Abstract
We examined Enterococcus faecalis strains clinically isolated from 100 patients with urinary tract infections (UTIs) for their susceptibility to levofloxacin (LVX) by measuring the MIC and investigated amino acid mutations by direct DNA sequencing, which were then correlated with LVX resistance. Next, we studied risk factors for LVX resistance, such as age, gender, and previous fluoroquinolone use, and investigated the statistical correlation of these risk factors with each amino acid mutation and LVX resistance. Of the 100 isolates tested, 14 isolates showed LVX resistance and all of these isolates had amino acid mutations. We demonstrated that 2 out of 4 mutations (Ser83-to-Ile in gyrA and Ser80-to-Ile in parC) had a significant correlation with LVX resistance. There was a significant relationship between isolates with 2 or 3 amino acid mutations and LVX resistance. In addition, we found a significant correlation between the previous use of fluoroquinolones and LVX resistance or the presence of mutations and also demonstrated that previous use of other types of antibiotics was significantly related to the presence of mutations by multivariate analysis. In conclusion, we found significant correlation between amino acid mutations in E. faecalis, LVX resistance, and risk factors such as previous use of fluoroquinolones.
INTRODUCTION
Enterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs) (7). Recently, enterococcal infections have increased, and E. faecalis accounts for the majority of enterococcal infections (7). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis (QREF) strains has recently been reported in several countries (12). In our institution, the proportion of QREF isolates gradually increased from 12/83 (14.4%) in 2004 to 10/49 (20.4%) in 2007, but this was not a statistically significant difference (P = 0.3755). Rudy et al. demonstrated in analyses of 130 E. faecalis strains isolated from urine that all strains were sensitive to glycopeptides (vancomycin [VAN] and teicoplanin), 96% were sensitive to penicillin, 43% to ciprofloxacin, and 28% to tetracycline (22). Muratani et al. reported a 38% frequency of levofloxacin (LVX)-resistant E. faecalis isolates from UTIs (17).
Mutations in the quinolone resistance-determining regions (QRDR) of gyrA coding for DNA gyrase and parC coding for DNA topoisomerase IV, the efflux system, antimicrobial-modifying resistance enzymes, and plasmid-mediated mechanisms are considered to contribute to resistance to fluoroquinolones (3, 10, 11, 18, 23, 25, 27). To our knowledge, no previous report has correlated risk factors with fluoroquinolone resistance and amino acid mutation in the QRDR in E. faecalis strains clinically isolated from the urine of UTI patients.
Rattanaumpawan et al. investigated the risk factors for fluoroquinolone resistance in enterococcal UTIs and demonstrated that recent exposure to antibiotics such as fluoroquinolones, extended-spectrum cephalosporins, and clindamycin was significantly associated with fluoroquinolone resistance in enterococcal uropathogens, including E. faecalis and Enterococcus faecium (21). In this study, we performed multivariate analyses using risk factors for LVX resistance in E. faecalis and used molecular biological methods to examine the correlation of risk factors with the presence of amino acid mutation in the QRDR in clinically isolated E. faecalis strains.
MATERIALS AND METHODS
Bacterial isolates.
We defined UTI as an infection of the urinary tract with >104 CFU cultured bacteria/spot in urine and at least one UTI symptom, such as fever or urinary frequency (4, 24). Urine cultures were collected consecutively from 100 patients with UTIs treated at 3 hospitals in Hyogo, Japan, from May to December 2009. In detail, 33 (33%) of these strains were collected in Kobe University Hospital, 32 (32%) in Akashi Municipal Hospital, and 35 (35%) were acquired in Miki City Hospital from all UTI patients treated in these hospitals. Posttreatment isolates and other repeat isolates from the same patients were excluded from this study. Specimens from all hospitals were transferred to Kobe University Hospital with Casitone medium (Eiken Chemical Co. Ltd., Tokyo, Japan) and maintained at room temperature.
Susceptibility testing.
