Antimicrobial Resistance in Haemophilus influenzae and Moraxella catarrhalis Respiratory Tract Isolates: Results of the Canadian Respiratory Organism Susceptibility Study, 1997 to 2002

MATERIALS AND METHODS

Between October 1997 and June 2002, a total of 7,566 unique patient isolates of H. influenzae and 2,314 unique patient isolates of M. catarrhalis were collected from 25 medical centers in major population centers in 9 of the 10 Canadian provinces. Each study site was asked to collect and submit 100 H. influenzae isolates and 30 M. catarrhalis isolates per year (one isolate per patient). All isolates were deemed to be significant by individual laboratory protocols and were collected from respiratory tract specimens only (22, 23). Organisms from each center were identified as H. influenzae by the criteria used at the local site and, where necessary, were further identified by the coordinating laboratory (Health Sciences Centre, Winnipeg, Manioba, Canada) by standard methodologies such as colonial morphology, Gram staining characteristics, and X- and V-factor requirements. Similarly, colonial morphology, Gram staining characteristics, as well as oxidase and DNase production were used by the coordinating laboratory to confirm the identity of each M. catarrhalis isolate. All isolates were sent to the coordinating laboratory on Amies semisolid transport medium containing charcoal (Difco Laboratories, Detroit, Mich.). Each isolate was then stocked in skim milk and stored at −70°C in preparation for antibiotic susceptibility testing. Production of β-lactamase was assessed by use of a cefinase disk test (Becton Dickinson Microbiology Systems, Cockeysville, Md.). Twenty-five antimicrobial agents (amoxicillin, amoxicillin-clavulanate, cefotaxime, cefuroxime, cefaclor, cefprozil, cefixime, imipenem, azithromycin, clarithromycin, telithromycin, doxycycline, trimethoprim-sulfamethoxazole [TMP-SMX], ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, gemifloxacin, chloramphenicol, linezolid, ertapenem, ABT-773, ABT-492, BMS284756 [garenoxacin], and GAR-936 [tigecycline]) were obtained as laboratory-grade powders from the respective manufacturers. Stock solutions were prepared and dilutions were made by the National Committee for Clinical Laboratory Standards (NCCLS) method (15, 16). Following two subcultures from frozen stocks, antimicrobial susceptibilities were determined by the NCCLS-approved broth microdilution method (15, 16). The plates were incubated in ambient air at 35°C for 24 h prior to reading of the results. MICs were interpreted by using NCCLS breakpoints (16), and colony counts were determined periodically to confirm the inocula. Quality control was ensured by using appropriate quality control organisms from the American Type Culture Collection.

RESULTS

The demographics of the patients whose isolates were used in CROSS are described in Table 1. The numbers of H. influenzae and M. catarrhalis isolates recovered from respiratory sources varied from 1,107 to 2,166 and 341 to 643 isolates per year, respectively, over the 5-year study period. In each year of the study, ≥90% of the isolates were isolated from sputum specimens, bronchoalveolar lavage specimens, or endotracheal secretions. Approximately 55 to 61% and 39 to 45% of the isolates were obtained from inpatients and outpatients, respectively, and approximately 40 and 60% of the isolates submitted were from females and males, respectively. The breakdowns of the isolates submitted by age group were approximately 22 to 24% from individuals ≤16 years of age, 33 to 39% from individuals 17 to 64 years of age, and 38 to 43% from individuals ≥65 years of age. Table 1 indicates that the demographics of the patients from whom isolates were recovered did not change over the 5-year study period.

Table 2 describes the incidence of β-lactamase-producing H. influenzae and M. catarrhalis isolates over the 5-year study period. Among the collection of 7,566 H. influenzae isolates, 22.5% were β-lactamase positive, while 92.4% of the 2,314 M. catarrhalis isolates were β-lactamase positive. The incidence of β-lactamase-producing H. influenzae isolates decreased significantly over the 5-year study period, from 24.2% in 1997-1998 to 18.6% in 2001-2002 (P < 0.01). In contrast, the incidence of β-lactamase-producing M. catarrhalis isolates did not change over the same period (94.0% in 1997-1998 to 90.0% in 2001-2002; P = 0.75). Table 2 also compares the incidence of β-lactamase-producing H. influenzae and M. catarrhalis isolates by province or region of Canada. The major observation arising from these data was that considerable variation in the prevalence of β-lactamase-producing H. influenzae and M. catarrhalis isolates occurred both within and between each province or region in any given year. General trends were also noted, including the observation that the incidence of β-lactamase-producing H. influenzae isolates declined in all regions of the country over the 5-year study period. In addition, the incidence of β-lactamase-producing M. catarrhalis isolates remained relatively unchanged in all regions of the country throughout the study period.

