Abstract
OBJECTIVE:
To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates.
METHODS:
E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6′) Ib-cr and qepA.
RESULTS:
2.1% and 1.1% of E coli harboured aac(6′)Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations.
CONCLUSION:
PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.
Keywords: Ciprofloxacin, Escherichia coli, Fluoroquinolone, Plasmid-mediated quinolone resistance, Quinolone resistance-determining region
Abstract
OBJECTIF :
Établir si les déterminants de la résistance aux quinolones à médiation plasmidique (RQMP) contribuent à la résistance croissante aux fluoroquinolones dans les isolats d’Escherichia coli des hôpitaux canadiens et déterminer les mécanismes de susceptibilité réduite à la ciprofloxacine dans un récent échantillonnage de 190 isolats cliniques d’E coli.
MÉTHODOLOGIE :
Les chercheurs ont colligé les isolats d’E coli (n=1 702) dans le cadre de l’étude de surveillance canadienne CANWARD de 2007 sur la résistance antibiotique dans les unités hospitalières. Le Clinical and Laboratory Standards Institute (CLSI) a effectué les tests de susceptibilité antimicrobienne au moyen de la technique de microdilution en milieu liquide. Au moyen d’un sous-groupe représentatif d’isolats (n=190), les chercheurs ont décelé les mécanismes de susceptibilité réduite à la ciprofloxacine à l’aide de la réaction en chaîne de la polymérase et du séquençage des régions responsables de la résistance aux quinolones (RRRQ) pour les gènes chromosomiques gyrA et parC, et à l’aide de la réaction en chaîne de la polymérase pour les gènes de RQMP, soit qnr, aac(6′)Ib-cr et qepA.
RÉSULTATS :
Les chercheurs ont constaté que 2,1 % et 1,1 % des E coli hébergent les gènes aac(6′)Ib-cr et qnrB, respectivement. Les chercheurs ont observé des substitutions d’acides aminés simples dans les RRRQ du gène gyrA d’isolats aux concentrations minimales inhibitrices de ciprofloxacine aussi basses que 0,12 μg/mL. Lorsque la concentration minimale inhibitrice de ciprofloxacine passait à 1 μg/mL (que le CLSI considère toujours comme susceptible), la majorité des isolats démontrait à la fois des mutations des gènes gyrA et parC.
CONCLUSION :
Les déterminants de la RQMP et les mutants des RRRQ d’isolats cliniques d’E coli ayant une susceptibilité réduite à la ciprofloxacine démontrent qu’il est nécessaire d’accroître la surveillance et de réévaluer les points de rupture du CLSI pour prévenir l’apparition d’une résistance aux fluoroquinolones.
In 2007, the Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study – an annual national surveillance study assessing antimicrobial resistance – identified Escherichia coli as the most commonly encountered pathogen within Canadian hospitals (1). In that study, 25.4% of E coli isolates were fluoroquinolone resistant (www.can-r.ca) (1).
Until recently, resistance to fluoroquinolones was considered to be only chromosomally encoded and most commonly involved amino acid substitutions in the quinolone resistance-determining regions (QRDR) of DNA gyrase (gyrA) and/or topoisomerase IV (parC), which are the main targets of fluoroquinolones (2). Reduced uptake by decreased expression of outer membrane porins and overexpression of efflux pumps also contributes to chromosomal fluoroquinolone resistance (3). Although originally considered to be improbable due to the plasmid curing effect of quinolones, plasmid-mediated quinolone resistance (PMQR) was first reported in 1998 and has become an emerging concern (4,5). Three different types of PMQR determinants have recently been reported. The first PMQR determinant includes the following quinolone resistance genes: qnrA, qnrB, qnrC, qnrD and qnrS. Qnr determinants are believed to bind to and protect DNA gyrase and/or topoisomerase IV from fluoroquinolone inhibition. The second type of PMQR determinant, aac(6′)Ib-cr, is an aminoglycoside-modifying enzyme that acetylates several fluoroquinolones including ciprofloxacin (3). Although both the qnr and aac(6′)Ib-cr genes only confer reduced susceptibility (increased minimum inhibitory concentration [MIC] but not elevated past the susceptible breakpoint) to the fluoroquinolones, they do provide a background for in vivo selection of chromosomal-borne mechanisms of resistance and result in the recovery of resistant mutants with higher levels of resistance than chromosomal changes alone (6). The third and most recently described PMQR determinant, qepA, is an efflux pump that extrudes hydrophilic fluoroquinolones such as ciprofloxacin (7). The purpose of the present study was to determine whether PMQR determinants play a role in increasing resistance to fluoroquinolones among E coli isolates in Canadian hospitals and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of E coli clinical isolates.