The susceptibility tests for any antibiotics were performed in each institution. The susceptibility tests for levofloxacin (LVX) were performed according to a Clinical and Laboratory Standards Institute (CLSI) guideline (6a) using the agar plate dilution method with Mueller-Hinton agar (Becton, Dickinson and Company, NJ). The isolates were inoculated with ≥104 CFU/spot, and fluoroquinolone susceptibilities were tested. The MIC ranges of LVX for E. faecalis strains were categorized by the CLSI guideline as mentioned above, and the LVX susceptibilities were defined as follows: sensitive, MICs of no more than 2 μg/ml; intermediate, MICs of 4 μg/ml; and resistant, MICs of no less than 8 μg/ml.
DNA extraction, PCR, and DNA sequencing.
The bacterial isolates were cultured on sheep blood agar plates for 14 h and then cultured in lysogeny broth medium for 8 h. DNA extraction was performed with a QIAamp DNA minikit (Qiagen, Tokyo, Japan). The oligonucleotide primers for the PCR amplification were as follows. For the gyrA QRDR, the forward primer was 5′-ATGAGTGAAGAAATTAAAGAAAACATTCA-3′ and the reverse primer was 5′-ACTCATACGTGCTTCGGTATAACGC-3′. For the parC QRDR, the forward primer was 5′-GTGACAATTTTGGAAAAACGCCAAG-3′ and the reverse primer was 5′-CACCACTTAACTGTGATAAACGAGC-3′ (18). PCR products were detected by 2.0% (wt/vol) agarose gel electrophoresis and then purified for sequencing with a QIAquick PCR purification kit (Qiagen, Tokyo, Japan). The products after thermal cycling were purified with Centri-Sep columns and analyzed using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Applied Biosystems, Warrington, United Kingdom). Database searches were conducted, and pairwise alignments of DNA sequences were carried out using BLAST.
Risk factors for resistance to LVX and amino acid mutations.
We examined the risk factors (age [>50 years old], gender [female], presence of underlying urinary tract disease, indwelling urinary catheter, presence of diabetes mellitus [DM], number of amino acid mutations, previous use of fluoroquinolone and other types of antibiotics, hospitalization, and treatment in a urological department) against resistance and sensitivity to LVX and against the presence of amino acid mutations in gyrA and parC. Previous use of antibiotics was defined as administration of an antibiotic for more than 48 h during the previous year (2) or previous 6 months (20). The risk factor of indwelling catheter was defined as catheterization at the time of bacterial isolation. The period for previous hospitalization was defined as hospitalization for a day or more and that of treatment in the urological department was defined as one or more consultations in the urological department to treat UTIs or other urinary tract diseases. Complicated and uncomplicated UTIs were classified by the presence (complicated) or absence (uncomplicated) of either underlying urinary tract disease or indwelling catheters. The correlations of these risk factors with amino acid mutations in the QRDR and LVX resistances were investigated by univariate and multivariate analyses.
Statistical analysis.
Statistical analyses were performed by univariate and multivariate logistic regression and the chi-square test using STATA (StataCorp LP, College Station, TX) with P values of <0.05 considered to indicate statistical significance.
RESULTS
Susceptibilities to LVX.
Among the isolates of E. faecalis examined in this study, 14 (14.0%) were resistant, 85 (85.0%) were sensitive, and only 1 (1.0%) showed intermediate susceptibility to LVX according to CLSI categories (Table 1). In our tested bacteria, the numbers of LVX-resistant isolates were 5/33 (14.7%) in Kobe University Hospital, 2/32 (6.25%) in Akashi Municipal Hospital, and 7/35 (20.0%) in Miki City Hospital. There was no significant difference in the rates of resistance in these institutions (P > 0.05) (data not shown).
Table 1.
Susceptibilities of 100 strains of E. faecalis to tested antimicrobial agentsa
| Antibioticb | MIC range examined (μg/ml) | Susceptibility in no. (%) of strains |
||
|---|---|---|---|---|
| Sensitive | Intermediate | Resistant | ||
| AMP | 2–16 | 99 (99.0) | 0 (0) | 1 (1.0) |
| IPM | 0.5–16 | 99 (99.0) | 0 (0) | 1 (1.0) |
| LVX | 0.25–8 | 85 (85.0) | 1 (1.0) | 14 (14.0) |
| SXT | 1–4 | 95 (97.9) | 0 (0) | 2 (2.1) |
Susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guideline M7-A8 (6a).
