1 Division of Farm Animal Science, Department of Clinical Veterinary Science, Langford BS40 5DU; 2 Department of Food and Environmental Safety, Veterinary Laboratories Agency (Weybridge), Woodham Lane, Addlestone, Surrey KT15 3NB, UK
Received 28 March 2003; returned 24 July 2003; revised 11 August 2003; accepted 29 October 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results: Our results showed that a single treatment failed to eradicate S. Typhimurium DT104, which continued to be isolated up to 35 days after treatment. We also provide evidence that treatment positively selects for S. Typhimurium DT104 strains that are already nalidixic acid resistant (gyrA Asn-87) or cyclohexane resistant, the latter being indicative of an up-regulated efflux pump. Emergence of fluoroquinolone resistance was not detected during treatment or post-treatment in any of the Salmonella strains monitored. However, the effect of enrofloxacin on the nalidixic acid-resistant and cyclohexane-resistant S. Typhimurium DT104 outlasted the current withdrawal time of 10 days for Baytril (commercial veterinary formulation of enrofloxacin).
Conclusions: In conclusion, our study has provided direct evidence that enrofloxacin-treated pigs could be entering abattoirs with higher numbers of quinolone-resistant zoonotic bacteria than untreated pigs, increasing the risk of these entering the food chain.
Keywords: zoonotics, antimicrobial resistance, animal models
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluoroquinolones act by directly inhibiting DNA gyrase and topoisomerase IV in susceptible bacteria. In Salmonella spp., quinolone resistance is conferred by mutations in the DNA gyrase, with the amino acid substitution at Asp-87 one of the most frequently observed.1 The topoisomerase IV is a secondary target of quinolones, and mutations in the parC and parE genes have been linked with high-level resistance to quinolones and reduced susceptibility to fluoroquinolones.2
In addition to mechanisms of resistance that are strictly specific to (fluoro)quinolones, Gram-negative bacteria can be resistant to these antibiotics by excluding them from the cell. Active efflux systems play a significant role in this. In S. Typhimurium DT104, the over-expression of the AcrAB efflux pump was linked with an increase in resistance to a number of unrelated antibiotics (chloramphenicol, tetracycline, ampicillin, nalidixic acid), disinfectants and organic solvents.3
The aim of this study was to investigate the link between a standard 5 day enrofloxacin treatment and emergence of fluoroquinolone resistance in S. Typhimurium DT104 in the pig. The effect of enrofloxacin treatment was tested on three phenotypes of S. Typhimurium DT104: quinolone-susceptible phenotype; cyclohexane-resistant phenotype, which is associated with up-regulated pumps involved in reduced susceptibility to fluoroquinolones;4 and finally, quinolone-resistant phenotype, where isolates had the common Asn-87 substitution.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thirty-six piglets from five litters of Saddleback-Duroc cross, weaned at 3 weeks, were housed as a single group for 2 weeks. They were then separated randomly, in groups of six, into pens with individual HEPA filtration, and fed a standard organic feed (Organic Feed Company, grower/finisher pellets, UK) ad libitum. All pigs were screened for the presence of Salmonella by selenite cysteine enrichment and plating out on Brilliant Green Agar (BGA) prior to the start of the study (detection limit: 1 cfu/g faeces)
Properties of S. Typhimurium DT104 strains
Nine field strains of S. Typhimurium DT104, isolated from asymptomatic pigs, were obtained from the culture collection at the Veterinary Laboratories Agency (VLA). Each had the classic penta-resistance phenotype conferring resistance to ampicillin, tetracycline, chloramphenicol, sulphonamide and streptomycin. Table 1 summarizes their resistance profiles to cyclohexane, the quinolone nalidixic acid and the fluoroquinolone, ciprofloxacin.
|
Each S. Typhimurium DT104 strain was streaked onto nutrient agar plates. A single colony was then grown separately overnight in nutrient broth (Oxoid) at 37°C. The cells were pelleted (14 000g, 15 min), washed in saline, re-suspended in antacid solution [composed of (g/L): MgCO3, 50 g; Mg2Si3O8, 50 g; NaHCO3, 50 g] and inoculated in the piglets by oral gavage as a single dose of 3 x 109 cfu, comprising three strains with identical phenotypes (Table 1).
Antibiotic treatment and sample collection
Twenty-four hours after inoculation, each group was subdivided into two subgroups (n = 6): one remaining untreated and the other treated with a standard therapeutic dose (15 mg/pig/day) of enrofloxacin (Baytril, Bayer plc, UK). Enrofloxacin was administered by individually oral dosing each pig as recommended by the manufacturer (pigs remained under 11 kg for the duration of the study). Faecal samples were collected by digital manipulation 24 h after inoculation, on day 3 during treatment and once a week thereafter.
Recovery and enumeration of S. Typhimurium
One gram of faeces from each pig was emulsified separately in 9 mL of PBS.
Group 1 was inoculated with three strains susceptible to nalidixic acid and ciprofloxacin. These were all furazolidone resistant, which was used as a selective marker. Triplicates of 10, 100 and 500 µL aliquots of PBS suspension were spread onto plates of MacConkey agar (Oxoid) supplemented with furazolidone 32 mg/L (Sigma). Non-lactose fermenting colonies were counted, picked and confirmed as Salmonella by colony morphology on BGA and Rambach media (Oxoid).
