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Antimicrobial Agents and Chemotherapy, January 2002, p. 119-124, Vol. 46, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.46.1.119-124.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Ortho-McNeil Pharmaceutical,1 The R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869,2 Focus Technologies, Herndon, Virginia 201713
Received 20 June 2001/ Returned for modification 13 August 2001/ Accepted 14 October 2001
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ABSTRACT |
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0.06 to 2 µg/ml) had a single parC or parE QRDR mutation. Although there has been an increase in the prevalence of single-step mutants, the increase may have been overestimated due in part to differences in geographical distribution for the two sets of isolates. |
INTRODUCTION |
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Despite the low incidence of fluoroquinolone resistance among pneumococci in the United States, there is concern that the incidence of this resistance will increase similarly to that which has occurred with the ß-lactams and macrolides. Several observations based on longitudinal analysis of surveillance data from North America hint at this potential. First, although the percentage of Canadian levofloxacin-resistant S. pneumoniae isolates remained constant (0.4%) from 1994 to 1998, the highest levofloxacin and ciprofloxacin MICs increased fourfold and eightfold, respectively, during this time period (2, 26). Secondly, Sahm et al., using data from TRUST surveillance, have shown that there was a slight increase in the incidence of levofloxacin resistance in the United States, from 0.1% in 1997–1998 to 0.6% in 1998–1999 and 0.5% in 1999–2000 (24; Selman et al., 40th ICAAC). Finally, recent reports have also documented higher prevalence of fluoroquinolone resistance (>1%) among clinical isolates of S. pneumoniae in various parts of the world including Hong Kong and Spain (8, 16).
Resistance to fluoroquinolones in pneumococci occurs in a stepwise fashion with mutations being observed first in either parC or gyrA (depending on the selecting fluoroquinolone) leading to decreased fluoroquinolone susceptibility (19). Strains usually become fully fluoroquinolone resistant with the addition of a mutation in the other target gene (either gyrA or parC) (19). Mutations in parE and gyrB and efflux also contribute to fluoroquinolone resistance but usually to a lesser extent (7, 19, 21).
Emergence of fluoroquinolone resistance during the course of antimicrobial therapy is most likely to develop from strains that already carry one quinolone resistance-determining region (QRDR) mutation, since they require only one additional mutation in one of the other target genes to become resistant. Fully fluoroquinolone-susceptible strains with a mutation in one of the primary targets have been observed (1, 11). Currently, the incidence of fluoroquinolone-susceptible clinical isolates containing single mutations in the QRDR region of any of the topoisomerase II genes is unknown.
In order to address the issue of the prevalence of mutations in the QRDR of topoisomerase IV and DNA gyrase among fluoroquinolone-susceptible pneumococci, we screened 528 levofloxacin-susceptible pneumococci (MICs, 0.5 to 2.0 µg/ml) collected during the TRUST 4 surveillance study (1999–2000 respiratory season) for the most common QRDR mutations, using an oligonucleotide probe assay in combination with DNA sequencing. This approach allowed for the analysis of a greater number of strains than would have been feasible by DNA sequencing alone. The results were compared to similar data obtained from 214 levofloxacin-susceptible clinical isolates collected between 1992 and 1996 to determine whether the mutation prevalence has changed over time.
(This work was presented in part at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., abstr. 702, December 2001.)
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MATERIALS AND METHODS |
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2.0 µg/ml). Isolates for which levofloxacin MICs were 0.5 and 1 µg/ml were chosen so that approximately one-third were penicillin susceptible, one-third were penicillin intermediate, and one-third were penicillin resistant. Isolates were selected from each of the nine U.S. census regions and comprised 128 institutions from 43 states. Eighty-seven (16.5%) of the isolates were from patients 17 years of age or younger, 430 (81.5%) isolates were from patients 18 years of age or older, and 11 (2%) were from patients whose ages were undetermined. For comparison, 214 levofloxacin-susceptible clinical S. pneumoniae strains (131 strains for which the levofloxacin MIC was 1 µg/ml and 83 strains for which the levofloxacin MIC was 0.5 µg/ml) isolated in the United States from 1992 to 1996 were also examined. Strains were from 46 institutions in 14 states.