PATIENTS AND METHODS
E coli isolates (n=1702) were collected as part of the CANWARD surveillance study. CANWARD is a laboratory-based surveillance study coordinated at the Health Sciences Centre in Winnipeg, Manitoba. From January 1 to December 31, 2007, inclusive, 12 sentinel hospital centres across Canada submitted pathogens from patients attending hospital clinics, emergency rooms, medical and surgical wards, and intensive care units, as previously described (1).
Following two subcultures from frozen stock, the in vitro activity of ciprofloxacin was determined by microbroth dilution in accordance with Clinical Laboratory and Standards Institute guidelines (8). Of the 1702 E coli isolates, a representative cohort of 190 E coli isolates with ciprofloxacin MICs ranging from ≤0.06 μg/mL to 8 μg/mL were further analyzed, including 20 of 1158 isolates with a ciprofloxacin MIC of ≤0.06 μg/mL (susceptible/wildtype; control group). These 20 isolates were selected to represent all geographical regions of the country. Thereafter, all 23 isolates with a ciprofloxacin MIC of 0.12 μg/mL, all 46 isolates with an MIC of 0.25 μg/mL, all 33 isolates with an MIC of 0.5 μg/mL, all 20 isolates with an MIC of 1 μg/mL, all five isolates with an MIC of 2 μg/mL, all nine isolates with an MIC of 4 μg/mL and all 34 isolates with an MIC of 8 μg/mL were studied. Reduced susceptibility to ciprofloxacin was defined as an MIC ranging from 0.12 μg/mL to 1 μg/mL (CLSI MIC breakpoints for ciprofloxacin are ≤1.0 μg/mL [susceptible], 2.0 μg/mL [intermediate], and ≥4.0 μg/mL [resistant]).
The molecular mechanisms of reduced susceptibility to ciprofloxacin were determined by polymerase chain reaction and sequencing of the QRDR of chromosomal gyrA and parC genes and the PMQR genes – qnrA, qnrB, qnrS, aac(6′)Ib-cr and qepA – using appropriate controls as previously described (9).
RESULTS
The distribution of ciprofloxacin MICs against the 1702 E coli isolates collected as part of CANWARD 2007 is as follows: 1158 (67.9%) with an MIC of ≤0.06 μg/mL, 23 (1.4%) with an MIC of 0.12 μg/mL, 46 (2.7%) with an MIC of 0.25 μg/mL, 33 (1.9%) with an MIC of 0.5 μg/mL and 20 (1.2%) with an MIC of 1 μg/mL. Thus, 8.4% of E coli demonstrated reduced susceptibility to ciprofloxacin (MIC 0.12 μg/mL to 1 μg/mL). Five (0.3%) isolates had a ciprofloxacin MIC of 2 μg/mL, nine (0.6%) with an MIC of 4 μg/mL, 34 (2.0%) with an MIC of 8 μg/mL and 374 (22.0%) with an MIC of >8 μg/mL.
The mechanisms of reduced susceptibility to ciprofloxacin in a cohort of 190 clinical E coli isolates are summarized in Table 1.
TABLE 1.
