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Clin Infect Dis. Author manuscript; available in PMC 2011 August 1.
Published in final edited form as:
PMCID: PMC2897836
NIHMSID: NIHMS202455

The Prevalence of Fluoroquinolone Resistance Mechanisms in Colonizing Escherichia coli Isolates from Hospitalized Patients

Ebbing Lautenbach, MD, MPH, MSCE,1,2,3,4 Joshua P. Metlay, MD, PhD,2,3,4,5 Xiangqun Mao, MD,6 Xiaoyan Han, MS,3 Neil O. Fishman, MD,1,4 Warren B. Bilker, PhD,2,3,4 Pam Tolomeo, MPH,3 Mary Wheeler, MBE,3 and Irving Nachamkin, Dr PH, MPH4,6

Abstract

Background

Fluoroquinolones (FQs) are the most commonly prescribed antimicrobials. The epidemiology of fecal colonization with Escherichia coli demonstrating reduced susceptibility to FQs remains unclear.

Methods

Over a three year period, all patients hospitalized >3 days were approached for fecal sampling. All E. coli with reduced susceptibility to FQs (MIC ≥0.125μg/mL to levofloxacin) were identified. We characterized gyrA and parC mutations and organic solvent tolerance. Isolates were compared using pulsed field gel electrophoresis.

Results

Of 353 subjects colonized with E. coli demonstrating reduced FQ susceptibility, 300 (85%) had ≥ 1 gyrA mutation, 161 (45.6%) had ≥ 1 parC mutation, and 171 (48.6%) demonstrated organic solvent tolerance. The mean number of total mutations (i.e., gyrA + parC) for E. coli isolates with a levofloxacin MIC ≥8 μg/ml vs. <8.0 μg/ml was 2.70 and 0.82, respectively (p<0.001). Of the 136 E. coli isolates with a levofloxacin MIC ≤8 μg/ml, 90 (66.2%) demonstrated a nalidixic acid MIC ≥16 μg/ml. There were significant differences over time in the proportion of E. coli isolates demonstrating gyrA mutation, parC mutation, and organic solvent tolerance. There was little evidence for clonal spread of isolates.

Conclusions

GI tract colonization with E. coli demonstrating reduced susceptibility to levofloxacin is common. Although 40% of study isolates exhibited a levofloxacin MIC <8 μg/ml (and would thus be missed by current CLSI breakpoints), nalidixic acid resistance may be a useful marker for detection of such isolates. Significant temporal changes occurred in the proportion of isolates exhibiting various resistance mechanisms.

Keywords: E. coli, fluoroquinolone, colonization, genotype, phenotype

INTRODUCTION

The fluoroquinolone (FQ) antibiotics have become the most commonly used class of antibiotics [1, 2]. As such, the increasing prevalence of FQ resistance in Escherichia coli, the most common gram negative pathogen, is most concerning [3, 4]. This is particularly true given the negative impact of FQ resistance on clinical outcomes [5].

The human gastrointestinal (GI) tract serves as a natural reservoir for E. coli [6] and E. coli isolates causing clinical infection are almost always derived from organisms colonizing the GI tract [7, 8]. In addition, the step-wise accumulation of FQ resistance determinants in E. coli (e.g., in response to selection pressure from antimicrobial use) in the clinical setting likely occurs at the level of the GI tract [9]. Despite this, most studies seeking to characterize FQ-resistant E. coli isolates have focused on isolates derived from clinical infections, rather than on fecal colonizing isolates [1014]. Studies that have focused on E. coli colonization in the clinical setting have typically assessed E. coli colonization with only one or a few fecal surveys at specific points in time or used sampling approaches that changed over time[12, 1416]. This approach limits the ability to assess secular changes and person to person transmission over time.

The goal of this study was to characterize GI tract colonization due to E. coli with reduced susceptibility to FQs among the hospitalized patient population using continuous enrollment of hospitalized patients over a three year time period. In addition, we sought to comprehensively characterize the resistance genotypes and phenotypes of fecal E. coli isolates with reduced susceptibility to FQs in this patient population.

METHODS

The study was performed at two University of Pennsylvania Health System hospitals: 1) The Hospital of the University of Pennsylvania (HUP), an academic tertiary care medical center with 725 patient beds; and 2) Penn Presbyterian Medical Center (PMC), a 344-bed urban community hospital. This study was reviewed and approved by the Institutional Review Board of the University of Pennsylvania.

