Fitness of Escherichia coli strains carrying expressed and partially silent IncN and IncP1 plasmids.

BACKGROUND: Understanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness. RESULTS: For the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was found to be beneficial for host fitness with RP1 but not for IncN plasmid pVE46. CONCLUSIONS: These findings suggest that the fitness impact of a given plasmid on its host cannot be inferred from results obtained with other host-plasmid combinations, even if these are closely related.

Determination of avilamycin A and B in pig faeces by solid phase extraction and reverse-phase HPLC assay.

A HPLC method is described for the simultaneous determination of avilamycin A and B in pig faeces, following extraction using acetonitrile and normal-phase solid phase extraction. The HPLC stationary phase was Kromosil 5 micro C-18 with a mobile phase of 48% acetonitrile and 52% 0.01N ammonium acetate buffer, pumped at a flow rate of 1 ml/min. Detection was by UV absorbance at 295 nm and an injection volume of 50 microl was used. Recovery from faeces was >98% and intra-assay precision (CV) was <9.0% for both compounds. The lowest limit of quantification was 0.9 mg/kg (avilamycin A) and 0.2 mg/kg (avilamycin B) with an accuracy of <15% error. No interference was seen from endogenous materials in pig faeces and commonly used veterinary antibiotics.

A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces.

A reverse-phase HPLC assay is described for the simultaneous assay of enrofloxacin (ENR) and ciprofloxacin (CPX) in pig faeces. Extraction used dichloromethane, 2-propanol and 0.3M ortho-phosphoric acid (1:5:4 v/v/v). Separation was achieved using a Spherisorb S5 C8 column, heated to 50 degrees C and a mobile phase of 0.16% ortho-phosphoric acid (adjusted to pH 3.0 with tetrabutylammonium hydroxide solution) with 20 ml acetonitrile per litre solution. The method used fluorescence detection (Ex 310 nm; Em 445 nm), a flow rate of 1 ml/min and a 20 microl injection volume. Retention times were approximately 6 min for ciprofloxacin and 10 min for enrofloxacin. The linearity range for both compounds was 0-20 mg/kg, lowest limit of quantification 0.3 mg/kg and recoveries were >92%.

Persistence of a wild type Escherichia coli and its multiple antibiotic-resistant (MAR) derivatives in the abattoir and on chilled pig carcasses.

The aim of this study was to evaluate the ability of an Escherichia coli with the multiple antibiotic resistance (MAR) phenotype to withstand the stresses of slaughter compared to an isogenic progenitor strain. A wild type E. coli isolate (345-2RifC) of porcine origin was used to derive 3 isogenic MAR mutants. Escherichia coli 345-2RifC and its MAR derivatives were inoculated into separate groups of pigs. Once colonisation was established, the pigs were slaughtered and persistence of the E. coli strains in the abattoir environment and on the pig carcasses was monitored and compared. No significant difference (P>0.05) was detected between the shedding of the different E. coli strains from the live pigs. Both the parent strain and its MAR derivatives persisted in the abattoir environment, however the parent strain was recovered from 6 of the 13 locations sampled while the MAR derivatives were recovered from 11 of 13 and the number of MAR E. coli recovered was 10-fold higher than the parent strain at half of the locations. The parent strain was not recovered from any of the 6 chilled carcasses whereas the MAR derivatives were recovered from 3 out of 5 (P<0.001). This study demonstrates that the expression of MAR in 345-2RifC increased its ability to survive the stresses of the slaughter and chilling processes. Therefore in E. coli, MAR can give a selective advantage, compared to non-MAR strains, for persistence on chilled carcasses thereby facilitating transit of these strains through the food chain.

Evidence of antibiotic resistance gene silencing in Escherichia coli.