Mechanisms of reduced susceptibility to ciprofloxacin (CIP) in a cohort of 190 clinical Escherichia coli isolates from Canadian hospitals
| CIP MIC | Isolates, n (%) | gyrA AA variants | parC AA variants | qnrA | qnrB | qnrS | aac(6′)Ib | qepA |
|---|---|---|---|---|---|---|---|---|
| CIP MIC ≤0.06 μg/mL; n=20; susceptible/wildtype | ||||||||
|
| ||||||||
| 0.015 | 9 (45) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.03 | 10 (50) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.06 | 1 (5) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| CIP MIC 0.12 μg/mL; n=23; reduced susceptible | ||||||||
|
| ||||||||
| 0.12 | 12 (52.2) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.12 | 4 (17.4) | S83L | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.12 | 3 (13.0) | D87G | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.12 | 2 (8.7) | D87N | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.12 | 1 | S83A | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.12 | 1 | No AA variants | D111Y | Negative | Negative | Negative | Negative | – |
| CIP MIC 0.25 μg/mL; n=46; reduced susceptible | ||||||||
|
| ||||||||
| 0.25 | 1 (2.2) | D87Y | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.25 | 1 (2.2) | D87N | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.25 | 3 (6.5) | D87G | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.25 | 4 (8.7) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.25 | 37 (80.4) | S83L | No AA variants | Negative | Negative | Negative | Negative | – |
| CIP MIC 0.5 μg/mL; n=33; reduced susceptible | ||||||||
|
| ||||||||
| 0.5 | 1 (3.0) | No AA variants | No AA variants | Negative | qnrB | Negative | Negative | – |
| 0.5 | 1 (3.0) | S83L | No AA variants | Negative | Negative | Negative | aac(6′)Ib-cr | – |
| 0.5 | 3 (12.1) | No AA variants | No AA variants | Negative | Negative | Negative | Negative | – |
| 0.5 | 28 (84.8) | S83L | No AA variants | Negative | Negative | Negative | Negative | – |
| CIP MIC 1 μg/mL; n=20; reduced susceptible | ||||||||
|
| ||||||||
| 1 | 1 (5.0) | S83L | S80I, E84V | Negative | Negative | Negative | Negative | – |
| 1 | 1 (5.0%) | D87N | No AA variants | Negative | Negative | Negative | Negative | – |
| 1 | 1 (5.0%) | S83L | S80I | Negative | Negative | Negative | aac(6′)Ib-cr | – |
| 1 | 2 (10.0) | S83L | S80I, E84G | Negative | Negative | Negative | Negative | – |
| 1 | 3 (15.0) | S83L | No AA variants | Negative | Negative | Negative | Negative | – |
| 11 | 4 (20.0) | S83L | E84G | Negative | Negative | Negative | Negative | – |
| 8 (40.0) | S83L | S80I | Negative | Negative | Negative | Negative | – | |
| CIP MIC 2 μg/mL; n=5; intermediate | ||||||||
|
| ||||||||
| 2 | 3 (60.0) | S83L, D87N | S80I | Negative | Negative | Negative | Negative | Negative |
| 2 | 1 (20.0) | S83L, D87N | S80I, E84V | Negative | Negative | Negative | Negative | Negative |
| 2 | 1 (20.0) | S83L, D87N | S80I, D111Y | Negative | Negative | Negative | Negative | Negative |
| CIP MIC 4 μg/mL; n=9; resistant | ||||||||
|
| ||||||||
| 4 | 1 (11.1) | S83A, D87Y | S80I | Negative | Negative | Negative | Negative | Negative |
| 4 | 1 (11.1) | S83L, D87N | S80I | Negative | Negative | Negative | Negative | Negative |
| 4 | 1 (11.1) | S83L | S80I | Negative | Negative | Negative | aac(6′)Ib-cr | Negative |
| 4 | 1 (1.11) | S83L, D87N | S80I | Negative | Negative | Negative | Negative | Negative |
| 4 | 2 (22.2) | S83L, D87N | S57T, S80I | Negative | Negative | Negative | Negative | Negative |
| 4 | 3 (33.3) | S83L, D87N | S80I, E84V | Negative | Negative | Negative | Negative | Negative |
| CIP MIC 8 μg/mL; n=34; resistant | ||||||||
|
| ||||||||
| 8 | 1 (2.9) | S83L, D87N | S80I, E84V | Negative | Negative | Negative | aac(6′)Ib-cr | Negative |
| 8 | 1 (2.9) | S83L, D87N | S80I, E84V | Negative | Negative | Negative | Negative | Negative |