MICs were determined by broth microdilution with panels manufactured by Trek Diagnostic Systems (Westlake, Ohio). MICs were performed in triplicate according to NCCLS recommendations using cation-adjusted Mueller-Hinton broth (MHB) with 5% lysed defibrinated horse blood (18). Quality control S. pneumoniae strain ATCC 49619 was included in each run of broth microdilution MICs. The antimicrobial agents tested were levofloxacin, ciprofloxacin, and penicillin.
PCR of quinolone resistance determinants. The QRDR of topoisomerase type II genes parC, parE, gyrA, and gyrB were amplified by PCR using primers and cycling conditions described by Pan et al. (19).
Colony growth and lysis. For each isolate one to two colonies were transferred into 1 well of a 96-well microtiter tray containing 100 µl of MHB with 5% lysed defibrinated horse blood and incubated 4 to 6 h at 35°C in 5% CO2. Approximately 3 µl of broth culture was spotted onto a 0.45-µm-pore-size Nytran SuPerCharge nylon membrane (Schleicher & Schuell, Keene, N.H.) that was placed on the surface of trypticase soy agar with 5% defibrinated sheep blood. The agar plates with the nylon membranes on the surface were incubated at 35°C in 5% CO2 overnight to allow cultures to grow. S. pneumoniae organisms growing on the membranes were lysed and prepared for hybridization with a protocol based on the method of Sambrook et al. (25). Briefly, cells were lysed by placing nylon membranes (colony side up) on filter paper saturated with 10% sodium dodecyl sulfate (SDS) solution and incubated for 5 min at room temperature. DNA was denatured by incubation of membranes on filter paper saturated with 0.5 M NaOH-1.5 M NaCl for 5 min. Membranes were then transferred to filter paper saturated with neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl [pH 7.4]) and incubated for 5 min. Membranes were then rinsed by incubation for 5 min on filter paper saturated with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). DNA was UV cross-linked to membranes using a UV Stratalinker 1800 (Stratagene, La Jolla, Calif.).
Oligonucleotide probe assay. Analysis of 25 levofloxacin-resistant pneumococci collected from a survey of 99 institutions throughout the United States (TRUST 3, 1998–1999) showed that most mutations occurred at Ser79 in ParC, Ser81 in GyrA, and Asp435 in ParE (Loeloff et al., 40th ICAAC). Oligonucleotide probes were designed to detect the wild-type nucleotide sequences corresponding to these amino acids (Table 1). Also, oligonucleotide probes were designed to detect the few mutations most often detected at these positions (Table 1). The probes parCwt and gyrAwt were used individually against all strains in the assay, whereas the probes parCmut1 and parCmut2 and the probes gyrAmut1 and gyrAmut2 were pooled together. Two other oligonucleotide probes were generated to detect the wild-type nucleotide sequences encoding Asp83 in ParC and Glu85 in GyrA, two amino acids which are also often mutated in levofloxacin-resistant pneumococci (Table 1). Since the nucleotide substitutions occurring in the genes corresponding to Asp83 in ParC and Glu85 in GyrA are multiple, probes were not designed to detect the mutant sequences at these positions. All strains were hybridized to the Asp83 ParC probe (parCwt2), and 140 strains were hybridized to the Glu85 GyrA probe (gyrAwt2).
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Hybridizations.
