Abstract
The clinical and molecular epidemiology of penicillin-resistant Streptococcus pneumoniae and the diagnostic accuracy of a six-primer PCR assay in identifying penicillin resistance were analyzed by using clinical isolates recovered over a 10-year period in middle Tennessee. The prevalence of non-penicillin-susceptible S. pneumoniae isolates (MIC, ≥0.1 μg/ml) increased from 10% in 1990 to 70% in 1999 (P < 0.001). Among S. pneumoniae isolates for which the penicillin MIC was ≥2 μg/ml (highly penicillin-resistant S. pneumoniae [PRSP]), 23 and 5% were resistant to at least three and at least five other antimicrobial classes, respectively. Pulsed-field gel electrophoresis identified 13 unique strain types, with type B accounting for 33% of PRSP isolates. The sensitivity, specificity, and negative and positive predictive values of the PCR assay in detecting PRSP were 99, 100, 99, and 100%, respectively. Penicillin resistance is rapidly increasing among S. pneumoniae isolates in Tennessee. The simultaneous detection of S. pneumoniae and high-level penicillin resistance can be accurately performed with the six-primer PCR assay.
Penicillin resistance among Streptococcus pneumoniae isolates is rapidly increasing in the United States (31). Mortality rates associated with highly penicillin-resistant S. pneumoniae (PRSP) infections are threefold higher than those associated with penicillin-susceptible S. pneumoniae (PSSP) (11). The emergence of resistance is of great concern since S. pneumoniae is frequently implicated in many serious infections, including meningitis, community-acquired pneumonia, and otitis media (16, 20, 25).
The state of Tennessee has the highest prevalence of penicillin-resistant S. pneumoniae isolates in the United States, with rates approaching 37% (31, 32). To describe the clinical and molecular epidemiology of penicillin-resistant S. pneumoniae isolates in Tennessee, 544 isolates recovered from 1990 through 1999 were analyzed. Trends in antimicrobial susceptibility, serotypes, and strain types were examined by using pulsed-field gel electrophoresis (PFGE). Alterations in the penicillin-binding protein 2B (pbp2B) gene, which confers penicillin resistance, were characterized for all isolates by using a six-primer PCR assay. This assay makes use of a set of primers which amplify a conserved S. pneumoniae-specific sequence in the pbp2B gene and four additional “resistance” primers which target altered areas of the pbp2B gene present in penicillin-resistant S. pneumoniae isolates (9, 27). The diagnostic accuracy of this PCR assay for rapidly detecting penicillin resistance among S. pneumoniae isolates was evaluated.
(This study was presented in part at the 101st General Meeting of the American Society for Microbiology, Orlando, Fla., 20 to 23 May 2001.)
MATERIALS AND METHODS
Definitions of penicillin resistance.
S. pneumoniae isolates were classified as PSSP, penicillin intermediate (PISP), or PRSP if the penicillin MIC was ≤0.06, 0.1 to 1, and ≥2 μg/ml, respectively (21).
Prevalence of non-penicillin-susceptible S. pneumoniae isolates.
To determine the prevalence of penicillin resistance among S. pneumoniae isolates over the 10-year study period, antibiograms generated by the Vanderbilt University Medical Center (VUMC) Clinical Microbiology Laboratory were reviewed. These rates included multiple patient isolates.
Collection of S. pneumoniae isolates.
From July 1990 through October 1999, all clinical isolates with presumed penicillin resistance were submitted to the VUMC Special Infectious Disease Laboratory for confirmatory susceptibility testing. These isolates were stored at −70°C. To avoid overrepresentation, only the first patient isolate was included in the clinical and molecular analyses of non-penicillin-susceptible S. pneumoniae isolates (PISP and PRSP). Patient demographics and clinical data were obtained from computerized medical records.
Identification and susceptibility testing.
Isolates were identified by conventional methods (10). Preliminary penicillin susceptibility testing was performed at the VUMC Clinical Microbiology Laboratory with a 1-μg oxacillin disk (21). For isolates which yielded a disk zone size of less than 20 mm, penicillin resistance was confirmed by broth microdilution from 1990 through 1997 and by the E-test method in 1998 and 1999 (18, 22).
