1 Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG; 2 Epidemiology Unit, Scottish Agricultural College, Drummondhill, Stratherrick Road, Inverness IV2 4JZ; 3 Scottish Agricultural College, Centre for Microbiological Research, Ferguson Building, Craibstone, Aberdeen AB21 9YA; 4 Scottish Agricultural College, Bush Estate, Penicuik, Midlothian EH26 0QE, UK
Received 29 October 2003; returned 8 January 2004; revised 9 February 2004; accepted 10 February 2004
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Abstract |
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Methods: Faecal samples were collected weekly from calves over a 4 month period and screened for E. coli resistant to ampicillin, apramycin and nalidixic acid at concentrations of 16, 8 and 8 mg/L, respectively. E. coli viable counts were performed on samples from a subset of calves.
Results: All calves acquired ampicillin- and nalidixic acid-resistant E. coli, while only 67% acquired apramycin-resistant E. coli during the study. Sixty-seven per cent of samples were resistant to at least one of the three antibiotics. Prevalence of ampicillin and nalidixic acid resistance was high initially and declined significantly with age (P < 0.001). No temporal or age-related pattern was observed in the prevalence of apramycin resistance. Housing the cohort had a significant effect on the prevalence of nalidixic acid resistance (P < 0.001). Total and ampicillin- and nalidixic acid-resistant E. coli counts declined with calf age (P < 0.001), with the rate of decline in ampicillin-resistant counts being greater than that for total counts (P < 0.001). The proportion of total E. coli counts that were resistant to ampicillin or nalidixic acid also declined with age (P < 0.001).
Conclusions: Cohort calves rapidly acquired antibiotic-resistant bacteria within days of birth. Carriage of resistant bacteria was associated with both age and housing status of the cohort.
Keywords: antibiotic resistance, E. coli, cattle
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Introduction |
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Current strategies to monitor the presence of antibiotic-resistant bacteria in food animals target mainly resistance in clinical specimens and involve only periodic cross-sectional evaluations of resistance in faecal flora on a larger scale.2,5,6 However, such surveys do not provide any information about the dynamics of antibiotic resistance in the normal flora. When resistant bacteria are acquired during the lifetime of a food animal and whether such acquisitions are transient or not, are fundamental issues that need to be explored. In this study we therefore investigated the acquisition of antibiotic-resistant commensal Escherichia coli in a cohort of newborn calves.
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Materials and methods |
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Faecal samples were kept at 4°C and screened within 48 h of collection on Chromocult tryptone bile X-glucuronide agar (TBX agar; Merck) containing antibiotics (Sigma) at the following breakpoint concentrations (based on the requirement to detect the potential for resistance within a veterinary context): ampicillin, 16 mg/L; apramycin, 8 mg/L; nalidixic acid, 8 mg/L. Antibiotics were selected as examples of ß-lactam, aminoglycoside and quinolone agents, antibiotic classes frequently used in farm animals. Samples were diluted 1:10 in maximum recovery diluent (MRD; Oxoid), 10 µL spread onto non-selective (antibiotic-free) and antibiotic-containing plates and incubated overnight at 44°C. Characteristic E. coli colonies of a dark-blue colour were recorded, indicative of the presence of the enzyme glucuronidase.7 Reference strains E. coli NCTC 10418, NCTC 11560, JR 225 and NCTC 12900 were used to confirm the activity of antibiotic-containing plates.
For a subset of animals (n = 24) sufficient faeces remained to permit viable count assays. Samples were diluted 1:10 in MRD/20% glycerol and stored at 70°C. Frozen faecal suspensions (101 dilution) were defrosted within 30 min at room temperature and further dilutions (103 and 105) prepared in MRD. Total and antibiotic-resistant counts were determined by duplicate 0.1 mL spread plates for all dilutions on unsupplemented TBX agar and agar containing the respective antibiotic at breakpoint concentration (as above). Glucuronidase-positive colonies were counted after incubation for 24 h at 44°C. Viable counts as colony forming units per g (cfu/g) were calculated as: plate count x 10 x dilution factor.