Patients were enrolled in this study from September 15, 2004 through October 19, 2007. We approached all patients hospitalized at the two study sites. All hospital floors and units were included. To be eligible, a patient had to have been hospitalized for at least three days and be deemed capable by research staff of providing consent. Research staff approached all patients on the third day of hospitalization to obtain informed consent. All eligible patients were enrolled if informed consent was provided. If a patient was unavailable, one additional attempt to approach the patient was made the following day. Each subject could only be enrolled once. For subjects who agreed to enroll, a peri-rectal swab was obtained by research staff. Of note, a peri-rectal swab has been shown to be highly sensitive and specific for detection of FQ-resistant E. coli when compared to stool culture [17].

Microbiological Methods

To detect E. coli isolates with reduced susceptibility to FQs, peri-rectal swabs were obtained from enrolled patients and samples were inoculated to MacConkey agar plates supplemented with levofloxacin (0.125 μg/ml). Levofloxacin was used as a marker for susceptibility to FQ antibiotics. Plates were streaked for isolation of colonies and incubated at 37°C in atmospheric air supplemented with 5% to 10% CO2 and were checked for growth at 24 and 48 hours. Colonies suspected of being E. coli based on morphological appearance were subcultured to blood agar plates (tryticase soy agar with 10% sheep blood) and MacConkey agar without levofloxacin. The subcultured isolates were examined for the appropriate colony morphology on the MacConkey agar (i.e., pink colonies) and tested for oxidase production on the blood agar plate. All oxidase negative colonies with the appropriate colony morphology were definitively identified using the semi-automated Vitek 2 identification and susceptibility system [18].

To determine the minimum inhibitory concentration (MIC) of levofloxacin between the concentrations of 0.002 μg/ml and 32 μg/ml, E. coli isolates were subsequently tested for susceptibility to levofloxacin using the E-test method [19]. Isolates were also tested for susceptibility to a variety other antimicrobials using the semi-automated Vitek 2 identification and susceptibility system (bioMerieux, Inc.) [18].

For all E. coli with decreased susceptibility to FQs, the quinolone resistance determining region of gyrA and parC were amplified and sequenced using previously described primers [20]. Sequencing was performed by the University of Pennsylvania DNA Sequencing Facility using an ABI 3730 DNA analyzer with BigDye Taq FS Terminator V 3.1. (Applied Biosystems, Foster City, CA). Sequence data were analyzed and compared to reference sequences using the LASERGENE software package (DNASTAR, Inc., Madison, WI).

Increased drug efflux via the AcrAB efflux pump was measured indirectly by the organic solvent tolerance assay [21, 22]. Overexpression of AcrAB was measured indirectly by the organic solvent tolerance assay [20] [23]. The appearance of confluent growth in the presence of a hexane:cylohexane (3:1) mixture was interpreted as positive for AcrAB overexpression.

The genetic relatedness of E. coli isolates was determined by molecular typing using pulsed field gel electrophoresis (PFGE). Chromosomal DNA was extracted and digested from isolates using the procedure described by Gautom [24]. Chromosomal DNA was digested with the XbaI enzyme and separated by PFGE using the CHEF Mapper XA System (Bio-Rad, Hercules, CA). All results were analyzed using the Fingerprinting II Informatix Software v 3.0 (Bio-Rad). The band patterns were compared by means of the Dice coefficient using the unweighted pair-group method to determine band similarity and interpreted according to established criteria [25]. Genetic relatedness was determined by isolates that had ≥80% similarity. Although there are no standard criteria to determine whether isolates are due to person-to-person transmission [26],we used the following criteria to classify isolates as related via person to person transmission: 1) the isolates were defined as similar on the basis of the PFGE type (≥80% similarity), and (2) they were defined as epidemiologically related on the basis of any overlap in the dates of hospitalization [27].

Statistical Methods

The proportion of subjects with fecal colonization due to E. coli with reduced susceptibility to levofloxacin was calculated. We also calculated the proportion of E. coli isolates with a levofloxacin MIC ≥8 μg/ml. We summarized the frequency of genetic mechanisms of resistance for all E. coli isolates exhibiting reduced susceptibility to FQs focusing specifically on mutations in gyrA and parC, as well as the presence of organic solvent tolerance. Finally, we analyzed the frequency of different resistance mechanisms by study hospital and calendar year, using Fisher's exact test.