| 8 | 1 (2.9) | S83L, D87N | E84V | Negative | Negative | Negative | Negative | Negative |
| 8 | 1 (2.9) | S83L, D87N | S80I, E84V | Negative | qnrB | Negative | Negative | Negative |
| 8 | 1 (2.9) | S83L, D87Y | S80I | Negative | Negative | Negative | Negative | Negative |
| 8 | 2 (5.9) | S83L, D87N | S57T, S80I | Negative | Negative | Negative | Negative | Negative |
| 8 | 2 (5.9) | S83L, D87N | S80I, E84G | Negative | Negative | Negative | Negative | Negative |
| 8 | 10 (29.4) | S83L, D87N | S80I | Negative | Negative | Negative | Negative | Negative |
| 8 | 15 (44.1) | S83L, D87N | S80I, E84V | Negative | Negative | Negative | Negative | Negative |
| n=190 | 149 (78.4%) | 65 (34.2%) | 0 | 2 (1.1%) | 0 | 4 (2.1%) | 0 | |
qepA polymerase chain reaction was only performed on resistant isolates (minimum inhibitory concentration [MIC] ≥2 μg/mL). – Not applicable; AA Amino acid
QRDR mutations in gyrA and parC
None of the 20 wildtype E coli contained mutations within the QRDR of gyrA and parC. However, single amino acid substitutions in the QRDR of gyrA were present in E coli with MICs as low as 0.12 μg/mL, in which 10 of 23 (43.5%) E coli had mutations in gyrA resulting in amino acid changes – S83L (40%), S83A (10%), D87G (30%) and D87N (20%). One isolate had a parC mutation outside of the QRDR, resulting in D111Y. At MICs of 0.25 μg/mL and 0.5 μg/mL, nearly all isolates had gyrA mutations, with 42 (91.3%) of the 46 E coli with a MIC of 0.25 μg/mL demonstrating amino acid changes – S83L (80.4%), D87G (6.5%), D87Y (2.2%) and D87N (2.2%); 29 (87.9%) of the 33 E coli with an MIC of 0.5 μg/mL had an S83L amino acid change. At an MIC of 1 μg/mL, mutations within the QRDR of both gyrA and parC were observed. gyrA mutations were present in all E coli with an MIC of 1 μg/mL, resulting in S83L (95%) and D87N (5%). parC single- and double-step mutations were observed in 16 of 19 (84.2%) isolates with gyrA mutations, and resulted in S80I (45%), E84G (20%), S80I and E84G (10%), and S80I and E84V (5%). Double-step gyrA mutations were observed in all but one (S83L; 2.1%) E coli with MICs of ≥2 μg/mL, resulting in S83L and D87N (91.7%), S83L and D87Y (2.1%), S83L and D87G (2.1%), and S83A and D87Y (2.1%). Both single-step and double-step parC mutations were observed among the same cohort of E coli with MICs of ≥2 μg/mL, resulting in S80I (37.5%), E84V (2.1%), S57T and S80I (8.3%), S80I and E84V (45.8%), S80I and E84G (4.2%), and S80I and D111Y (2.1%). All mutations observed within the QRDR of gyrA and parC have been previously observed for clinical isolates and are known to increase fluoroquinolone MICs (10). Figure 1 demonstrates the stepwise accumulation of mutations in the QRDR of gyrA, followed by parC QRDR changes as the ciprofloxacin MIC increases in E coli.
Figure 1).
gyrA and parC quinolone resistance-determining regions amino acid (AA) variants among Escherichia coli with increasing ciprofloxacin minimum inhibitory concentrations (MIC).*Any AA variant
PMQR genes:
Four (2.1%) of the 190 isolates were found to carry the aac(6′)Ib-cr gene. Two of the four isolates had reduced susceptibilities (0.5 μg/mL and 1 μg/mL) to ciprofloxacin and had mutations within the QRDR of gyrA (S83L) and one also had a parC mutation (S80I). The remaining two isolates harbouring aac(6′)Ib-cr gene had resistant MICs (4 μg/mL and 8 μg/mL) and had mutations within the QRDR of gyrA and parC.
Two (1.1%) of the 190 isolates were found to carry the qnrB gene. Of the two isolates harbouring qnrB, one isolate had reduced susceptibility to ciprofloxacin (MIC 0.5 μg/mL) with no QRDR mutations, and the other isolate was resistant to ciprofloxacin and contained double mutations in both gyrA (S83L, D87N) and parC (S80I, E84V). No qnrA, qnrS or qepA PMQR genes were observed among the cohort. Demographic data for PMQR positive isolates are summarized in Table 2.
TABLE 2.