Ten 17-bp oligonucleotide probes (Table 1) were end labeled with [
-32P]ATP (specific activity, 6,000 Ci/mmol) (NEN Life Science, Boston, Mass.) as described by Sambrook et al. (25). Hybridizations to lysed colonies fixed on membranes or PCR dot blots were carried out in the presence of 3 M tetramethyl ammonium chloride (TMAC) to make the melting temperature of the oligonucleotides dependent on length and not on base composition (9, 28). Hybridizations were carried out at 43°C overnight, and washing occurred at 48°C for 1 h. Membranes were prehybridized in TMAC hybridization buffer (3 M TMAC, 0.1 M NaPO4 [pH 6.8], 1 mM EDTA [pH 8.0], 5x Denhardt’s reagent, 0.6% SDS, 100 µg of yeast tRNA per ml) at 43°C for 2 h. The buffer was discarded, and fresh TMAC hybridization buffer along with labeled probe was added and incubated at 43°C overnight. Membranes were washed for 15 min at room temperature in TMAC wash buffer (3 M TMAC, 50 mM Tris-HCl [pH 8.0], 0.2% SDS) followed by a washing for 1 h at 48°C in TMAC wash buffer. Membranes were then washed three times for 10 min at room temperature in 2x SSC–0.1% SDS. Membranes were exposed to X-OMAT XAR-2 film (Kodak) at -70°C for 1 to 7 days.
DNA sequencing analysis. To confirm the oligonucleotide probe assay results, DNA sequencing of the specific QRDR was performed on strains that hybridized to a probe designed to detect QRDR mutations and not to the corresponding wild-type probe. Additionally, the QRDR of strains that did not hybridize to any probe was sequenced to detect the mutation present. DNA sequencing was done on the parC, parE, and gyrA QRDRs of 15 strains that hybridized only to the wild-type probes to confirm the sensitivity of the oligonucleotide probe assay. Finally, since there were only 14 strains with levofloxacin MICs of 2 µg/ml, the QRDRs of parC, gyrA, parE, and gyrB were sequenced for each strain. For all DNA sequencing, PCR products from two separate reactions were sequenced in the forward and reverse directions by ACGT, Inc. (Northbrook, Ill.) or Genome Therapeutics Corporation (Waltham, Mass.).
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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4 µg/ml) in adults, especially older adults (65 years old) between 1993 and 1998 (2). Thirteen (46%) of the 28 single-step mutants in the present study were from adults 65 years of age or older. None of the 28 mutants were susceptible to ciprofloxacin (MIC, 2 µg/ml), and for 20 (71%) mutants the ciprofloxacin MIC was 4µg/ml.
Among the 214 levofloxacin-susceptible isolates collected during 1992 to 1996, only one strain had a mutation. For this strain, the levofloxacin MIC was 1 µg/ml and there was a mutation leading to a Ser79-Phe substitution in ParC. This strain was susceptible to penicillin (penicillin MIC, 0.03 µg/ml) and nonsusceptible to ciprofloxacin (ciprofloxacin MIC, 2 µg/ml).
An analysis of the penicillin MICs for the 25 TRUST 4 strains having a single parC mutation showed that 17 (68%) were penicillin nonsusceptible (11 strains were penicillin intermediate, 6 strains were penicillin resistant). This observation is most likely a reflection of the constitution of the sample population, which was chosen so that approximately two-thirds were penicillin nonsusceptible. The three strains with a single parE mutation were all penicillin susceptible (Table 3). Due to the small number of parE mutations detected, it is unclear if there is a real association of first-step parE mutations and penicillin-susceptible strains.
The sample population did not represent the real incidences of penicillin-susceptible, penicillin-intermediate, and penicillin-resistant strains among all levofloxacin-susceptible TRUST 4 isolates, which were 66, 18, and 16%, respectively (Selman et al., 40th ICAAC). Accordingly, the prevalence of single-step QRDR mutations was determined by the distribution of levofloxacin MIC and penicillin susceptibility (Table 3).