We then investigated the relationship between the mechanism(s) of resistance and the level of reduced susceptibility. We compared E. coli isolates with a levofloxacin MIC ≥8 μg/ml vs. MIC <8 μg/ml on the basis of: 1) median number of gyrA mutations; 2) median number of parC mutations; 3) median number of total mutations (i.e., gyrA + parC mutations); and 4) presence of organic solvent tolerance. We also investigated the association between specific FQ resistance mechanisms (i.e., gyrA muatation, parC mutation, organic solvent tolerance) and susceptibility to the following antibiotics: chloramphenicol, trimethoprim-sulfamethoxazole (TMP-SMZ), amikacin, gentamicin, imipenem, tetracycline, and tobramycin.

Categorical variables were compared using the Fisher's exact test while continuous variables were compared using the Student's t test or the Wilcoxon rank sum test, depending on the validity of the normality assumption [28]. For all calculations, a two-tailed P value of <0.05 was considered significant. All statistical calculations were performed using standard programs in STATA v 10.0, (Stata Corp, College Station TX).

RESULTS

During the study period, a total of 353 subjects were identified as colonized with E. coli demonstrating reduced FQ susceptibility. These 353 represented 15.1% of all subjects who agreed to have a sample obtained. Among the 353 subjects, the median age was 56 (interquartile range = 48–65) and 187 (53.0%) were male. With regard to race and ethnicity, 140 (39.7%) were white, 100 (28.3%) were African-American; 3 (0.9%) were Native American, 6 (1.7%) were Asian, 3 (0.9%) were Hispanic, 5 (1.4%) were classified as “other”, and 99 (28.1%) were unknown. Of the 353 subjects, 271 (76.7%) were hospitalized at Hospital 1 while 82 (23.2%) were hospitalized at Hospital 2.

Of the 353 study isolates, 217 (61.5%) demonstrated a levofloxacin MIC ≥8 μg/ml. Among these 353 isolates, the mean number of gyrA mutations per isolate was 1.45 (range 0–4), while the mean number of parC mutations per isolate was 0.51 (range 0–2). The mean number of total mutations (gyrA + parC) per isolate was 1.98 (range 0–4).

The number of gyrA and parC mutations among study isolates is noted in Table 1. Among all E. coli isolates with reduced susceptibility to FQs, the total number of mutations (gyrA + parC) was as follows: zero (n=48); one (n=77); two (n=85); three (n=121); and four (n=22). Of note, no isolate exhibited a parC mutation without also having a gyrA mutation.

Table 1
Mutations in gyrA and parC

For E. coli isolates with a levofloxacin MIC ≥8 μg/ml, the mean number of gyrA mutations per isolate was 1.93 compared to 0.70 mutations for isolates with a levofloxacin MIC <8.0 μg/ml (p<0.001). Similarly, the mean number of parC mutations for E. coli isolates with a levofloxacin MIC ≥8.0 μg/ml vs. <8.0 μg/ml was 0.77 and 0.12, respectively (p<0.001). Finally, the mean number of total mutations (i.e., gyrA + parC) for E. coli isolates with a levofloxacin MIC ≥8.0 μg/ml vs. <8.0 μg/ml was 2.70 and 0.82, respectively (p<0.001).

For E. coli isolates with a levofloxacin MIC ≥2.0 μg/ml, the mean number of gyrA mutations per isolate was 1.93 compared to 0.69 mutations for isolates with a levofloxacin MIC <2.0 μg/ml (p<0.001). Similarly, the mean number of parC mutations for E. coli isolates with a levofloxacin MIC ≥2.0 μg/ml vs. <2.0 μg/ml was 0.75 and 0.11, respectively (p<0.001). Finally, the mean number of total mutations (i.e., gyrA + parC) for E. coli isolates with a levofloxacin MIC ≥2.0 μg/ml vs. <2.0 μg/ml was 2.72 and 0.81, respectively (p<0.001). Presence of gyrA or parC mutations was not significantly associated with resistance to other antibiotics.