Demographic data of plasmid-mediated quinolone resistance (PMQR)-positive isolates
| Stock number | PMQR determinant | City, province | Location | Age, years | Sex | Source |
|---|---|---|---|---|---|---|
| 74886 | qnrB | Toronto, Ontario | ICU | 74 | Male | Blood |
| 76265 | qnrB | London, Ontario | Medicine general | 62 | Male | Blood |
| 74912 | aac(3′)1b-cr | Toronto, Ontario | Medicine general | 63 | Female | Urine |
| 77206 | aac(3′)1b-cr | Montreal, Quebec | Emergency room | 66 | Female | Wound |
| 70720 | aac(3′)1b-cr | Vancouver, British Columbia | Clinic | 30 | Female | Urine |
| 75797 | aac(3′)1b-cr | Montreal, Quebec | Medicine general | 75 | Female | Blood |
ICU Intensive care unit
DISCUSSION
Increasing ciprofloxacin resistance among clinical isolates of E coli in Canadian hospitals is worrisome because ciprofloxacin is often used empirically in Canada and around the world to treat various types of infections. Although it is well known that resistance to the quinolones in E coli most commonly results from the accumulation of mutations primarily occurring in gyrA followed by parC, we tried to determine the prevalence of PMQR determinants and mechanisms of resistance among clinical isolates of E coli with reduced ciprofloxacin MICs. Because the most common PMQR determinants, qnr and aac(6′)Ib-cr, only provide low levels of resistance (or reduced susceptibility) to the fluoroquinolones, we decided to use a cohort of E coli ranging in ciprofloxacin MICs from susceptible to resistant (≤0.12 μg/mL to 8 μg/mL). All available E coli isolates with reduced susceptibility to ciprofloxacin were studied. PMQR determinants are clinically important because they increase the mutant prevention concentration of ciprofloxacin, thus facilitating the recovery of mutants with higher levels of resistance to quinolones (5,11). Therefore, it has been suggested that such isolates provide a background for in vivo selection of additional chromosomal mechanisms of resistance to occur during or after treatment with fluoroquinolones (3,12). As a consequence, E coli isolates with reduced susceptibility to ciprofloxacin exhibit the potential for developing complete resistance and effectively eliminating the possibility of ciprofloxacin-based treatment of E coli infections. Thus, we set out to test the hypothesis that increasing fluoroquinolone resistance observed in E coli in Canada may be due to the presence of PMQR determinants among clinically significant E coli isolates with reduced ciprofloxacin susceptibilities.
Many PMQR prevalence studies in E coli focus on populations of ciprofloxacin intermediate/resistant E coli or populations of extended spectrum beta-lactamase (ESBL)-producing E coli (9,13,14). There are very few studies that have focused on an E coli population with ciprofloxacin MICs ranging from susceptible to resistant (15–17). We demonstrate that the prevalence of PMQR determinants among this cohort remains low in Canada. Only 2.1% of isolates harboured the aac(6′)Ib-cr gene and 1.1% carried a qnrB gene, in which equal proportions were observed among E coli with reduced susceptible and resistant MICs. No other PMQR determinants were identified. Similar rates of qnr (1.1%) and aac(3′)Ib-cr (3.2%) were detected among E coli and Klebsiella species from two sets of consecutive isolates collected from 2004 to 2005 in Norway and Sweden, with resistance to nalidixic acid and/or reduced susceptibility/resistance to ciprofloxacin (17). It is not surprising that aac(6′)Ib-cr was the most common PMQR determinant because two of the four isolates were ESBL producers, and many studies have shown its predominance among other populations of E coli, especially among ESBL producers (9,14,17–19). With only a limited number of isolates harbouring PMQR determinants, it is difficult to link demographic data that may predict their presence (Table 2). However, because both qnrB-positive isolates were collected from the province of Ontario and isolated from blood, this provides increased awareness of the presence and potential spread among Ontario hospitals. The aac(6′)Ib-cr harbouring isolates from the present study were obtained only from female patients, but were present across Canada. Thus, it appears that PMQR determinants do not have much of an impact on the increasing ciprofloxacin resistance among E coli in Canada because their prevalence remains low. Their presence, however, should be concerning because dissemination may occur and potentially fuel rapid and further increases in fluoroquinolone resistance among E coli (14). This was observed in the clinical setting when a fluoroquinolone-susceptible, qnrA-producing E coli developed chromosomal mutations in the QRDR of gyrA and parC, resulting in ensuing resistance after treatment with norfloxacin (12).
