Activity of antibiotics used in human medicine for Campylobacter jejuni isolated from farm animals and their environment in Lancashire, UK

Laura J. V. Piddocka,*, V. Riccia, K. Stanleyb and K. Jonesa

a Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, B15 2TT; b Division of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A retrospective study of 96 Campylobacter jejuni isolated from farm animals and the environment showed that most were less susceptible than the NCTC type strain to nalidixic acid (MICs 4–32 mg/L), ciprofloxacin (MICs 1–2 mg/L) and erythromycin (MICs 16–64 mg/L), but had similar susceptibility to tetracycline (MICs 4–8 mg/L) and kanamycin (MICs 4–8 mg/L). None had the high MICs of ciprofloxacin (>32 mg/L) or erythromycin (1024 mg/L) typically associated with clinical resistance in this species. Some farms used antimicrobial agents, but there was no obvious association between the use of agents and the susceptibility of the isolates.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There has been considerable interest in the role of antibiotics for growth promotion and therapy of infections in food-producing animals, especially as a potential transfer route of antibiotic-resistant pathogens, or the genes encoding antibiotic resistance, into the human food chain. Most reports on antimicrobial susceptibility are for bacteria isolated from sick and dying animals1 with few from bacteria isolated from healthy animals or retail meats. There are no data on antibiotic resistance among environmental isolates of Campylobacter jejuni.

Campylobacter is the predominant cause of bacterial food-borne enteritis in humans, and causes low mortality. Most cases are thought to arise from the handling and consumption of poultry,2 although there is molecular evidence that non-poultry sources, especially bovine ones, have been underestimated.3 As the infection is usually self-limiting, many would consider antimicrobial therapy to be unjustified unless the patient has unresolving diarrhoea or is immunocompromised, or if the infection is extra-intestinal. Erythromycin and ciprofloxacin are the first and second drugs of choice, for treatment in humans, but C. jejuni strains resistant to both have been isolated from clinical samples in the UK.2 Despite the recommendation that antibiotics should only be used in those patients with a clinical need, antibiotics, especially fluoroquinolones, are prescribed increasingly for the treatment of enteritis in normal, otherwise healthy, individuals.

Against this background, we sought to examine the susceptibility of C. jejuni, previously isolated from animals and their environment, to antibiotics used in human medicine.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates were cultured from faeces of healthy livestock from five Lancashire dairy farms (farms 1–4 and 7) and two sheep farms (farms 7 and 8) during studies on the carriage of Campylobacter spp. in farm animals between 1992 and 1995.4,5 Slurry strains were isolated from slurries disposed of on land at farm 4.6 Faeces of starlings were sampled on farm 4, and faeces from gulls at the municipal refuse site at Lancaster, Lancashire, UK. Groundwater isolates were cultured from a spring on farm 6.7 Information on antibiotic use before and during the sampling period was sought retrospectively from the farmers (TableGo).


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Table. Antimicrobial susceptibility of Campylobacter strains isolated from farm animals and their environment
 
Ninety-six isolates of C. jejuni from dairy cows (n = 19), sheep (n = 20), calves (n = 11), birds (11 from starlings and nine from gulls), slurry (n = 20) or ground water (n = 6) were included in this study. Four control strains were used: the NCTC type strain of C. jejuni subspecies jejuni NCTC 11351, Campylobacter coli NCTC 11366, Campylobacter lari NCTC 11352 and C. jejuni subspecies doyleii NCTC 11951. All bacteria were isolated, maintained and cultivated as described previously;2 they were identified by the modified Preston biotyping scheme and PCR-based amplification of 23S ribosomal RNA gene fragments.8 The MIC of each antibiotic for each strain was determined as described previously.2


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of isolates and history of antimicrobial use