AMP, ampicillin; IPM, imipenem; LVX, levofloxacin; SXT, trimethoprim-sulfamethoxazole.
Sequence changes of gyrA and parC in the QRDRs in E. faecalis strains.
Among 100 isolates tested, 18 isolates had amino acid mutations and 14 isolates were resistant to LVX. Of the 18 strains with amino acid mutations, all 18 strains had amino acid mutations in gyrA and 14 strains had amino acid mutations in parC. We demonstrated 4 different kinds of mutations in the QRDRs of gyrA and parC: the alterations of amino acid serine 83 to isoleucine (94.4%) and glutamic acid 87 to lysine (5.6%) in gyrA and the alterations of serine 80 to isoleucine (77.8%) and glutamic acid 84 to valine (5.6%) in parC in the QRDR. The combinations of gyrA and parC mutations were classified into 4 different patterns (Table 2).
Table 2.
Amino acid mutation patterns of gyrA and parC in E. faecalis and the rate of resistance to LVX
| No. of mutations | Mutation ina: |
No. of strains with resistance to LVX/total no. (%)b | |||
|---|---|---|---|---|---|
|
gyrA |
parC |
||||
| Ser83-to-Ile | Glu87-to-Lys | Ser80-to-Ile | Glu84-to-Val | ||
| 0 | 0/82 (0) | ||||
| 1 | Ile | 1/3 (33.3) | |||
| 2 | Lys | 1/1 (100) | |||
| 3 | Ile | Ile | 11/13 (84.6) | ||
| 4 | Ile | Ile | Val | 1/1 (100) | |
| Total | 14/100 (14.0) | ||||
Ser, serine; Glu, glutamic acid; Ile, isoleucine; Lys, lysine; Val, valine; LVX, levofloxacin.
Percentages were calculated as the number of LVX-resistant strains with each mutation pattern.
Correlation of amino acid mutations in the QRDR with resistance to LVX.
We demonstrated that 2 mutations (Ser83-to-Ile in gyrA and Ser80-to-Ile in parC) had a significant correlation to higher MICs of LVX by multivariate analysis (Table 3). All LVX-resistant isolates had amino acid mutations in the QRDR. All 82 isolates with no mutation, 2 out of 4 isolates with one mutation, and 2 out of 13 isolates with 2 mutations were sensitive to LVX. One isolate with 3 mutations was resistant to LVX. Multivariate analyses demonstrated a significant relationship between isolates with 2 or 3 mutations involving both gyrA and parC and resistance to LVX. An inverse correlation was shown between isolates with no mutation and higher MICs of LVX (β-coefficient [β-coef.] = −5.14; P < 0.01) (Table 4).
Table 3.
Correlation of MICs of LVX with each amino acid mutation by multivariate analysis
| Amino acid mutationa | MIC range (μg/ml) | OR (95% CI) | P valueb |
|---|---|---|---|
| Ser83-to-Ile in gyrA | 0.25–8 | 6.41 (5.80–7.02) | <0.01 |
| Glu87-to-Lys in gyrA | 8 | NA | NA |
| Ser80-to-Ile in parC | 1–8 | 6.89 (6.50–7.28) | <0.01 |
| Glu84-to-Val in parC | 8 | NA | NA |
There is one isolate with Glu87-to-Lys in gyrA and also with Glu84-to-Val in parC. CI, confidence interval; NA, not applicable.
Boldface indicates statistical significance.
Table 4.
Correlation of MICs of LVX with the number of mutations by multivariate analysis
| No. of mutations | MIC range (μg/ml) | β-coef. (95% CI)b | P value |
|---|---|---|---|
| 0 | 0.25–4 | −5.14 (−5.95–4.32) | <0.01 |
| 1 | 0.25–8 | −1.72 (−4.30–0.86) | 0.19 |
| 2 or 3a | 1–8 | 5.89 (1.00–10.8) | 0.02 |
There is only one isolate with 3 amino acid mutations.