Based on the prevalence of single mutations determined by analysis of the 528 levofloxacin-susceptible TRUST 4 isolates, the incidence of single mutations that would be found among all 9,438 levofloxacin-susceptible TRUST 4 isolates was estimated as follows (Table 4). First, all the TRUST 4 isolates with levofloxacin MICs of 2 µg/ml were checked for single QRDR mutations. Second, in order to estimate the incidence of single QRDR mutations in the rest of the levofloxacin-susceptible strains, the following assumptions were made: (i) the prevalence of single QRDR mutations determined for the strains for which levofloxacin MICs were 1 µg/ml (grouped by penicillin susceptibility) would be the same for all TRUST 4 isolates for which the levofloxacin MIC was 1 µg/ml (3,318 penicillin susceptible, 655 penicillin intermediate, 663 penicillin resistant) and (ii) the incidence of single QRDR mutations among the 4,788 TRUST 4 isolates for which levofloxacin MICs were 0.5 µg/ml was assumed to be negligible and was dismissed since no single QRDR mutations were found among the 270 strains analyzed from this group. The possible error introduced by this assumption is <0.4% (1/270).
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Surveillance remains a key component in monitoring the development of fluoroquinolone resistance among clinical isolates of S. pneumoniae. Currently, most published surveillance studies focus on the characterization of QRDR mutations in topoisomerase IV and DNA gyrase of fluoroquinolone-resistant isolates. Fluoroquinolone resistance develops in a stepwise fashion. Therefore, knowing the incidence of fluoroquinolone-susceptible pneumococci with single QRDR mutations in topoisomerase IV or DNA gyrase provides a measure for the potential of resistance development, since these strains represent possible precursors to fluoroquinolone resistance. While strains carrying QRDR mutations in either topoisomerase IV or DNA gyrase have been observed occasionally among fluoroquinolone-susceptible pneumococci (1, 11, 23), determination of their incidence has not been feasible by traditional DNA sequencing methods. This study provides an estimate of the current level of strains carrying QRDR mutations in topoisomerase IV or DNA gyrase in a levofloxacin-susceptible population.
The strains in this study were analyzed only for the most frequent topoisomerase IV and DNA gyrase QRDR mutations known to contribute to increased fluoroquinolone MICs that occur for fluoroquinolone-resistant clinical pneumococci (1, 3, 11, 12, 14, 15, 22). Thus, the present detection procedure inherently underestimates the total number of single mutational events. Nonetheless, among a collection of 25 levofloxacin-resistant strains collected in the TRUST 3 (1998–1999) study from 99 different participating hospitals throughout the United States, the QRDR probes used in this study would have detected 71 and 78% of all ParC and GyrA observed amino acid changes, respectively (Loeloff et al., 40th ICAAC). It also would have detected 100% of all amino acid changes in ParE that are associated with decreased susceptibility to fluoroquinolones. The incidence of the frequent (13 of the 25 strains) mutation resulting in the Ile460 to Val change in ParE was not investigated, since thus far it has not been associated with decreased susceptibility to fluoroquinolones (1, 22). Therefore, the estimated values for the incidence of single QRDR mutations leading to amino acid changes (associated with decreased fluoroquinolone susceptibility) in DNA gyrase or DNA topoisomerase IV provided by the herein used detection procedure were close to the values obtained by sequencing all relevant regions of the analyzed strains.
The high incidence of mutations affecting the Ser79 position relative to other sites of ParC (Table 2) is consistent with results from sequencing the appropriate QRDR regions from clinical isolates of S. pneumoniae nonsusceptible to fluoroquinolones (1, 11, 14; Loeloff et al., 40th ICAAC). However, the distribution of the Ser79 substitutions among levofloxacin-susceptible TRUST 4 strains determined by the probe assay is biased in favor of Phe over Tyr in a ratio of 8.5 to 1. In contrast, among U.S. clinical isolates of S. pneumoniae resistant to levofloxacin, the ratio of Ser79-Phe to Ser79-Tyr substitutions is less than 2 (1, 11, 14; Loeloff et al., 40th ICAAC). The bias in favor of Ser79-Phe may simply result from the more frequent transition TCT
TTT, that replaces one pyrimidine for another, over the less frequent transversion TCTTAT, which replaces a pyrimidine with a purine. An additional selective advantage for the Ser79-Phe substitution cannot be excluded.