Of the 353 study isolates, 171 (48.6%) demonstrated organic solvent tolerance. Of the 171 isolates, 101 (59.1%) had a levofloxacin MIC ≥8 μg/ml while, 116 of 181 (64.1%) isolates without organic solvent tolerance had a levofloxacin MIC ≥8 μg/ml (p=0.38). E. coli isolates exhibiting organic solvent tolerance were significantly more likely to be resistant to chloramphenicol (17.5% vs 6.6%, respectively; p=0.002). However, presence of organic solvent tolerance was not associated with increased resistance to other antibiotics tested.

As noted previously, 48 isolates exhibited no mutations in gyrA or parC. Among these isolates, 45 (94%) had a levofloxacin MIC <0.25ug/ml). Also, 37 (77%) of these 48 isolates demonstrated organic solvent tolerance.

Among all 353 E. coli isolates, 306 (86.7%) demonstrated a nalidixic acid MIC in the non-susceptible range (i.e., ≥16 μg/ml). Of the 217 E. coli isolates with a levofloxacin MIC ≥8 μg/ml, 216 (99.6%) exhibited a nalidixic acid MIC ≥16 μg/ml. Of the 136 E. coli isolates with a levofloxacin MIC ≤8 μg/ml, 90 (66.2%) demonstrated a nalidixic acid MIC ≥16 μg/ml.

There were no significant differences when comparing isolates from the two study sites. For Hospitals 1 and 2, respectively, the proportion of isolates demonstrating organic solvent tolerance was 48.0% and 50.0% (p=0.42) and the proportion of isolates exhibiting a levofloxacin MIC of >8ug/ml was 60.9% and 63.4% (p=0.39). Similarly the proportion of isolates at Hospitals 1 and 2 demonstrating at least one gyrA mutation was 84.5% and 86.6% (p=0.40) while the proportion of isolates exhibiting at least one parC mutation was 45.0% and 47.6% (p=0.39).

Among E. coli with reduced susceptibility to levofloxacin, the annual proportion of isolates with a levofloxacin MIC ≥8 μg/ml did not change significantly over time: 15/20 (75%) in 2004; 46/71 (64.8%) in 2005; 86/146 (58.9%) in 2006; and 70/116 (60.3%) in 2007 (p=0.50). However, there were significant differences across study years in the proportion of E. coli isolates demonstrating various mechanisms of resistance. For example, the proportion of isolates with at least one gyrA mutation was 18/20 (90%) in 2004; 67/71 (94.4%) in 2005; 113/146 (77.4%) in 2006; and 102/116 (87.9%) in 2007 (p=0.005). Similarly, the proportion of isolates with at least one parC mutation was 11/20 (55.0%) in 2004; 37/71 (52.1%) in 2005; 74/146 (50.7%) in 2006; and 39/116 (33.6%) in 2007 (p=0.02). Finally, the proportion of isolates exhibiting organic solvent tolerance was 5/20 (25%) in 2004; 26/71 (36.6%) in 2005; 79/145 (54.5%) in 2006; and 61/116 (52.6%) in 2007 (p=0.02).

Among all E. coli isolates with reduced susceptibility to FQs, there were 49 PFGE types. Within these, there was one large cluster of related isolates (i.e., PFGE types 12a through 12f) comprising 48 isolates. However, within this cluster, only 2 of 48 subjects also met epidemiologic criteria for relatedness (i.e., overlapping period of hospitalization with another subject from the cluster). There was also a smaller related large cluster of PFGE type 16 (16a through 16c) comprising 17 isolates. Within this cluster, only three met epidemiologic criteria for relatedness. Thus, there were only five subjects (1.5%) whose isolates met criteria for person-to-person transmission.

DISCUSSION

Of 353 subjects colonized with E. coli with reduced susceptibility to FQs, 217 (61.5%) were colonized with an E. coli meeting the Clinical and Laboratory Standards Institute (CLSI) breakpoint for FQ resistance (i.e., a levofloxacin MIC ≥8 μg/ml). Thus, 136 isolates (or nearly 40%) would not have been identified as having reduced susceptibility to FQs by current CLSI standards. Of these 136 E. coli isolates, 90 (66.2%) were non-susceptible to nalidixic acid. This suggests nalidixic acid may be a useful marker for reduced FQ susceptibility among fecal E. coli isolates. These data support recent suggestions that routinely reporting nalidixic acid susceptibilities might effectively identify many isolates already harboring an early gyrA with or without a parC mutation (i.e., early mutations that result in an increased fluoroquinolone MIC, but an MIC which nevertheless does not meet the established threshold for resistance) [14, 29]. Indeed, these are precisely those isolates most likely to become fully resistant in the presence of antimicrobial selective pressure [30]. Efforts to study the potential for optimizing FQ resistance surveillance efforts and/or FQ prescribing based on nalidixic acid susceptibilities should be pursued.