Boldface indicates statistical significance.
Correlation of risk factors with resistance and sensitivity to LVX and amino acid mutations.
The proportion of inpatients to outpatients was 53:47. Regarding the risk factors, we found that previous use of fluoroquinolones was significantly related to LVX resistance (odds ratio [OR] = 6.36; P = 0.015) and the presence of amino acid mutations (OR = 4.49; P = 0.040). We also demonstrated that previous use of other types of antibiotics was significantly related to the presence of mutations (OR = 4.08; P = 0.039). There was an inverse correlation between LVX sensitivity and previous use of fluoroquinolones (OR = 0.19; P = 0.037) or previous use of other types of antibiotics (OR = 0.23; P = 0.041), and LVX sensitivity was significantly related to treatment in the urological department (OR = 16.1; P = 0.036) based on multivariate analyses (Table 5).
Table 5.
Correlation of risk factors with LVX resistance and presence of amino acid mutations in all UTI patients by multivariate analysisa
| Risk factor | LVX resistance |
LVX sensitivity |
Mutation |
|||
|---|---|---|---|---|---|---|
| OR (95% CI) | P value | OR (95% CI) | P value | OR (95% CI) | P value | |
| Previous use of fluoroquinolones | 6.36 (1.44–28.2) | 0.015 | 0.19 (0.04–0.90) | 0.037 | 4.49 (1.07–18.8) | 0.040 |
| Previous use of other type of antibiotics | 3.57 (0.92–13.9) | 0.066 | 0.23 (0.06–0.94) | 0.041 | 4.08 (1.07–15.5) | 0.039 |
| Underlying urinary tract disease | 3.66 (0.63–21.3) | 0.149 | 0.32 (0.05–1.89) | 0.209 | 1.62 (0.38–6.80) | 0.512 |
| Indwelling catheter | 1.94 (0.52–7.32) | 0.327 | 0.42 (0.11–1.70) | 0.225 | 3.08 (0.89–10.6) | 0.075 |
| DM | 1.08 (0.23–5.10) | 0.921 | 0.83 (0.17–4.13) | 0.816 | 1.83 (0.44–7.55) | 0.404 |
| Age (>50 yr) | 0.99* (0.96–1.03) | 0.791 | 1.01* (0.97–1.05) | 0.690 | 0.97* (0.94–1.00) | 0.076 |
| Gender (female) | 0.58 (0.12–2.68) | 0.482 | 2.06 (0.40–10.7) | 0.389 | 0.45 (0.11–1.92) | 0.282 |
| Hospitalization | 0.48 (0.07–3.56) | 0.474 | 5.74 (0.56–59.3) | 0.142 | 0.94 (0.18–4.88) | 0.940 |
| Treatment in urological department | 0.15 (0.02–1.37) | 0.093 | 16.1 (1.19–217) | 0.036 | 0.32 (0.05–1.91) | 0.212 |
Boldface indicates statistical significance. An asterisk indicates β-coef.
Presence of underlying urological disease (complicated or uncomplicated UTIs) as a risk factor for LVX resistance.
In addition, the isolates in our study were classified as 77 isolates from patients with complicated UTIs and 23 isolates from patients with uncomplicated UTIs based on the presence of underlying urinary tract disease. The frequency of resistant strains from patients treated in a urological department (OR = 4.23; P < 0.01) was significantly higher in complicated UTI cases than in uncomplicated UTI cases, and the frequency of complicated UTIs was lower than that of uncomplicated UTIs in females (OR = 0.33; P = 0.02). The patients with complicated UTIs were significantly older than those with uncomplicated UTIs (β-coef. = 1.04; P = 0.01). The median number of mutations in complicated UTI cases was 0.39 compared to 0.13 in uncomplicated UTI cases, but the difference did not reach statistical significance (β-coef. = 1.92; P = 0.16) (Table 6).
Table 6.