Statistical analyses were performed using S-Plus (Insightful, Seattle, WA, USA), with P < 0.05 taken as significance. Changes in the cohort prevalence of resistance to the three antibiotics, and the proportion of the total counts of a sample resistant to a particular antibiotic, were analysed using generalized linear mixed-effects models with binomial errors. Calf identity was entered as a random effect to account for inherent differences between calves and the lack of independence between samples.8 Time, calf age and housed status were entered as fixed effects. Proportions were taken as the arithmetic mean count of an antibiotic count plate divided by that of the total count plate. Two samples where no total counts were detected were excluded; for seven samples where the ampicillin -resistant count exceeded the total count the proportion was set to 1. The freezethaw procedure was assumed to have an equal effect on antibiotic-resistant and -susceptible bacteria.
Changes in the total and antibiotic-resistant E. coli population log-transformed counts were analysed using generalized linear mixed-effects models with normal errors on data (both positive and undetected) from those calves that had at least one positive count for a particular antibiotic. Calves where no positive counts were found for a given antibiotic (ampicillin, one; apramycin, nine; nalidixic acid, four) were excluded. No qualitative differences were observed (results not shown) using positive counts only or all counts.
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Results and discussion |
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The cohort prevalence of antibiotic-resistant E. coli changed with age and time (Figure 1). The prevalence of AmpRE.coli declined linearly with age from high levels at calf birth (P < 0.001). Neither the decline in the prevalence of AmpRE.coli with time nor changes due to housed status were significant after the effects of calf age had been taken into account (P > 0.172). However, there was a significant interaction between calf age and housed status (P = 0.015), with no change in prevalence of AmpRE.coli prior to housing (P = 0.183), but a significant decline during the housed period (P < 0.001). Similarly, the prevalence of NalRE.coli significantly declined with age (P < 0.001), but there was no significant change with time once calf age had been taken into account (P = 0.858). There was a significant effect of being housed, even after taking into account age effects (P < 0.001): prior to housing, the prevalence of NalRE.coli remained around 50% (P = 0.661), but dropped at housing and remained low. The prevalence of AprRE.coli remained low with no overall age-related, temporal or housing pattern detected (P > 0.330).
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Changes in viable count profiles of total and antibiotic-resistant E. coli in the cohort are shown in Figure 2. Total counts declined as calves aged (P < 0.001). While total counts did not decline with time after age had been accounted for (P = 0.069), the decline in total counts with age was greater in those calves born in the early part of the study (P = 0.005). There was no relationship between total counts and being housed (P > 0.078). AmpRE.coli counts also declined with age (P < 0.001), but were lower than total counts (P < 0.001), with the rate of decline greater than for total counts (P < 0.001). AmpRE.coli counts were also lower during the housed period (P < 0.001), with the age-related decline in AmpRE.coli counts greater during the non-housed period (P < 0.001). There was no change with time or interaction between time and age once the effects of housing and age had been taken into account (P = 0.869). The same age, time and housing status patterns were observed with NalRE.coli counts. However, there was no difference in the rate of decline in NalRE.coli counts compared with total counts (P = 0.996). AprRE.coli counts did not change with either age or time (P > 0.075).
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Overall, viable count analysis demonstrated that while total E. coli counts do decline with age, the rate of decline of AmpRE.coli and NalRE.coli counts was significantly greater, with the proportion of total counts that were both ampicillin and nalidixic acid resistant also declining rapidly with age. These results indicate that cohort calves preferentially lost resistant relative to susceptible bacteria as they aged.
In conclusion, it has been demonstrated that cohort calves were rapidly colonized by antibiotic-resistant E. coli shortly after birth. The prevalence of AmpRE.coli and NalRE.coli within the cohort declined over the study period. Age-related changes within an animal, together with housing the cohort, had a significant negative effect upon gut carriage of resistant bacteria by cohort calves.
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Acknowledgements |
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Footnotes |
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