Farms 1–4 and 6 were primarily dairy farms with some grazing sheep. Farms 7 and 8 bred sheep only. All dairy farms had used antibiotics previously (TableGo). Four of the five dairy farms used oxytetracycline and at least two (probably four) used cepravin (cephalonium), a semisynthetic broad-spectrum cephalosporin, throughout the study. No retrospective information was available for farms 1 (which was a dairy farm with sheep) and 2. Farm 3 had a medium-sized closed dairy herd, i.e. no animals were bought in. Farm 4 also reared turkeys during June to December, had a medium-sized closed dairy herd and kept surplus calves born to the dairy herd in the shed where the starlings were roosting. Framycetin sulphate (neomycin sulphate) and dihydrostreptomycin sulphate were used in the dairy herd. ‘Lincospectin’ was used for foot-dipping of cattle. Strains were isolated from dairy slurries that had been spread on land in late winter/early spring; these persisted on land for up to 20 days.6 The groundwater strains were isolated from a polluted spring which resurged on farm 6.7 The hydrological evidence suggested that the source of contamination was a cracked slurry pit within the catchment area of the spring. The microbiological evidence was consistent with the Preston biotypes of some C. jejuni isolates from the dairy herd, and those from the groundwater being similar.7 The sheep sampled on farms 7 and 8 were part of a closed flock, were never exposed to antibiotics on the farm and were never housed indoors. Site 10 was the local refuse tip. The C. jejuni strains included in this study were isolated from seagulls feeding at the tip.

Antimicrobial susceptibility

There are no recommended antibiotic breakpoint concentrations (or an agreed susceptibility testing method) for Campylobacter spp., so these data were analysed with reference to the available data from the National Council for Clinical Laboratory Standards and British Society for Antimicrobial Chemotherapy for the agents tested and bacteria other than Campylobacter spp. Owing to the small numbers of isolates from each farm, the MIC data are shown as the range of values obtained for each agent (TableGo). Most isolates were less susceptible than NCTC 11351 to nalidixic acid, ciprofloxacin and erythromycin, but had similar susceptibilities to tetracycline and kanamycin. Only one isolate was inhibited by the same concentration of nalidixic acid as NCTC 11351. All others required a higher concentration, with seven of the 11 calf isolates and four of the 20 slurry isolates from farm 4 and three of the nine sheep isolates from farm 7 requiring 32 mg/L for inhibition. All isolates were less susceptible than NCTC 11351 to ciprofloxacin, but none had the high MICs of ciprofloxacin (>32 mg/L) typically associated with fluoroquinolone resistance in this species.2 Half (48/96) required 32–64 mg/L erythromycin for inhibition, suggesting low level resistance, but none had the high MICs (1024 mg/L) typically associated with erythromycin resistance in this species. A quarter (21/96) required tetracycline 8 mg/L for inhibition, and the majority (68/96) of isolates, as with the NCTC type strain, required kanamycin 8 mg/L for inhibition. There was no obvious association between use of the agents on the farms and the susceptibilities of strains.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study focused upon C. jejuni, as this species is the predominant cause of food-borne enteritis in humans. Molecular subtyping of a number of these strains showed them to be indistinguishable from human clinical isolates from the Lancaster area.9 The strains isolated from free-living birds (starlings), farm slurries and a local spring had similar susceptibilities to those of strains isolated from farm livestock. Compared with the recommended breakpoint concentrations for each agent, the farm isolates were essentially susceptible to quinolones, tetracycline, chloramphenicol and kanamycin. A surprising amount of low-level macrolide (erythromycin) resistance was observed even though there was no apparent macrolide, lincosamide or streptogramin use on any of the farms before isolation of these strains.

Isolates included in this study were less susceptible to nalidixic acid and ciprofloxacin than NCTC 11351, although there was no history of fluoroquinolone use on the farms. Therefore, it may be that the NCTC type strain is not representative of C. jejuni in general and that the susceptibility of non-clinical strains, of animal or environmental origin, to this class of agents is less than has previously been supposed. No fluoroquinolone-resistant strains were observed, but with such inherent ‘borderline’ susceptibility to fluoroquinolones, the emergence of a resistant strain after fluoroquinolone exposure could occur quite readily. The majority of strains, if they entered the food chain and caused an infection in humans, would not be susceptible to erythromycin treatment, which many general practitioners still consider to be the first-line agent.

Occasional use of oxytetracycline in the dairy herds had little effect upon susceptibility to this agent. Similarly, the use of an aminoglycoside on farm 4 did not affect kanamycin MICs.