The primary mechanism of reduced susceptibility to ciprofloxacin in E coli was due to the stepwise accumulation of mutations in the QRDR of gyrA, followed by parC QRDR changes as previously reported (2,3,10,20). Disturbingly, 43.5% and 91.3% of E coli with ciprofloxacin MICs of 0.12 μg/mL and 0.25 μg/mL, respectively, demonstrated QRDR changes in gyrA, which is well below the CLSI ciprofloxacin intermediate and resistant breakpoints of 2 μg/mL and 4 μg/mL, respectively. In addition, the vast majority of E coli with ciprofloxacin MICs of 1 μg/mL and defined as susceptible by CLSI breakpoints demonstrated QRDR changes in both gyrA and parC. Consequently, the risk for development of high-level resistance to the fluoroquinolones is greatly underestimated by the current CLSI breakpoints, and they do not adequately detect these mutants.
There were a few limitations regarding the present study. We did not assess for reduced uptake of ciprofloxacin by measuring decreased expression of outer membrane porins, nor did we assess the presence of overexpression of efflux pumps (other than qepA). In particular, reduced uptake may play a role in the decreased susceptibility to ciprofloxacin in the one isolate harbouring qnrB with a ciprofloxacin MIC of 0.5 μg/mL, with no amino acid variants within the QRDR of gyrA and parC. However, from a clinical perspective, it is clear that the most common and important mechanisms conferring reduced susceptibility and resistance to ciprofloxacin among these isolates involved amino acid substitutions in the QRDR of gyrA and parC.
Clinically significant E coli isolates from the present study were isolated from blood, urine, wound and respiratory specimens. Isolates obtained from the urinary tract had ciprofloxacin MICs ranging from susceptible to resistant (≤0.06 μg/mL to 8 μg/mL). For the treatment of lower urinary tract infections, because the fluoroquinolones ciprofloxacin and levofloxacin attain very high concentrations in the urine (eg, approximately 300 μg/mL), these high concentrations may theoretically overcome E coli with reduced susceptibility or even resistance to fluoroquinolones (21). However, because fluoroquinolone MICs are known to be elevated in acidic urine (pH approximately 5.5 versus broth at approximately 7.2), and the concern that strains with a first-step QRDR mutation or PMQR determinants may rapidly develop resistance on therapy, we would not recommend fluoroquinolone therapy for urinary infections or any other infections due to E coli with known fluoroquinolone-resistance determinants. In addition, no data are available describing whether once-daily dosing or twice-daily dosing of fluoroquinolones for treatment of such strains is the preferred dosage. Thus, treatment of these strains with fluoroquinolones is not recommended for fear of microbiological failure.
CONCLUSION
Reduced susceptibilities to ciprofloxacin in Canadian clinical isolates of E coli were primarily due to the accumulation of mutations in the QRDR of gyrA followed by parC. Although the prevalence of PMQR determinants among this cohort was low, their presence and the presence of gyrA and parC QRDR mutants among E coli with reduced susceptibility to ciprofloxacin has clinical implications and may fuel the development of high-level fluoroquinolone resistance, effectively eliminating the possibility of ciprofloxacin-based treatment of E coli infections. The present study demonstrates the need for increased monitoring of PMQR determinants among clinical isolates of E coli demonstrating susceptibility and resistance to fluoroquinolones to determine whether these determinants will continue to emerge among these isolates. There appears to be a need to reassess the current CLSI breakpoints because susceptible isolates with MICs that approach the susceptible breakpoint possess gyrA and parC resistance mechanisms, and continued use of fluoroquinolones to treat infections caused by these isolates may accelerate the development of resistance.
Acknowledgments
The current article was presented, in part, at the 2009 American Society for Microbiology 109th General Meeting in Philadelphia (USA). Ms Patricia Baudry-Simner is supported by the Natural Sciences and Engineering Research Council and the Manitoba Health Research Council. The CANWARD study was supported, in part, by Abbott, Affinium, Janssen, Merck, Ortho/Ortho McNeil, Pfizer and Wyeth. The authors thank the participating centres, investigators and laboratory staff for their continued support.
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