Susceptibility to chloramphenicol, clindamycin, tetracycline, levofloxacin, erythromycin, and vancomycin was determined by the Kirby-Bauer disk diffusion method. Susceptibility to broad-spectrum cephalosporins was determined by the E-test method in 1999 and by broth microdilution during other years (18, 22). Established NCCLS breakpoints were used (21). Susceptibility data for these antibiotics for 1998 and 1999 isolates were available through review of the computerized microbiology database.
PCR determination of pbp2B alterations.
PCR assays were performed on all stored PISP and PRSP isolates. Primer selection was based on the published gene sequences of the pbp2B transpeptidase-encoding region of South African penicillin-resistant S. pneumoniae strains (27). Six primers were used: P5 (5"-CTG ACC ATT GAT TTG GCT TTC CAA-3") and P6 (5"-TTT GCA ATA GTT GCT ACA TAC TG-3"), which are specific for S. pneumoniae, and R1 (5"-GCC TTT TCT AGG CCA ATG CCG ATT AC-3"), R2 (5"-GCC TAC GAT TCA TTC CCG ATT-3"), R3 (5"-AAA TTG GCA TAT GGA TCT TTT CCT-3"), and R4 (5"-GTT TTA ACT AAC AAT TTA GAA TCC-3"), which are specific for four alterations in the pbp2B gene of PRSP. For each isolate, four PCRs were performed, each containing primers P5 and P6 plus R1, R2, R3, or R4. Amplification was carried out with a PE 9700 thermal cycler (Applied Biosystems Inc., Foster City, Calif.) as previously described (9).
To document the absence of the R1, R2, R3, or R4 amplification product among PSSP isolates, PCR assays were performed on a set of PSSP isolates obtained from a collection at VUMC.
Serotyping and genomic DNA analysis by PFGE.
A subset of PRSP isolates was chosen for serotyping and PFGE analysis. The subset included the first isolate recovered at every 3-month interval from 1990 through 1998 and the first three isolates recovered at every 3-month interval in 1999, if available.
Serotyping was performed by using the Quellung reaction (10). For PFGE, genomic DNA was extracted from logarithmic-phase cultures grown in brain heart infusion broth (Becton Dickinson Microbiology Systems, Sparks, Md.), prepared in low-melting-point agarose plugs, and digested with SmaI enzyme (New England Biolabs, Beverly, Mass.) for 24 h as previously described (29). The DNA size standards used were a bacteriophage lambda ladder consisting of concatemers starting at 48.5 kbp and increasing to approximately 1,000 kbp (Bio-Rad Laboratories, Hercules, Calif.). Electrophoresis was performed with a GenePath system (Bio-Rad) by using dedicated program 2 for 20 h. The gels were stained with ethidium bromide, rinsed, and photographed under UV light by using a Gel Doc 2000 computerized documentation system (Bio-Rad). Isolates differing in restriction patterns by one to six bands were considered closely or possibly related and were assigned to a subtype (e.g., A1). Isolates whose restriction patterns differed by ≥7 bands were considered unrelated and were assigned to a different strain type (30).
Statistical analysis.
Associations between PISP and PRSP and patient demographics, city of residence, and culture site were analyzed by using the chi-square test or the Wilcoxon rank-sum test. Temporal trends in the prevalence of penicillin resistance over the 10-year study period were analyzed by using the chi-square test for linear trends. P values of ≤0.05 were considered statistically significant. All analyses were performed by using STATA 7.0 (Stata Corporation, College Station, Tex.). The sensitivity, specificity, and positive and negative predictive values of the PCR assays in detecting penicillin resistance among all S. pneumoniae isolates were calculated by using standard definitions (12).
RESULTS
Temporal trends in penicillin resistance among S. pneumoniae isolates.
The percentage of non-penicillin-susceptible (MIC, ≥0.1 μg/ml) S. pneumoniae isolates increased significantly over the 10-year study period (P < 0.001), according to antibiogram data generated by the VUMC Clinical Microbiology Laboratory. Prior to 1997, 10% (mean) (range, 3 to 16%) of isolates were resistant to penicillin. This percentage increased progressively to 26% in 1997, 56% in 1998, and 70% in 1999 (Fig. 1).
FIG. 1.
Prevalence of non-penicillin-susceptible S. pneumoniae isolates (MIC, ≥0.1 μg/ml) recovered in middle Tennessee from 1990 through 1999.