The possibility that unexpressed antibiotic resistance genes are carried by bacterial genomes is seldom investigated. Potential silencing of the resistance genes bla(OXA-2), aadA1, sul1, and tetA carried on the plasmid pVE46 in a recent porcine isolate of Escherichia coli was investigated following oral inoculation of the strain into organic piglets. A small proportion of isolates recovered from feces did not express one or more resistance genes, despite retaining the pVE46 plasmid. Different combinations of unexpressed resistance genes were observed, and 12 representative isolates were selected for further study. Surprisingly, in most cases the resistance genes and their promoters, although not expressed, were intact, with fully wild-type sequences. Apart from four isolates exhibiting intermediate-level tetracycline resistance, no mRNA for the unexpressed genes was detected. Silencing of resistance genes was reversible at low frequencies between 10(-6) and 10(-10). Introduction of the plasmid from silenced isolates to another strain restored expression, indicating that gene silencing was a property of the host chromosome rather than the plasmid itself. When the same recent porcine E. coli strain carrying the unrelated plasmid RP1 was inoculated into piglets, three isolates (of 9,492) that no longer expressed RP1-encoded resistance genes were recovered. As with pVE46, in most cases the coding sequences and promoter regions of these genes were found to be intact, but they were not transcribed. Such gene silencing indicates a previously unrecognized form of transcriptional control that overrides standard expression signals to shut down gene expression. These findings suggest that unexpressed resistance genes may occur in the wild and hence may have clinical implications.

Effect of a 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in the pig.

OBJECTIVES: There are concerns that the use of enrofloxacin in livestock production may contribute to the development of fluoroquinolone resistance in zoonotic bacteria. The objective of our study was to investigate the effect of a single 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in a pig model. 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.

Emergence of fluoroquinolone resistance in the native Campylobacter coli population of pigs exposed to enrofloxacin.

OBJECTIVE: The effect of a single 5 day enrofloxacin treatment on the native Campylobacter coli population in conventionally weaned 5-week-old pigs was investigated. MATERIALS: Twelve pigs were split into two groups of six: one group was treated with a therapeutic dose (15 mg/pig/day) of enrofloxacin and the other remained untreated to act as the control. Campylobacter coli were isolated from faecal samples and tested for ciprofloxacin resistance by measuring MIC values. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene of resistant isolates were identified by sequencing and denaturing HPLC. Levels of enrofloxacin and its primary metabolite ciprofloxacin in the pig faeces were also measured by HPLC. RESULTS: No quinolone-resistant C. coli (n = 867) were detected in any of the pigs prior to treatment, indicating <0.1% resistance in the group. Resistant C. coli were isolated from pigs for up to 35 days after treatment with a therapeutic dose. These resistant C. coli had MIC values of 128 mg/L and 8-16 mg/L for nalidixic acid and ciprofloxacin, respectively, and the same single point mutation causing a Thr-86 to Ile substitution in the QRDR was identified in each. The concentration of enrofloxacin in the pig faeces was 2-4 micro g/g faeces for the duration of the 5 day therapeutic treatment and was detected up to 10 days post-treatment. Ciprofloxacin was also measured and peaked at 0.6 micro g/g faeces in the treated group. CONCLUSION: This study provides evidence that a single course of enrofloxacin treatment contributes directly to the emergence and persistence of fluoroquinolone resistance in C. coli.

Determination by HPLC of chlortetracycline in pig faeces.

An HPLC assay used to determine chlortetracycline (CTC) in pig faeces is reported. Prodigy ODS3 (4.6 x 150 mm) was used for the stationary phase, whereas the mobile phase comprised oxalic acid, sodium oxalate and sodium decane sulfonate (66%)--each of 4 mM, and 34% acetonitrile. The mobile phase was pumped at a flow rate of 1 mL/min. Detection of CTC was by ultraviolet absorbance at 370 nm, and a 20 micro L injection volume was used. Recovery from faeces was >90%, and coefficients of variability between runs were <10%. The lowest limit of quantification was 3.5 mg/kg, with an accuracy of <7% error. There was no interference from endogenous materials in the pig faeces, or commonly used antibiotics, and the method is suitable for use in drug disposition studies.