In Vivo Selection of Campylobacter Isolates with High Levels of Fluoroquinolone Resistance Associated with gyrA Mutations and the Function of the CmeABC Efflux Pump

Abstract

Enrofloxacin treatment of chickens infected with fluoroquinolone(FQ)-sensitiveCampylobacter promoted the emergence of FQ-resistant Campylobacter mutants which propagated in the intestinal tract and recolonized the chickens. The recovered isolates were highly resistant to quinolone antibiotics but remained susceptible to non-FQ antimicrobial agents. Specific single-point mutations in the gyrA gene and the function of the CmeABC efflux pump were linked to the acquired FQ resistance. These results reveal that Campylobacter is hypermutable in vivo under the selection pressure of FQ and highlight the need for the prudent use of FQ antibiotics. Fluoroquinolone (FQ)-resistantCampylobacter jejuni strains are rapidly increasing throughout the world, which has posed a serious threat to public health (19,20). Although FQ resistance in Campylobacter can occur following the treatment of humans with the antibiotic (4, 17, 22), poultry are considered a significant source for FQ-resistant Campylobacter (1,5,7,18). Laboratory studies have demonstrated the emergence of FQ-resistant Campylobacter in experimental chickens treated with FQ antibiotics (10, 12). However, the previously published works revealed little information on the in vivo dynamics of the emergence of FQ-resistant Campylobacter in chickens. Specifically, it is unknown how Campylobacter populations shift in individual birds in response to the antibiotic treatment and how extensively the infected chickens are colonized by the resistant organisms. In addition, the genetic mechanisms responsible for the in vivo-acquired resistance to FQ in Campylobacter strains are not known. Answering these questions will greatly improve our understanding of the development and mechanisms of FQ resistance in Campylobacter and may facilitate the design of means to prevent the occurrence of FQresistant Campylobacter in vivo.In this study, we examined the dynamics of changes of Campylobacter populations in chickens treated with enrofloxacin and determined the molecular mechanisms associated with the acquired FQ resistance in the in vivo-selected resistant isolates. C. jejuni strain S3B was originally isolated from chicken feces in our laboratory. Bacterial cultures were routinely grown in Mueller-Hinton (MH) broth or plates (Becton Dickinson and Company, Sparks, Md.) at 42°C under microaerophilic conditions generated by the CampyPak Microaerophilic System (BBL). Day-old broiler chickens were obtained from a commercial hatchery. Prior to use, the birds were tested negative for Campylobacter by conventional culture methods. Two independent experiments (A and B) were conducted whose designs are detailed in Table 1. Each group of chickens was maintained in a sanitized wire-floored cage. Feed and water were provided ad libitum. Infection of the chickens with C. jejuni strain S3B (ciprofloxacin MIC ϭ 0.125 g/ml) and treatment with enrofloxacin (Baytril; Bayer Corporation) are detailed in Table 1. Cloacal swabs were collected periodically, resuspended in MH broth, and plated onto MH plates con...

Campylobacter

“…For example environmental selection following enrofloxacin treatment of chickens infected with fluoroquinolone-sensitive Campylobacter spp. resulted in the emergence of the corresponding resistant isogenic strains suggesting that this organism is hypermutatable under these conditions [87]. Horizontal transfer of such resistance determinants (acquisition) together with any genetic modification of pre-existing genes through point mutations (intrinsic) or some other genetic event, are thought to be the main mechanisms contributing to bacterial resistance [2,10,140].…”

“…Active efflux pumps are known to contribute to intrinsic and acquired resistance to a range of antimicrobial agents [79,87,91]. These pumps reduce the intracellular accumilation of antimicrobial agents and other compounds and this feature is now recognised as a major mechanism of resistance in pathogenic organisms [110,130,178].…”

“…Susceptibility studies by Lin et al [79,80] using wild type and isogenic mutants in C. jejuni demonstrated that inactivation of the CmeABC pump by insertional mutagenesis substantially increased the susceptibility of C. jejuni to several classes of antimicrobial agent(s) and also to heavy metals and bile salts [79]. Resistance to bile salts may be a necessary step for successful colonization of animal intestines, contributing to bacterial pathogensis [87].…”

Erratum: Can landscape ecology untangle the complexity of antibiotic resistance?

“…Much of this assumption is based on the expected fitness cost of maintaining the resistance gene(s). Recent studies indicate that the maintenance of resistance might not impose a significant fitness cost [23][24][25][26] or that this cost can be overcome [19][20][21]27 . However, there is also a large background pool of resistance, probably a result of the fact that many resistance genes protect the bacterial cell in such a way that they are likely to be functional against other compounds in the environment.…”

“…This assumption has been questioned [19][20][21] , and recent evidence demonstrates that, in the absence of selection pressure due to antibiotic use, resistance levels in bacterial populations are often slow to decline, if at all 5,7,22 . In fact, some organisms with antibiotic-resistant phenotypes and genotypes seem to be fitter than their susceptible counterparts [23][24][25][26] , and compensatory mutations have been observed that allow the resistant organism to retain its fitness level 27 . Many potential selection forces for resistance have been described, including antibiotics, metals and other compounds such as disinfectants.…”