PISP and PRSP.
Among the 406 nonsusceptible S. pneumoniae isolates submitted to the VUMC Special Infectious Disease Laboratory during the 10-year study period, 168 (41%) were PISP and 238 (59%) were PRSP.
Patient characteristics and culture sites.
Demographic data were available for 303 of the 406 patients with either PISP or PRSP isolates. The remaining 103 specimens were sent from other Tennessee hospitals to VUMC for confirmatory testing. PISP and PRSP were recovered from 125 and 178 of the 303 patients, respectively. Demographic data are shown in Table 1. There were no significant differences in characteristics between patients harboring PISP and PRSP isolates (P > 0.05) (data not shown).
TABLE 1.
Characteristics of 303 patients colonized or infected with PISP or PRSP isolates
| Characteristic | No. (%) of patients |
|---|---|
| Age (yr) | |
| <5 | 165 (55) |
| 5-17 | 10 (3) |
| 18-64 | 103 (34) |
| ≥65 | 25 (8) |
| Male | 166 (56) |
| Caucasian | 247 (82) |
| Nashville resident | 98 (32) |
Body culture sites from which the 406 patient isolates were obtained were as follows: sputum or nasopharynx, 29%; blood, 25%; ear fluid, 16%; sinus aspirate, 11%; cerebrospinal fluid, 6%; bronchoalveaolar lavage, 5%; and other sites, 7%. There were no significant differences in culture sites between PISP and PRSP isolates (P > 0.05) (data not shown).
Susceptibility profiles.
The profiles of susceptibility of PISP and PRSP isolates recovered from 1998 through 1999 to seven different antimicrobial agent classes are shown in Table 2.
TABLE 2.
In vitro activities of antimicrobial agents for PISP and PRSP isolates with the R3 amplification product and collected from 1998 through 1999
| Antibiotic | PISP
|
PRSP
|
|||||||
|---|---|---|---|---|---|---|---|---|---|
| No. of isolates | % of isolates that werea:
|
No. of isolates | % of isolates that werea:
|
||||||
| S | I | R | S | I | R | ||||
| Ceftriaxone | 29 | 72 | 17 | 10 | 74 | 5 | 42 | 53 | |
| Chloramphenicol | 28 | 78 | 0 | 22 | 68 | 82 | 2 | 16 | |
| Clindamycin | 29 | 76 | 7 | 17 | 71 | 86 | 1 | 13 | |
| Levofloxacin | 27 | 100 | 0 | 0 | 76 | 97 | 0 | 3 | |
| Erythromycin | 29 | 62 | 0 | 38 | 70 | 30 | 0 | 70 | |
| Tetracycline | 29 | 69 | 0 | 31 | 69 | 75 | 0 | 25 | |
| Vancomycin | 28 | 100 | 0 | 0 | 70 | 100 | 0 | 0 | |
S, I, and R, susceptible, intermediate, and highly resistant to the listed antibiotics, respectively.
Susceptibility data were available for all seven classes of antimicrobial agents for 27 PISP and 68 PRSP isolates. For the PISP isolates, 25, 14, and 7% were resistant to at least three, at least four, and at least five antibiotic classes. For the PRSP isolates, 23, 10, and 5% were resistant to at least three, at least four, and at least five antibiotic classes, respectively. All isolates were susceptible to vancomycin, and ≥97% were susceptible to levofloxacin.
For 29 (38%) of the 76 PRSP isolates, the penicillin MIC was ≥4 μg/ml; for 7 (24%) of these 29 isolates, the ceftriaxone MIC was ≥4 μg/ml.
Capsular serotypes.
Among 42 isolates submitted for serotyping, 4 (10%) were nontypeable. Serotypes 14 and 23F were the most common, representing 14 (31%) and 9 (21%) of the isolates, respectively. Other serotypes represented ≤10% of the isolates. Serotypes included in the 23- and 7-valent vaccines accounted for 100 and 92% of the isolates, respectively.
Molecular typing.
PFGE was performed on 42 PRSP isolates. All of these isolates contained the R3 amplification product (see below). Over the 10-year study period, 13 unique PFGE patterns were identified. Type Q (8 isolates [19%]) and type B (14 isolates [33%]) were the most common PFGE patterns identified (Table 3). During the last 5 years of the study, type B strains accounted for 53% of the PRSP strains (14 of 27).