We noted that the large majority of isolates had at least one gyrA mutation, with many demonstrating additional gyrA and/or parC mutations. These findings suggest that, in colonization in the clinical setting, the first step in the evolution of FQ-resistant E. coli is a gyrA mutation with subsequent steps likely including additional gyrA or parC mutations and/or enhanced efflux [31, 32]. Prospective studies with serial fecal sampling are needed to confirm the nature of longitudinal changes in E. coli GI colonization over time.

We also found that nearly 50% of isolates demonstrated efflux pump overexpression as indicated by organic solvent tolerance. This percentage is somewhat higher than that of prior reports including our own [12, 1416] and suggests this mechanism of resistance may be becoming more widespread over time. The clinical implications of widespread organic solvent tolerance are clear in the fact that efflux overexpression typically confers resistance to multiple other antimicrobial agents [33]. Despite the many recognized substrates of efflux pumps, we found presence of organic solvent tolerance was associated with a greater likelihood of resistance to chloramphenicol, but not other antibiotics.

Finally, we found several temporal changes in isolate characteristics. In particular, we found significant differences across study years for the presence of gyrA and parC mutations as well as organic solvent tolerance. As this study enrolled patients continuously over time, these results extend considerably findings from our earlier work which only assessed colonization through several point prevalence surveys [12, 1416]. Our results suggest substantial changes over time in the prevalence of organic solvent tolerance among E. coli with reduced susceptibility to FQs [16]. While an outbreak of a specific E. coli strain might be one explanation, results of the PFGE analysis argue against substantial person-to-person spread. Likewise, there were no major changes in the antimicrobial formulary in the two hospitals which might explain these results. Given the consistent findings across studies, future investigations of temporal changes in resistance mechanisms may provide valuable insights into the evolution of these resistant pathogens.

Our study had a few potential limitations. While selection bias is of potential concern, we sought to enroll all eligible subjects. Although only 51% of eligible subjects were enrolled, participants and non-participants were similar with regard to available data (i.e., age, sex, hospital location, duration of hospitalization prior to invitation to enroll) suggesting no substantial bias was introduced by non-participation.

In sampling subjects, only one colony was selected for evaluation. However, recent work has noted that subjects may on occasion be colonized with multiple distinct strains of FQ-resistant E. coli [34, 35]. Despite these recent findings, our goal in the current study was to identify subjects colonized with E. coli with reduced susceptibility to fluoroquinolones, regardless of the number of strains with which a given subject was colonized. As our goal was not to examine strain diversity, we believe obtaining only one strain person was reasonable. However, this approach clearly limits the ability to comment on the phenotypic and genotypic characterization of multiple isolates per subject.

In addition, our study focused only on identifying the most common, and clinically important, mechanisms of FQ resistance. As such, we did not identifying less common mechanisms (e.g., qnr, aac(6')-lb-cr). For those E. coli with reduced susceptibility to fluoroquinolones that did not manifest either gyrA/parC mutations or efflux overexpression, it is possible that one of these less common resistance mechanisms may have contributed to reduced susceptibility. In addition, our study was conducted in a large tertiary care medical center and a smaller urban community hospital; the results may not be generalizable to other dissimilar institutions.

In summary, GI tract colonization with E. coli demonstrating reduced susceptibility to FQs is common in hospitalized patients. Although approximately 40% of study isolates exhibited a levofloxacin MIC <8 μg/ml (and would thus be missed by current CLSI breakpoints), nalidixic acid resistance may be a useful marker for detection of such isolates. Significant differences occurred across study years in the proportion of isolates exhibiting various resistance mechanisms, suggesting future research should more clearly elucidate potential evolution of FQ resistance mechanisms in the clinical setting over time.