For 14 S. pneumoniae strains (0.15% of the TRUST 4 isolates), the levofloxacin MIC was 2 µg/ml. Upon sequencing of all the QRDR regions, nine had single parC mutations, one had both a parC and a gyrB mutation, and four others did not show any QRDR alterations resulting in amino acid changes. For these four isolates no evidence indicating that a reserpine-sensitive efflux pump was acting upon levofloxacin was observed (data not shown). Thus, in at least four of the isolates (representing 0.04% of the TRUST 4 isolates) a levofloxacin MIC of 2 µg/ml was not associated with QRDR mutations or PmrA effects (7). Factors other than QRDR mutations may contribute significantly to the fluoroquinolone MIC observed for a clinical isolate.
Prior to 2000, most fluoroquinolones approved for use in the United States (levofloxacin, ofloxacin, ciprofloxacin, trovafloxacin, and grepafloxacin) had topoisomerase IV (parC/E) as their primary S. pneumoniae target (5, 17). The exception was sparfloxacin, targeting DNA gyrase, but this drug was not widely used in the United States (20). Accordingly, all but one of the QRDR mutations were found only in parC or parE among the levofloxacin-susceptible strains from TRUST 4 (1999–2000) and from the group of isolates obtained from 1992 to 1996. GyrA QRDR mutations were not found among TRUST 4 levofloxacin-susceptible strains (levofloxacin MICs, 2 µg/ml) but were detected only in strains for which levofloxacin MICs were 4 µg/ml and in combination with at least one topoisomerase IV mutation (data not shown). This observation has also been reported by others (1, 11).
The comparison of the prevalence of single-step mutants among a collection of isolates from 1992 to 1996 and the TRUST 4 isolates (1999–2000) shows that the incidence of single-step mutants has increased. The cause of the observed increase in the prevalence of single-step mutants is unknown. However, it may be related to the greater use of fluoroquinolones today than in the early to mid-1990s, prompted by increasing ß-lactam and macrolide resistance in S. pneumoniae.
An increase in the prevalence of single-step mutants was not unexpected. However, the actual magnitude of that increase was somewhat surprising since the incidence of levofloxacin resistance among S. pneumoniae strains in the United States has remained relatively constant over the past 3 years: 0.6% in 1998–1999, 0.5% in 1999–2000, and 0.8% in 2000–2001 (24; L. J. Kelly et al., 41st ICAAC). Thus, at present it cannot be excluded that the prevalence of first-step mutants in the set of isolates from 1992 to 1996 may have been underestimated. The sample was smaller (214 isolates versus 528 isolates) and less diverse (46 institutions from 14 states compared to 128 institutions from 46 states) than the set of TRUST 4 isolates examined. Only 5 of the 14 states in which isolates were collected from the 1992 to 1996 set of isolates were represented by the first-step mutants (nine mutants) found in the TRUST 4 isolates. Thus, 19 of the 28 mutants (68%) from TRUST 4 were found in states not represented in the 1992 to 1996 set of isolates. Additional studies looking at another representative set of isolates from an intermediate time frame (1997–1998) are under way.
In summary, by analyzing 528 levofloxacin-susceptible S. pneumoniae clinical isolates for single QRDR mutations, it was estimated that approximately 4.5% of all 9,438 levofloxacin-susceptible TRUST 4 (1999–2000) isolates contained a QRDR mutation. In contrast, only one isolate from 214 representative levofloxacin-susceptible strains from 1992 to 1996 carried a single QRDR mutation. Continued monitoring is warranted to see how the incidence of levofloxacin-susceptible strains carrying QRDR mutations will change over time. Of particular interest is whether the recent introduction into the clinic of gatifloxacin, a fluoroquinolone that has DNA gyrase (gyrA/B) as its primary target (5), and moxifloxacin, which may target DNA gyrase (6), will affect the prevalence and profile of single QRDR mutants. According to their mechanisms of action, gatifloxacin and moxifloxacin would be expected to select preferentially for gyr (A/B) mutations (5, 6). It remains to be determined how the addition of DNA gyrase single mutants into a population that already contains topoisomerase IV single mutants will affect the emergence of fluoroquinolone-resistant strains within S. pneumoniae, a species known to efficiently exchange DNA by transformation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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