The in vitro activities of 25 antibiotics against 7,566 H. influenzae isolates are presented in Table 3. The overall rates of resistance to amoxicillin and amoxicillin-clavulanate for H. influenzae were 19.3 and 0.1%, respectively. The proportions of strains found to be β-lactamase negative but amoxicillin resistant (BLNAR) and β-lactamase positive and amoxicilin-clavulanate resistant (BLPACR) were 0.1 and 0.2%, respectively. The rank order of cephalosporin activity on the basis of the MICs at which 90% of isolates are inhibited (MIC₉₀s) was cefotaxime > cefixime > cefuroxime > cefprozil > cefaclor. On the basis of the present NCCLS breakpoints, the overall proportions of isolates found to be resistant to cefotaxime, cefixime, cefuroxime, cefprozil, and cefaclor were 0.2, 0.3, 0.5, 1.0, and 7.1%, respectively. No imipenem-resistant strains were isolated. On the basis of the MICs, azithromycin was more active than clarithromycin (14-OH clarithromycin was not tested); however, on the basis of the NCCLS breakpoints, resistance rates were 2.1 and 1.6%, respectively. Rates of resistance to other agents were as follows: doxycycline, 1.5%; TMP-SMX, 14.2%; and chloramphenicol, 0.2%. All fluoroquinolones tested, including the investigational fluoroquinolones BMS284756 (garenoxacin) and ABT-492, displayed potent activities against the H. influenzae isolates, with MIC₉₀s of ≤0.03 μg/ml. The MIC₉₀s of the investigational ketolides telithromycin and ABT-773 were 2 and 4 μg/ml, respectively. Linezolid demonstrated limited activity, and the MIC₉₀ of the investigational glycylcycline GAR-936 (tigecycline) was 4 μg/ml. The distributions of the MICs of selected antibiotics for H. influenzae are displayed in Table 4. As can be observed, a shift to the left in the MIC (a decrease in the MIC) of amoxicillin concurs with the finding that the incidence of β-lactamase-producing H. influenzae is decreasing. No other MIC shift patterns were observed.

For M. catarrhalis, the rates of resistance derived by using the NCCLS breakpoint criteria for H. influenzae were <1% for all antibiotics tested except amoxicillin and TMP-SMX (1.5%) (Table 5). No strains were found to be resistant to amoxicillin-clavulanate. The rank order of the activities of the cephalosporins on the basis of the MIC₉₀s was cefotaxime = cefixime > cefuroxime = cefprozil > cefaclor. On the basis of present NCCLS breakpoints for H. influenzae, the overall proportions of isolates found to be resistant to cefotaxime, cefixime, cefuroxime, cefprozil, and cefaclor were 0, 0, 0.3, 0.2, and 0.5%, respectively. No imipenem-resistant strains were isolated. On the basis of the MICs, azithromycin and clarithromycin (14-OH clarithromycin was not tested) were equipotent, and no resistant strains were isolated. The rates of resistance to the other agents tested were as follows: doxycycline, 0.2%; TMP-SMX, 1.5%; and chloramphenicol, 0%. All fluoroquinolones tested, including the investigational fluoroquinolones BMS284756 (garenoxacin) and ABT-492, displayed potent activities against M. catarrhalis, with MIC₉₀s of ≤0.06 μg/ml. The MIC₉₀s of the investigational ketolides telithromycin and ABT-773 were 0.12 μg/ml. Linezolid demonstrated moderate activity, and the MIC₉₀ of the investigational glycylcycline GAR-936 (tigecycline) was 0.5 μg/ml.

DISCUSSION

CROSS is an ongoing longitudinal surveillance program that studies the incidence of antibiotic resistance in respiratory pathogens across all regions of Canada (22, 23). As such, it represents a unique opportunity for comparison of rates of antibiotic resistance among isolates from various geographically distributed medical centers, among isolates from patients with different demographic profiles, and by antimicrobial class. From 1997 to 2002, the same 25 medical centers participated in this study. Each year, large numbers of respiratory tract isolates of H. influenzae (1,107 to 2,166) and M. catarrhalis (341 to 643) were isolated and collected during the same time of year (during the winter months). Over the 5-year study period, study demographics remained constant; the specimen type was primarily sputum specimens, bronchoalveolar lavage specimens, and endotracheal secretions. The service breakdown was approximately 55 to 61% inpatients and 39 to 45% outpatients, while the gender breakdown was approximately 40 to 41% female and 59 to 60% male. Breakdown of isolates by patient age was approximately as follows: ≤16 years of age, 20%; 17 to 64 years of age, 40%; and ≥65 years of age, 40% (Table 1).