Group 2 was inoculated with cyclohexane-resistant strains. The same procedure as above was followed, but Iso-Sensitest agar submerged in cyclohexane was used as the selective medium (see cyclohexane resistance).
Group 3 was inoculated with nalidixic acid-resistant strains. The same procedure as above was followed but MacConkey agar supplemented with nalidixic acid (50 mg/L) was used as the selective medium.
Determination of antibiotic resistance
MIC values were determined by an agar doubling dilution method following NCCLS guidelines, but with Iso-Sensitest agar (Oxoid CM471), as conducted by Randall & Woodward.4
Cyclohexane resistance
Cyclohexane resistance was determined as described by Randall & Woodward.4
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
S. Typhimurium DT104 was re-isolated up to 35 days after treatment from all pigs in all three groups, although in declining numbers. When comparing S. Typhimurium DT104 shed in the faeces from the untreated pigs of each of the three groups, a consistent pattern was observed whereby after an initial sharp decline in Salmonella counts from 104 to 102 cfu/g of faeces, cfu counts stabilized at around 102 9 days post-treatment. There were no significant differences between the untreated pigs in any of the three groups (P = 0.96).
Effect of enrofloxacin treatment on S. Typhimurium DT104 colonization
In group 1, there was no significant difference (P > 0.05) in the recovery of Salmonella between treated and untreated pigs inoculated with quinolone-susceptible S. Typhimurium DT104. Treated pigs inoculated with cyclohexane-resistant S. Typhimurium DT104 (group 2) and treated pigs inoculated with gyrA mutant S. Typhimurium DT104 (group 3) consistently shed higher numbers of Salmonella than the untreated pigs from their respective control groups (P < 0.01 for both). In group 2, Salmonella counts were 100-fold higher in treated than the untreated pigs for 2 weeks post-treatment, and remained higher than the untreated pigs up to 35 days post-treatment; in group 3, treated pigs were shedding 10-fold higher numbers than untreated pigs for 14 days post-treatment (Figure 1).
|
In group 1, MIC values for inoculated S. Typhimurium DT104 remained at 4 mg/L for nalidixic acid and 0.015 mg/L for ciprofloxacin (n = 443). Similarly, the MIC values for the cyclohexane-resistant strains isolated from group 2 (n = 200) and nalidixic-resistant strains isolated from group 3 (n = 491) remained at 16 mg/L and 128 mg/L for nalidixic acid, and 0.06 mg/L and 0.25 mg/L for ciprofloxacin, respectively (Table 1). The strains that were cyclohexane susceptible (strains inoculated in groups 1 and 3) remained susceptible for the duration of this study.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We did not detect an increase in fluoroquinolone resistance in the bacterial populations monitored, suggesting that they were able to withstand the selective pressure created by the enrofloxacin treatment. Both nalidixic acid-resistant and cyclohexane-resistant Salmonella populations already had an effective mechanism of resistance recognized to support quinolone resistance1,4 and known to play a role in Salmonella survival of ciprofloxacin treatment in human patients.5 This would have facilitated their survival during treatment, perhaps giving them a competitive advantage over the susceptible enteric population. The high numbers of S. Typhimurium DT104 shed in the faeces seen in the treated pigs (Figure 1) lends support to this hypothesis. These data, therefore, emphasize the threat such a phenotype can pose to human health, particularly as cyclohexane resistance has been strongly linked with the up-regulation of efflux pumps, which confer multiple antibiotic resistance. These results provide evidence to support the view that where nalidixic acid or cyclohexane resistant S. Typhimurium DT104 are recovered from pigs needing antimicrobial treatment, a compound other than a fluoroquinolone should be used.
The 5 day treatment of enrofloxacin failed to eliminate the quinolone- and cyclohexane-susceptible S. Typhimurium DT104, although during and 1 day post-treatment, treated pigs were shedding less Salmonella than the untreated pigs. This correlates with other studies that have reported failure of fluoroquinolone treatment at eradicating Salmonella infections in animals6 and humans.5
In conclusion, antimicrobial resistance in Salmonella spp. can be the direct outcome of antimicrobials used in animal production, and the use of fluoroquinolones in the pig may have important public health implications.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Khodursky, A. B., Zechiedrich, E. L. & Cozzarelli, N. R. (1995). Topoisomerase IV is a target of quinolones in Escherichia coli. Proceedings of the National Academy of Sciences, USA 92, 118015.[Abstract]
3
.
Piddock, L. J. V., White, D. G., Gensberg, K. et al. (2000). Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrobial Agents and Chemotherapy 44, 311821.
4
.
Randall, L. P. & Woodward, M. J. (2001). Multiple antibiotic resistance (mar) locus in Salmonella enterica serovar Typhimurium DT104. Applied and Environmental Microbiology 67, 119097.
5
.
Threlfall, E. J. (2000). Epidemic Salmonella typhimurium DT104a truly international multiresistant clone. Journal of Antimicrobial Chemotherapy 46, 710.
6 . Wilkinson, M. J., Taylor, D. J., Laurie, J. et al. (2000). Attempted eradication of salmonellosis from a colony of short-tail grey opossums (Monodelphis domestica). Laboratory Animals 34, 21722.[ISI][Medline]