Assessment of the fitness impacts on Escherichia coli of acquisition of antibiotic resistance genes encoded by different types of genetic element.

OBJECTIVES: Little is known of the fitness cost that antibiotic resistance exerts on wild-type bacteria, especially in their natural environments. We therefore examined the fitness costs that several antibiotic resistance elements imposed on a wild-type Escherichia coli isolate, both in the laboratory and in a pig gut colonization model. METHODS: Plasmid R46, Tn1 and Tn7 and a K42R RpsL substitution were separately introduced into E. coli 345-2 RifC, a rifampicin-resistant derivative of a recent porcine isolate. The insertion site of Tn1 was determined by DNA sequencing. The fitness cost of each resistance element was assessed in vitro by pairwise growth competition and in vivo by regularly monitoring the recovery of strains from faeces for 21 days following oral inoculation of organic piglets. Each derivative of 345-2 RifC carrying a resistance element was grown in antibiotic-free broth for 200 generations and the experiments to assess fitness were repeated. RESULTS: RpsL K42R was found to impose a small fitness cost on E. coli 345-2 RifC in vitro but did not compromise survival in vivo. R46 imposed a cost both before and after laboratory passage in vitro, but only the pre-passage strain was at a disadvantage in vivo. The post-passage isolate had an advantage in pigs. Acquisition of Tn7 had no impact on the fitness of E. coli 345-2 RifC. Two derivatives containing Tn1 were isolated and, in both cases, the transposon inserted into the same cryptic chromosomal sequence. Acquisition of Tn1 improved fitness of E. coli 345-2 RifC in vitro and in vivo in the case of the first derivative, but in the case of a second, independent derivative, Tn1 had a neutral effect on fitness. CONCLUSIONS: The fitness impact imposed on E. coli 345-2 RifC by carriage of antibiotic resistance elements was generally low or non-existent, suggesting that once established, resistance may be difficult to eliminate through reduction in prescribing alone.

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig.

AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.

Rifampicin resistance and its fitness cost in Enterococcus faecium.

OBJECTIVES: The genetic basis of rifampicin resistance and the associated fitness cost in Enterococcus faecium were investigated. METHODS: Twelve spontaneous rifampicin-resistant E. faecium mutants were selected from four parent strains recently isolated from porcine faecal material. The DNA sequence of the complete rpoB gene from the parent strains and of nucleotides -189 to +1785 from the mutants was determined from PCR amplicons. The fitness of the mutants was assessed by determining growth rate, by direct growth competition and by the ability of some of the mutants to survive in the pig intestine. RESULTS: The rpoB genes of the parent strains diverged from each other by 1-10% and each encoded proteins that were 1208 amino acids in length. All mutants had a single amino acid substitution in the region implicated in rifampicin resistance in other organisms. Six mutants carried the substitution H489Y/Q, two mutants carried the substitution R492H, one mutant carried the substitution Q480H, two mutants carried the substitutions S494L and V224I, and one mutant carried the substitutions G485D and V224I. Per generation fitness costs of the mutants ranged from a gain of 2.5% to a cost of 10%. Mutants with the substitution H489Y/Q were the most fit, whereas the double mutants were the least fit. The mutant with the substitution H489Q was able to survive in the pig gut for 12 days. There was some correlation between the rifampicin MIC and fitness cost, with higher MICs being associated with higher fitness costs. CONCLUSIONS: Substitutions in RpoB are associated with rifampicin resistance in E. faecium. The fitness cost of resistance is variable and can sometimes be absent.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig.

AIMS: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracycline-resistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. METHODS AND RESULTS: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l-1 and a 10% increase in E. coli with an MIC > 50 mg l-1 during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 microg g-1 faeces during treatment. CONCLUSION: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.