technician, Anna-Kaisa Keskinen, for her excellent laboratory work. We thank all of the workers at the farms for participating in this project. This study was funded by the Finnish Ministry of Agriculture and Forestry and was performed at the Centre of Excellence on Microbial Food Safety Research, Academy of Finland. P. Juntunen was funded by the Graduate School of the Veterinary Faculty of the University of Helsinki and the Finnish Veterinary Association.

Disclosure Statement

No competing financial interests exist.

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Campylobacter coli Isolates from Finnish Farrowing Farms Using Aminopenicillins: High Prevalence of bla OXA-61 and β-Lactamase Production,But Low MIC Values

Abstract

Introduction

Methods

Antimicrobial therapies at the farms

Sampling, isolation, confirmation, and antimicrobial susceptibilities of C. coli

Detection of blaOXA-61 and production of β-lactamase

Detection of gyrA and 23S rRNA gene mutations

Statistical analysis

Results

Antimicrobial susceptibility

Presence of blaOXA-61 and production of β-lactamase

Mutations in gyrA and 23S rRNA genes

Discussion

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Detection of bla_OXA-61 and production of β-lactamase

Presence of bla_OXA-61 and production of β-lactamase