TABLE 3.
PFGE types of PRSP isolates with the R3 amplification product over a 10-year period
| Study yr | No. of isolates | PFGE type(s) (no. of isolates) |
|---|---|---|
| 1990 | 1 | Q (1) |
| 1991 | 3 | A (1), Q (2) |
| 1992 | 4 | G (1), H (1), Q (1), R (1) |
| 1993 | 3 | I (1), Q (2) |
| 1994 | 4 | B (1), I (1), L (1), Q (1) |
| 1995 | 4 | B (2), O (1), Q (1) |
| 1996 | 4 | B (3), L (1) |
| 1997 | 4 | B (2), L (2) |
| 1998 | 3 | A (1), B (1), P (1) |
| 1999 | 12 | A (2), B (5), C (1), D(2), E(2) |
R1, R2, R3, and R4 amplification products of PISP and PRSP.
The six-primer PCR assay was performed on 544 isolates, of which 138 were PSSP, 168 were PISP, and 238 were PRSP. S. pneumoniae DNA was detected among all isolates. R1, R2, R3, and R4 amplification products were not detected among the PSSP isolates.
The R3 amplification product was detected in 300 (73%) of the 406 PISP and PRSP isolates. This “resistant” product was present among the majority of PRSP isolates (233 of 238 [98%]) compared to PISP isolates (67 of 168 [40%]) (P < 0.001). Amplification products obtained by using primers R1 to R4 were not detected in 87 PISP and PRSP isolates (21%). Among these isolates, intermediate resistance to penicillin (85 of 168) was more common than high-level resistance (2 of 238) (P < 0.001) (Table 4).
TABLE 4.
Susceptibility of 544 S. pneumoniae isolates to penicillin according to resistant amplification products determined with primers R1 to R4
| Amplification product | No. (%) of isolates
|
||||
|---|---|---|---|---|---|
| Tested | That were:
|
||||
| PSSP (n = 138) | PISP (n = 168) | PRSP(n = 238) | |||
| R1 | 8 (2) | 0 | 8 (5) | 0 | |
| R2 | 11 (2) | 0 | 8 (5) | 3 (1) | |
| R3 | 300 (55) | 0 | 67 (40) | 233 (98) | |
| R4 | 0 | 0 | 0 | 0 | |
| No resistant product | 225 (41) | 138 (100) | 85 (50) | 2 (1) | |
There were no statistically significant differences in age, gender, sex, and residence among patients whose PISP or PRSP isolates harbored the R3 amplification product versus R1, R2, or no amplification product (data not shown).
The diagnostic performance of the PCR assay for detecting both PISP and PRSP was as follows: specificity, 100%; sensitivity, 79%; positive predictive value, 100%; and negative predictive value, 61%. When the analysis was limited to PRSP, the sensitivity and negative predictive value increased to 99%, and the specificity and positive predictive value remained at 100% (Table 5).
TABLE 5.
Diagnostic performance of a six-primer PCR assay for the simultaneous detection of S. pneumoniae and penicillin resistance
| S. pneumoniae | %
|
||||
|---|---|---|---|---|---|
| Sensitivity | Specificity | Predictive value
|
|||
| Negative | Positive | ||||
| PISP and PRSP | 79 | 100 | 61 | 100 | |
| PISP only | 49 | 100 | 82 | 100 | |
| PRSP only | 99 | 100 | 99 | 100 | |
DISCUSSION
This study documents a dramatic increase in penicillin resistance among S. pneumoniae isolates recovered over a 10-year study period in Tennessee. From 1990 through 1996, only 10% of S. pneumoniae isolates were resistant to penicillin. This percentage increased to 70% by 1999, the last year of the study. A substantial proportion of these resistant isolates recovered during the last 2 years of the study had high-level resistance, with penicillin and ceftriaxone MICs of ≥4 μg/ml (3). At this level of resistance, therapeutic tissue concentrations of penicillin or ceftriaxone cannot be achieved, mandating the use of non-β-lactam antibiotics (1, 15). A total of 23% of PRSP isolates (MIC, ≥2 μg/ml) were also resistant to at least three other antimicrobial classes, and 5% of isolates were resistant to at least five other antimicrobial classes. The increasing rates of penicillin resistance among S. pneumoniae isolates, coupled with the magnitude of resistance in individual isolates and the occurrence of multidrug resistance, raise serious concerns about the therapeutic options available for common and life-threatening S. pneumoniae infections, particularly at standard drug doses.