Risk factors in complicated and uncomplicated UTI cases
| Risk factor | No. (%) of cases ofa: |
OR | P value | |
|---|---|---|---|---|
| Complicated UTI (n = 77) | Uncomplicated UTI (n = 23) | |||
| Underlying urinary tract disease | 69 (89.6) | |||
| Indwelling catheter | 29 (37.7) | |||
| Treatment in urological department | 50 (64.9) | 7 (30.4) | 4.23 | <0.01 |
| Hospitalization | 37 (48.1) | 16 (69.6) | 0.40 | 0.08 |
| Gender (female) | 23 (29.9) | 13 (56.5) | 0.33 | 0.02 |
| Previous use of other types of antibiotics | 22 (28.6) | 5 (21.7) | 1.44 | 0.52 |
| DM | 18 (23.4) | 1 (4.4) | 6.71 | 0.07 |
| Previous use of fluoroquinolones | 17 (22.1) | 3 (13.0) | 1.89 | 0.35 |
For the groups with complicated and uncomplicated UTI, the median (range) ages were 72 (18 to 104) and 61 (0 to 81) years (β-coef., 1.04; P = 0.01), respectively, and the median (range) numbers of mutations were 0.39 (0 to 3) and 0.13 (0 to 2) (β-coef., 1.92; P = 0.16), respectively.
Of the isolates from the 77 complicated UTI cases, 16 (20.8%) had amino acid mutations and 13 (16.9%) were resistant to LVX. Of the 23 isolates from uncomplicated UTI cases, on the other hand, 2 (8.70%) had amino acid mutations and only 1 (4.3%) was resistant to LVX. We demonstrated a significant relationship between LVX resistance and previous use of fluoroquinolones (OR = 8.70; P = 0.011) or other types of antibiotics (OR = 4.80; P = 0.040) in complicated UTI cases (Table 7).
Table 7.
Correlation of risk factors with LVX resistance in complicated UTI by multivariate analysisa
| Risk factor | OR (95% CI) | P value |
|---|---|---|
| Previous use of fluoroquinolones | 8.72 (1.65–46.1) | 0.011 |
| Previous use of other type of antibiotics | 4.81 (1.08–21.5) | 0.040 |
| Indwelling catheter | 1.51 (0.33–6.85) | 0.590 |
| Underlying urinary tract disease | 1.22 (0.08–18.2) | 0.887 |
| DM | 1.06 (0.21–5.21) | 0.945 |
| Age (>50 yr) | 0.99* (0.95–1.04) | 0.759 |
| Gender (female) | 0.71 (0.14–3.62) | 0.681 |
| Hospitalization | 0.45 (0.05–3.96) | 0.473 |
| Treatment in urological department | 0.17 (0.02–1.69) | 0.130 |
There were a total of 77 cases of complicated UTIs. Boldface indicates statistical significance. An asterisk indicates β-coef.
DISCUSSION
An increase in antibiotic-resistant E. faecalis isolates has been encountered (16), and the emergence of QREF has recently been reported in several countries (18, 27, 22). It may be useful for both basic and clinical research to investigate the relationship between risk factors for fluoroquinolone resistance and its related mechanisms in order to prevent progressively increasing resistance to antibiotics.
The use of aminoglycosides in the previous 30 days was significantly correlated with ciprofloxacin (CIP) resistance in analyses of risk factors of vancomycin-resistant E. faecalis (VREF) (9). We found that previous use of fluoroquinolones was significantly correlated with LVX resistance and the presence of amino acid mutations and demonstrated a significant correlation between previous use of other types of antibiotics and the presence of mutations by multivariate analyses, indicating that previous exposure to antibiotics was related to mutation in the QRDR and, therefore, may cause fluoroquinolone resistance. This is supported by previous reports demonstrating that underlying urinary tract diseases predispose patients to repeated UTIs and exposure to antibiotics such as fluoroquinolones, leading to the selection of resistant E. faecalis strains and the development of UTIs which may be caused by QREF (28). To our knowledge, there is no report demonstrating the correlation of underlying urinary tract disease with mutation in E. faecalis isolates from UTIs. From a molecular biology viewpoint, our demonstration of the correlation between risk factors such as previous use of fluoroquinolones or other kinds of antibiotics and significant mutations in fluoroquinolone-resistant strains may be a new finding.
