In this 3-year study, we found that 49.4% of a total of 89 FQREC isolates were OST-positive. Furthermore, the proportion of OST-positive isolates changed over time, such that the proportion of FQREC isolates that were OST-positive was notably lowest in the last year of the study (ie, 2004). In the final multivariable model, OST positivity was significantly associated with location on the surgical service and receipt of a first-generation cephalosporin in the 30 days prior to sampling. Finally, 19 of 71 isolates that were characterized by PFGE demonstrated clonal relatedness, the majority of which were OST-negative.
Previous studies have demonstrated conflicting results in regard to risk factors for efflux pump overexpression in FQREC. A study evaluating 153 clinical E. coli
isolates (approximately 80% of which were FQREC) from a tertiary care hospital in the United States [18
] found no association between efflux pump overexpression and patient age, sex, hospital location, or culture source. However, this study was limited to bivariable analyses and did not evaluate prior use of antimicrobial agents as potentially selecting for specific resistance mechanisms. Furthermore, selection bias was a potential concern as the study was largely limited to evaluation of clinical urinary cultures (ie, approximately 80% of isolates). Another study performed in Japan [17
] evaluated urinary FQREC isolates from 38 patients with urinary tract infections; results of the study implicated female sex and treatment in the urologic department on multivariable analysis as risk factors for overexpression of the marA
gene, a transcriptional activator associated with increased expression of the AcrAB efflux pump. However, this study was limited by a small sample size as well as potential selection bias given evaluation solely of clinical urinary isolates.
In contrast, our study evaluated risk factors for efflux pump overexpression in a large sample of patients with gastrointestinal tract colonization with FQREC as assessed via fecal cultures performed hospital-wide. FQ resistance likely arises at the level of gastrointestinal tract colonization, particularly in response to selection pressure from use of specific antimicrobial agents (eg, FQs) [11
]. We found a significant association between efflux pump overexpression and recent use of a first-generation cephalosporin in our study, with patients who received cephalexin or cefazolin less likely to have subsequent recovery of FQREC isolates that were OST-positive. To our knowledge, this is the first study to demonstrate such an association. Interestingly, in an intact cell model of E. coli
containing the AcrAB-TolC complex [28
], Nagano and Nikaido were able to determine the kinetic behavior for several cephalosporins and thereby evaluate the degree of drug efflux. In this model, the investigators demonstrated that although extensive efflux occurred with many cephalosporins, cefazolin specifically (a cephalosporin with 2 hydrophilic substituents) showed very little evidence for active efflux [28
]. Of the patients who received a first-generation cephalosporin in our study, the majority (>90%) were prescribed cefazolin. It is possible that given the lack of active efflux of cefazolin in E. coli,
the use of this antibiotic results in a selective disadvantage for FQREC with efflux pump overexpression as the predominant mechanism of FQ resistance. However, further studies are needed to elucidate this relationship, including the kinetic behavior of other commonly used antimicrobials and selection for specific mechanisms of FQ resistance. Nevertheless, given the association between efflux overexpression and resistance to other antimicrobials [14
], consideration of this finding in institutions with high rates of cefazolin use will be important.
Proton-pump inhibitors and aspirin are commonly prescribed agents in both the outpatient and inpatient healthcare settings [30
]. As such, we postulated that the use of these drugs, which act as efflux pump inhibitors and activators, respectively, may be associated with efflux pump overexpression. However, our study did not demonstrate such an association, although this may have been due to small numbers of patients who were prescribed proton-pump inhibitors and aspirin (12 in each group).
The results of our study also demonstrated an increased risk of efflux pump overexpression in FQREC isolates recovered from patients who were on a surgical service at either study hospital. It is unclear why there was a significant association between location on a surgical service and OST positivity. Although 19 isolates were found to be clonally related by PFGE analysis, the patients from whom these isolates were recovered were approximately evenly distributed between surgical and medical services, therefore arguing against an outbreak of a specific, OST-positive E. coli strain among a particular service. It is possible that efflux pump overexpression as a mechanism of resistance may decrease transmissibility. However, further studies are needed to evaluate differences in transmissibility among specific resistance mechanisms in FQREC.
Finally, of the 19 isolates that were genetically related by PFGE analysis, 7 demonstrated temporal overlap in dates of hospitalization of the associated patients (ie, from 31 December 2003 through 18 March 2004). Although all of the patients from whom the 7 isolates were recovered were hospitalized at HUP, building and floor locations differed among the group at the time of fecal sampling. Nevertheless, given the close temporal overlap among these isolates, as well as their OST-negative status, it is possible that acquisition from other patients or the environment may have played a role in these findings.
There are potential limitations of our study. Selection bias is a potential concern; although only approximately 60% of eligible subjects were enrolled as detailed in the original study, participants and nonparticipants had similar demographic characteristics apart from median age [14
]. Similarly, misclassification bias is a concern in case-control studies. However, testing for specific mechanisms of FQ resistance, including efflux pump overexpression, was performed prior to data collection, and cases and controls were classified solely based on OST. Finally, the present study was conducted in a single healthcare system, and these results may not be generalizable to other institutions.
In conclusion, the results of our study demonstrate a significant association between efflux pump overexpression as the mechanism of resistance in FQREC colonization and prior use of first-generation cephalosporins and location on a surgical service. It is clear that the clinical and molecular epidemiology of FQ resistance is complex, and future studies will need to focus on elucidating the relationship between specific antimicrobial use and resulting selection pressure for various resistance mechanisms. Furthermore, it will be important for future studies to evaluate whether differences in risk factors exist depending on the underlying mechanism of resistance among other antimicrobial-resistant bacteria.