Figure 1
Levofloxacin MIC and Susceptibility to Other Antimicrobial Agents All statistically significant associations shown

ACKNOWLEDGEMENTS

This study was primarily supported by National Institutes of Health (NIH) grant R01-AI055008 (EL). Support was also provided by NIH Grant K24-AI080942 (EL), K24-AI073957 (JPM), as well as an Agency for Healthcare Research and Quality (AHRQ) Centers for Education and Research on Therapeutics cooperative agreement (U18-HS10399).

Dr. Lautenbach has received research support from Merck, Ortho-McNeil, Cubist, and AstraZeneca.

Footnotes

Presented in part at the 19th Annual Meeting of the Society for Healthcare Epidemiology of America (SHEA) of March 19–22, 2009 San Diego, CA.

All other authors report no potential conflict of interest.

REFERENCES

1. Linder JA, Huang ES, Steinman MA, Gonzales R, Stafford RS. Fluoroquinolone prescribing in the United States: 1995 to 2002. Am J Med. 2005;118:259–68. [PubMed]
2. Hooper DC. New uses for new and old quinolones and the challenge of resistance. Clin Infect Dis. 2000;30:243–54. [PubMed]
3. Lautenbach E, Strom BL, Nachamkin I, et al. Longitudinal trends in fluoroquinolone resistance among Enterobacteriaceae isolates from inpatients and outpatients, (1989–2000): Differences in the emergence and epidemiology of resistance across organisms. Clin Infect Dis. 2004;38:655–62. [PubMed]
4. Neuhauser MM, Weinstein RA, Rydman R, Danziger LH, Karam G, Quinn JP. Antibiotic resistance among gram-negative bacilli in US intensive care units. JAMA. 2003;289:885–8. [PubMed]
5. Lautenbach E, Metlay JP, Bilker WB, Edelstein PH, Fishman NO. Association between fluoroquinolone resistance and mortality in Escherichia coli and Klebsiella pneumoniae infections: Role of inadequate empiric antimicrobial therapy. Clin Infect Dis. 2005;41:923–9. [PubMed]
6. Bettelheim KA. The genus Escherichia. In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH, editors. The prokaryotes:A handbook on the biology of bacteria. Ecophysiology, isolates, identification, applications. Springer-Verlag; New York: 1991.
7. Arbeit RD, Arthur M, Dunn R, Kim C, Selander RK, Goldstein R. Resolution of recent evolutionary divergence among Escherichia coli from related lineages: the application of pulsed field electrophoresis to molecular epidemiology. J Infect Dis. 1990;161:230–5. [PubMed]
8. Brumfitt W. Progress in understanding urinary infections. J Antimicrob Chemother. 1991;27:9–22. [PubMed]
9. Oethinger M, Jellen-Ritter AS, Conrad S, Marre R, Kern WV. Colonization and infection with fluoroquinolone-resistant Escherichia coli among cancer pateints: clonal analysis. Infection. 1998;26:379–84. [PubMed]
10. Everett MJ, Jin YF, Ricci V, Piddock LJV. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Agents Chemother. 1996;40:2380–6. [PMC free article] [PubMed]
11. McDonald LC, Chen FJ, Lo HJ, et al. Emergence of reduced susceptibility and resistance to fluoroquinolones in Escherichia coli in Taiwan and contributions of distinct selective pressures. Antimicrob Agents Chemother. 2001;45:3084–91. [PMC free article] [PubMed]
12. Oethinger M, Kern WV, Goldman JD, Levy SB. Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli. J Antimicrob Chemother. 1998;41:111–4. [PubMed]
13. Ozeki S, Deguchi T, Yasuda M, et al. Development of a rapid assay for detecting gyrA mutations in escherichia coli and determination of incidence of gyrA mutations in clinical strains isolated from patients with complicated urinary tract infections. Journal of Clinical Microbiology. 1997;35:2315–2319. [PMC free article] [PubMed]
14. Komp Lindgren P, Karlsson A, Hughes D. Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections. Antimicrob Agents Chemother. 2003;47:3222–32. [PMC free article] [PubMed]
15. Park YH, Yoo JH, Huh DH, Cho YK, Choi JH, Shin WS. Molecular analysis of fluoroquinolone resistance in Escherichia coli on the aspect of gyrase and multiple antibiotic resistance (mar) genes. Yonsei Med J. 1998;39:534–40. [PubMed]