In comparing risk factors in complicated and uncomplicated UTI cases, we found that for females, the frequency of complicated UTIs was lower than the frequency of uncomplicated UTIs. Females may have a greater likelihood of developing cystitis, even though they have fewer underlying urinary tract diseases than males. Also, the patients with complicated UTIs were significantly older than those with uncomplicated UTIs. This is because older individuals tend to have more underlying urinary tract diseases than younger ones (5), and young females in particular tend to present with simple cystitis in most cases (6).
In this study, we found that 2 out of 4 mutations (Ser83-to-Ile in gyrA and Ser80-to-Ile in parC) were significantly correlated with the MICs of LVX. A previous report demonstrated the amino acid mutation of Ser83-to-Ile in gyrA in 15 strains (28.3%) and the amino acid mutation of Ser80-to-Ile in parC in 9 strains (17.9%) of 53 clinical strains of E. faecalis (14). There are regional and national geographic differences in levels of fluoroquinolone resistance by E. faecalis. This may be related partly to different methods of antibiotic use (2). We had fewer gyrA mutations (18.0%) and parC mutations (14.0%) but more wild-type strains (82.0%) than previously reported (14), and this may be partly because our study included fewer strains from complicated UTI cases than other reports (14, 10). Our mutation results differed in part from other reports, which may be significant from an epidemiological viewpoint.
Our statistical data revealed a correlation between the number of mutations and fluoroquinolone resistance. Previous reports demonstrated that even strains with a single amino acid mutation in gyrA showed a decreased susceptibility to fluoroquinolones (26). We demonstrated a significant relationship between isolates with 2 or 3 mutations involving both gyrA and parC and MICs of LVX; however, the presence of significant mutations was more highly correlated with fluoroquinolone resistance than the raw number of mutations (Table 5). Two out of 13 strains with 2 mutations were susceptible to LVX, and this may suggest that another mechanism may also be involved in fluoroquinolone resistance, such as the efflux system (3), requiring further research for the elucidation of this mechanism (8, 15). Previous works showed that the efflux pump in E. faecalis is involved in resistance to tetracycline and erythromycin (ERY) (11) and plasmid-mediated fluoroquinolone resistance (presence of the qnrA gene) in E. faecalis (23).
The frequency of LVX-resistant strains was 13/77 (16.9%) strains in complicated UTI cases and 1/23 (4.3%) strains in uncomplicated UTI cases in this study. We found that more QREF strains were isolated from patients with complicated UTIs than from those with uncomplicated UTI cases, although the difference was not significant (P = 0.40). In complicated UTI cases, E. faecalis, Escherichia coli, and Pseudomonas aeruginosa tended to be the most frequently isolated organisms, and these 3 organisms accounted for 44.6% of cases (17). In catheter-associated UTIs (CA-UTIs), E. coli, Klebsiella pneumoniae, and E. faecalis constituted 66.0, 11.4, and 5.4% of cases, respectively (1). The characterization of efflux pumps may lead to the development of new weapons against antibiotic resistance, efflux pump inhibitors (EPIs) (13, 19). EPIs induce a significant reduction in resistance to antibiotics to which bacterial strains were initially resistant (19), indicating that the development of new EPIs would be an effective treatment of infections caused by multidrug-resistant bacterial strains. This is our next task to further our understanding of the emergence of antibiotic-resistant E. faecalis.
In conclusion, we demonstrated that amino acid mutations in the QRDR were related to fluoroquinolone resistance in E. faecalis isolated from UTI patients and found that the previous use of fluoroquinolones was significantly related to LVX resistance and the presence of mutations and also that the more significant the mutations were in gyrA and parC, the more likely the strain was to be resistant to LVX based on multivariate analyses. This study demonstrated the mechanism and risk factors for fluoroquinolone resistance in E. faecalis in UTIs and, thus, sheds light on both the basic science and clinical aspects of this growing problem.
ACKNOWLEDGMENTS
We thank Shouhiro Kinoshita for collecting bacterial isolates, and Gary Mawyer for English editing.
We have no financial support or conflicts of interest to report for this paper.
Footnotes
Published ahead of print on 14 September 2011.
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