Most penicillin-resistant S. pneumoniae infections can be adequately treated with quinolones. However, the efficacy of this class of antimicrobial agents may be temporary, since rates of quinolone resistance among S. pneumoniae isolates are increasing throughout the United States and the in vitro emergence of resistance with exposure to quinolones has been documented (7, 31). In addition, quinolones are not approved for use in children, in whom infections caused by penicillin-resistant S. pneumoniae are most prevalent (15). Vancomycin continues to remain active against S. pneumoniae. However, limiting the use of this antibiotic is paramount due to the emergence of Staphylococcus aureus isolates with reduced susceptibility to vancomycin (26, 28) and the persistent increase in the rates of vancomycin-resistant enterococci in Tennessee and throughout the United States (5, 23). Preventing infections caused by penicillin-resistant S. pneumoniae with vaccines would decrease the overuse of these antibiotics and limit the emergence of resistance. In this study, serotypes included in the 7- and 23-valent vaccines accounted for 92 and 100% of isolates, respectively.
In the United States, rates of penicillin resistance among S. pneumoniae isolates range from 15 to 35% (31). These various rates reflect different geographical locations and underlying risk factors, including age of less than 5 years and Caucasian race (8, 14, 32). The high rates of resistance documented in the present study may reflect a predominance of Caucasian children or study location.
The dissemination of PRSP throughout Tennessee was polyclonal, with 13 unique strains identified over 10 years. However, a single clone accounted for over half of the isolates recovered during the last 5 years of the study. This epidemiological pattern suggests that PRSP spread across Tennessee due to both horizontal transfer of altered penicillin-binding protein genes between S. pneumoniae isolates (4, 17) and clonal dissemination. Prior surveillance studies described the global dissemination of a small number of S. pneumoniae clones (2, 6, 13, 19).
Rapid and accurate diagnosis of penicillin-resistant S. pneumoniae infections may improve patient outcomes and guide the selection of appropriate antimicrobial therapy. The six-primer PCR assay described in the present study was highly sensitive (99%) and specific (100%) for the simultaneous detection of S. pneumoniae and high-level penicillin resistance. This assay was previously shown to have 100% negative and positive predictive values in detecting penicillin-resistant S. pneumoniae in cerebrospinal fluid (9). This PCR-based method can provide information on penicillin susceptibility within hours, resulting in optimal prescribing of antimicrobial agents and avoidance of the unnecessary use of broad-spectrum antimicrobial agents. In addition to providing rapid information on the optimal choice of antimicrobial agents, this assay may also have a role in clinical situations where cultures are negative as a result of prior antibiotic therapy, since PCR-based methods do not rely on viable organisms.
The present study had several limitations. First, data on prior antibiotic exposure were not available; hence, the contribution of this risk factor to the high rates of penicillin resistance could not be assessed (14). Second, the diagnostic accuracy of the six-primer PCR assay was based on a high prevalence of penicillin-resistant S. pneumoniae isolates with a predominance of the resistant R3 amplification product. The diagnostic accuracy of this assay may therefore differ in geographical areas with lower rates of penicillin resistance or in areas in which different pbp2 gene mutations confer penicillin resistance. Last, because of the suboptimal detection of PISP, this assay may not be useful for meningitis.
Antimicrobial agent-resistant pathogens continue to emerge and disseminate (24). If Tennessee is a sentinel state for PRSP, then this study provides a grim outlook for future rates of penicillin resistance of S. pneumoniae in other states and countries. Rapid methods for the diagnosis of pneumococcal infections and the detection of antimicrobial resistance, in addition to ongoing efforts to optimize vaccination strategies, are necessary to decrease the morbidity and mortality associated with penicillin-resistant S. pneumoniae infections.
Acknowledgments
We thank Rosemary Verrall and Narinder Midha for excellent assistance and Kathryn Edwards and Charles Stratton for thoughtful review of the manuscript.
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