16. Lautenbach E, Fishman NO, Metlay JP, et al. Phenotypic and genotypic characterization of fecal Escherichia coli isolates with decreased susceptibility to fluoroquinolones: Results from a large hospital-based surveillance initiative. J Infect Dis. 2006;194:79–85. [PubMed]
17. Lautenbach E, Harris AD, Perencevich EN, Nachamkin I, Tolomeo P, Metlay JP. Test characteristics of perirectal and rectal swab compared to stool sample for detection of fluoroquinolone-resistant Escherichia coli in the gastrointestinal tract. Antimicrob Agents Chemother. 2005;49:798–800. [PMC free article] [PubMed]
18. Ling TK, Tam PC, Liu ZK, Cheng AF. Evaluation of VITEK 2 rapid identification and susceptibility testing system against gram-negative clinical isolates. J Clin Microbiol. 2001;39:2964–6. [PMC free article] [PubMed]
19. Baker CN, Stocker SA, Culver DH, Thornsberry C. Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. J Clin Microbiol. 1991;29:533–8. [PMC free article] [PubMed]
20. Wang H, Dzink-Fox JL, Chen M, Levy SB. Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from china: role of acrR mutations. Antimicrobial Agents Chemother. 2001;45:1515–21. [PMC free article] [PubMed]
21. Okusu H, Ma D, Nikaido H. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol. 1996;178:306–8. [PMC free article] [PubMed]
22. Oethinger M, Kern WV, Jellen-Ritter AS, McMurry LM, Levy SB. Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the acrAB efflux pump. Antimicrobial Agents Chemother. 2000;44:10–13. [PMC free article] [PubMed]
23. White DG, Goldman JD, Demple B, Levy SB. Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J Bacteriol. 1997;179:6122–6. [PMC free article] [PubMed]
24. Gautom RK. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. J Clin Microbiol. 1997;35:2977–80. [PMC free article] [PubMed]
25. Goering RV, Tenover FC. Epidemiological interpretation of chromosomal macro-restriction fragment patterns analyzed by pulsed-field gel electrophoresis. J Clin Microbiol. 1997;35:2432–3. [PMC free article] [PubMed]
26. Lautenbach E. Antimicrobial Resistance in Gram-Negative Pathogens: Crafting the Tools Necessary to Navigate the Long Ascent out of the Abyss. J Infect Dis. 2009;200:838–40. [PMC free article] [PubMed]
27. Johnson JK, Smith G, Lee MS, et al. The Role of Patient-to-Patient Transmission in the Acquisition of Imipenem-Resistant Pseudomonas aeruginosa Colonization in the Intensive Care Unit. J Infect Dis. 2009;200:900–5. [PMC free article] [PubMed]
28. Kleinbaum DG, Kupper LL, Morgenstern H. Epidemiologic research: principles and quantitative methods. Van Nostrand Reinhold; New York: 1982.
29. Ruiz J, Gomez J, Navia MM, et al. High prevalence of nalidixic acid resistant, ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae. Diagn Microbiol Infect Dis. 2002;42:257–61. [PubMed]
30. Tavio MM, Vila J, Ruiz J, Ruiz J, Martin-Sanchez AM, Jimenez de Anta MT. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. J Antimicrob Chemother. 1999;44:735–42. [PubMed]
31. Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis. 2005;40(Suppl 7):S432–8.
32. Lehn N, Stower-Hoffman J, Kott T, et al. Characterization of clinical isolates of Escherichia coli showing high levels of fluoroquinolone resistance. J Clin Microbiol. 1996;34:597–602. [PMC free article] [PubMed]
33. Poole K. Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrobial Agents Chemother. 2000;44:2233–41. [PMC free article] [PubMed]
34. Lautenbach E, Bilker WB, Tolomeo P, Maslow JN. Impact of diversity of colonizing strains on strategies for sampling Escherichia coli from fecal specimens. J Clin Microbiol. 2008;46:3094–6. [PMC free article] [PubMed]
35. Lautenbach E, Tolomeo P, Black N, Maslow JN. Risk factors for fecal colonization with multiple distinct strains of Escherichia coli among long-term care facility residents. Infect Control Hosp Epidemiol. 2009;30:491–3. [PMC free article] [PubMed]