This study shows that the incidence of β-lactamase-producing H. influenzae decreased from 24.2% in 1997-1998 to 18.6% in 2001-2002 (Table 2). This finding was confirmed with a shift to the left in the MICs of amoxicillin (Table 4). Previously published studies that have assessed the prevalence of β-lactamase-producing H. influenzae in Canada have reported prevalences of 31.3% (1997), 28.4% (1992-1993), 26.0% (1991), and 16.9% (1985 to 1987) (8, 10, 17, 19, 23). Numerous studies assessing the prevalence of β-lactamase-producing H. influenzae isolates in the United States have also been published (7, 8, 10-12, 18). A 1994-1995 study examining the prevalence of β-lactamase-positive H. influenzae isolates among 1,537 clinical isolates obtained from 30 medical centers in the United States reported a prevalence of 36.4% (7). Doern et al. (8) studied the prevalence of β-lactamase-producing H. influenzae isolates in the United States in 1997 as part of the SENTRY program, and they reported a prevalence of 34.2%. Doern et al. (8) further commented that the incidence of β-lactamase-producing H. influenzae strains in North America had leveled off at approximately 30%. In this study, we report that the incidence of β-lactamase-positive H. influenzae isolates is declining in Canada. One reason why this may be occurring is that Canadian clinicians in outpatient practice are using fewer oral β-lactams, such as penicillin, amoxicillin, cephalosporins, and amoxicillin-clavulanate, and are preferentially prescribing more new macrolides, such as azithromycin and clarithromycin, and fluoroquinolones, such as ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin (3).

As in previous studies, we noticed a marked variation in the prevalence of β-lactamase-producing H. influenzae isolates per province or region from year to year (Table 2). This underscores the importance of performing ongoing surveillance studies on an annual basis so as not to overreact to an increase or a decrease in resistance rates over a 1-year period. It is only through systematic, annual, and ongoing surveillance that one can truly assess the patterns of resistance over time and understand the impacts of interventions on antibiotic resistance rates. Although regional variation occurred, some general patterns were observed. This included the observation that the highest prevalence of β-lactamase-producing H. influenzae isolates occurred in the Maritime provinces and Quebec, while the lowest prevalence of β-lactamase-producing H. influenzae isolates occurred in western Canada.

The proportions of strains found to be BLNAR and BLPACR were 0.1 and 0.2%, respectively (Table 3). Doern et al. (7) have also previously reported a low prevalence of BLNAR (2.5%) and BLPACR (1.1%) strains, and it appears that the incidence of these isolates is not increasing in North America. Although some have questioned the clinical predictive value of the MICs of oral cephalosporins used to treat localized respiratory tract infections caused by H. influenzae (4), the most active cephalosporins included cefotaxime, cefixime, cefuroxime, and cefprozil, with the rate resistance to cefaclor being 7.1%. Among the new macrolides, the rates of resistance to azithromycin and clarithromycin were 2.1 and 1.6%, respectively. The rates of resistance to the other agents were relatively low, with the rates of resistance to doxycycline, TMP-SMX, and chloramphenicol being 1.5, 14.2, and 0.2%, respectively (Table 3). These rates did not change over the 5-year study period. As would be expected (24), all of the fluoroquinolones consistently possessed excellent activities against H. influenzae. In fact, only one fluoroquinolone-resistant H. influenzae strain was isolated over the 5-year study period.

The prevalence of β-lactamase-producing M. catarrhalis isolates was 92.4% and did not change over the study period (Table 2). This is consistent with the findings of other North American surveillance studies that have assessed the prevalence of β-lactamase-producing M. catarrhalis isolates (6, 10, 12, 18, 20, 23), including the SENTRY study, which reported prevalences of β-lactamase-positive M. catarrhalis isolates of 92.0% in the United States and 92.2% in Canada (8). As with H. influenzae, significant year-to-year variations in resistance were observed between provinces or regions, again emphasizing the importance of annual ongoing surveillance to assess long-term patterns of resistance. With the exception of TMP-SMX (resistance rate, 1.5%), the rates resistance of M. catarrhalis isolates to antibiotics other than amoxicillin were very low (Table 5). As reported previously (8), many β-lactamase-producing M. catarrhalis strains display only low-level resistance to amoxicillin, likely because of low-level expression of the BRO-2 enzyme (2, 20, 21).

In conclusion, the mean prevalence of β-lactamase-producing H. influenzae isolates was 22.5% over the study period and decreased significantly from 24.2 to 18.6% over the 5 years. The mean prevalence of β-lactamase-producing M. catarrhalis isolates was 92.4% and did not change significantly over the 5-year study period.