Wider ranges of fluoroquinolone, erythromycin and tetracycline susceptibility were found in a larger study of Campylobacter spp. that included isolates from humans, cattle (n = 29), broilers and pigs in Denmark, and some strains were more susceptible than those in the present study.1 However, greater nalidixic acid resistance was seen. It is possible that these differences were caused by methodological variations or reflect differences in antibiotic use between countries.

Although the handling and consumption of chicken has been implicated as the most common risk for Campylobacter spp. infection, recent molecular studies have shown that a significant proportion of human clinical strains are similar to cattle strains.3 However, the significance of colonization of animal livestock with antibiotic-resistant bacteria relates not only to the potential for contamination of the carcass at slaughter or of milk at the farm, but also environmental and water contamination during disposal of abattoir effluents and slurries to land. The environmental transmission cycle of Campylobacter spp. is not fully understood. Recent studies have implicated free-living birds10 and aquatic sources as the origin of near-universal colonization of commercial poultry flocks. Our studies show that the avian and aquatic strains of C. jejuni isolated from the farm environment had similar susceptibilities to the antibiotics tested as strains isolated from the animal livestock. These studies provide evidence that antibiotic-resistant bacteria may be isolated from farm animals and the environment. These may enter the environmental contamination cycle and, even in the absence of antibiotic exposure, continue to infect further animal hosts.


    Notes
 
* Corresponding author. Tel: +44-121-4146969; Fax: +44-121-4146966; E-mail: l.j.v.piddock{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Aarestrup, F. M., Nielsen, E. M., Madsen, M. & Engberg, J. (1997). Antimicrobial susceptibility patterns of thermophilic Campylobacter spp. from humans, pigs, cattle and broilers in Denmark. Antimicrobial Agents and Chemotherapy 41, 2244–50.[Abstract]

2 . Gaunt, P. N. & Piddock, L. J. V. (1996). Ciprofloxacin resistant Campylobacter spp. in humans: an epidemiological and laboratory study. Journal of Antimicrobial Chemotherapy 37, 747–57.[Abstract]

3 . On, S. L. W., Nielsen, E. M., Engberg, J. & Madsen, K. (1998). Validity of SmaI-defined genotypes of Campylobacter jejuni examined by SalI, KpnI, and BamHI polymorphisms: evidence of identical clones infecting humans, poultry and cattle. Epidemiology and Infection 120, 231–7.[ISI][Medline]

4 . Stanley, K. N., Wallace, J. S., Currie, J. E., Diggle, P. & Jones, K. (1998). Seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves. Journal of Applied Microbiology 85, 472–80.[ISI][Medline]

5 . Stanley, K. N., Wallace, J. S., Currie, J. E., Diggle, P. J. & Jones, K. (1998). Seasonal variation of thermophilic campylobacters in lambs at slaughter. Journal of Applied Microbiology 84, 1111–6.[ISI][Medline]

6 . Stanley, K. N., Wallace, J. S. & Jones, K. (1998). Thermophilic campylobacters in dairy slurries of Lancashire farms: seasonal effects of storage and land application. Journal of Applied Microbiology 85, 405–9.[ISI]

7 . Stanley, K. N., Cunningham, R. & Jones, K. (1998). Isolation of Campylobacter jejuni from groundwater. Journal of Applied Microbiology 85, 187–91.[ISI][Medline]

8 . Eyers, M., Chapelle, S., van Camp, G., Goossens, H. & De Wachter, R. (1993). Discrimination among thermophilic Campylobacter species by PCR amplification of 23S rRNA gene fragments. Journal of Clinical Microbiology 31, 3340–3.[Abstract]

9 . Owen, R. J., Sutherland, K., Fitzgerald, C., Gibson, J., Borman, P. & Stanley, J. (1995). Molecular subtyping scheme for serotypes HS1 and HS4 of Campylobacter jejuni. Journal of Clinical Microbiology 33, 872–7.[Abstract]

10 . Gregory, E., Barnhart, H., Dreesen, D. W., Stern, N. J. & Corn, J. L. (1997). Epidemiological study of Campylobacter spp. in broilers: Source, time of colonization, and prevalence. Avian Diseases 41, 890–8.[ISI][Medline]

Received 17 December 1999; returned 12 March 2000; revised 17 April 2000; accepted 2 May 2000