Abstract
Canadian invasive Haemophilus influenzae isolates from 1990 to 2006 were examined for antibiotic susceptibility: 42 strains (17.8%) were resistant to ampicillin by β-lactamase production, 5.5% were β-lactamase negative ampicillin intermediate, and 2.5% were intermediate to only the 2-μg ampicillin disk. An increase in β-lactamase-negative ampicillin-intermediate strains has been found.
Before the introduction of the Haemophilus influenzae serotype b (Hib) conjugate vaccine, Hib was a common cause of childhood meningitis and other invasive infections, such as septicemia and epiglotittis (6). Since the introduction of the Hib conjugate vaccine in North America and Europe, disease due to Hib has become rare (1, 9). Recent studies suggest that non-type b H. influenzae, particularly nontypeable (NT) strains, is now responsible for most H. influenzae disease (2, 10). Currently, there is no vaccine available for non-type b H. influenzae, and antibiotic treatment is the only effective method to combat disease caused by these strains. Antibiotic resistance in H. influenzae was first documented in 1972 with the discovery of an ampicillin-resistant isolate (4). Since then, H. influenzae has developed resistance to several other antibiotics, and resistance rates continue to change over time. Therefore, it is important to monitor for antibiotic susceptibility in H. influenzae. Here, we report the antibiotic susceptibility profiles of invasive H. influenzae isolates collected over the last 17 years in Canada against antibiotics commonly used for treatment of both respiratory and invasive infections.
A total of 236 H. influenzae isolates from individual invasive-disease cases from 1990 to 2006 were included in this study. These isolates were submitted from provincial public health laboratories across Canada, as well as from the Clinical Microbiology Laboratories at the Health Sciences Centre and St. Boniface General Hospital in Winnipeg, Manitoba.
The isolates were serotyped by slide agglutination using commercial antisera (Difco, Oakville, Ontario, Canada; Denka Seiken, Tokyo, Japan) and confirmed by PCR detection of serotype-specific capsular-polysaccharide synthesis genes using a previously described method (3).
β-Lactamase production was detected using Dryslide nitrocefin (BBL, Becton Dickinson, Oakville, Ontario, Canada). Disk diffusion was carried out as described by the Clinical Laboratory Standards Institute (8). H. influenzae strain ATCC 49247 was used as a control in each experiment. The following antibiotics (Oxoid, Nepean, Ontario, Canada) were tested: ampicillin (2 and 10 μg), amoxicillin-clavulanic acid (30 μg), cefaclor (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clarithromycin (15 μg), moxifloxacin (5 μg), and trimethoprim-sulfamethoxazole (25 μg). Detection of β-lactamase-negative ampicillin-resistant (BLNAR) strains was accomplished by using two concentrations of ampicillin (7).
Seventy-one isolates (30%) were obtained from the years 1990 to 1999, and the remaining 165 isolates (70%) were from 2000 to 2006. The majority of isolates (62.3%) were NT, followed by serotype a (Hia) (21.6%), Hib (7.6%), serotype f (4.2%), serotype e (2.1%), serotype c (1.7%), and serotype d (0.4%). Half of the Hib isolates were from 1990 (nine isolates), one was from 1991, and the remaining eight isolates were from five different years between 2000 and 2006. From 1990 to 1999, 64.8% of the isolates were NT and 12.5% were Hia. For 2000 to 2006, the percentages of NT and Hia isolates were 61.2% and 25.5%, respectively.
Overall, 42 isolates were resistant to ampicillin by β-lactamase production. Of the serotypeable strains, 44% of the 18 Hib isolates were resistant to ampicillin, including 1 showing multiple-drug (ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole) resistance. Three (60%) serotype e and only one (2%) Hia isolates were resistant to ampicillin. No ampicillin resistance was found in the small numbers of serotype c, serotype d, and serotype f strains studied. There was no association between encapsulation and resistance to ampicillin, as 12 (13.5%) of the 89 serotypeable strains versus 30 (20.5%) of the 147 NT strains were resistant to ampicillin (t test; P > 0.1). Significantly more NT strains (19 out of 147; 12.9%) than serotypeable strains (2 out of 89; 2.3%) showed resistance to trimethoprim-sulfamethoxazole (t test; P < 0.001). The antibiotic susceptibility patterns of the H. influenzae isolates according to serotype are summarized in Table 1.
TABLE 1.
Antibiotic susceptibility patterns of 236 H. influenzae isolates according to serotype
| Antibiotic susceptibility patterna | No. of strains/serotype
|
Total | ||||||
|---|---|---|---|---|---|---|---|---|
| a | b | c | d | e | f | NT | ||
| Amp2 I | 4 | 4 | ||||||
| Amp Ib | 1 | 1 | 1 | 3 | ||||
| Amp2 I, Sxt R | 2 | 2 | ||||||
| Amp I, Sxt R | 5 | 5 | ||||||
| Amp I, Cec I | 4 | 4 | ||||||
| Amp I, Cec R | 1 | 1 | ||||||
| Amp R | 1 | 7 | 2 | 21 | 31 | |||
| Amp R, Amc R | 1 | 1 | ||||||
| Amp R, Cec I | 1 | 1 | ||||||
| Amp R, Clr R | 1 | 2 | 3 | |||||
| Amp R, Sxt R | 4 | 4 | ||||||
| Amp R, Sxt R, Amc R, Cec R | 1 | 1 | ||||||
| Amp R, Chl R, Sxt R | 1 | 1 | ||||||
| Cec I | 1 | 1 | ||||||
| Clr R | 1 | 1 | 1 | 3 | ||||
| Sxt R | 1 | 6 | 7 | |||||
| Sxt R, Cec I | 1 | 1 | ||||||
| Susceptible to all | 50 | 9 | 4 | 1 | 1 | 7 | 91 | 163 |
| Total | 51 | 18 | 4 | 1 | 5 | 10 | 147 | 236 |
I, intermediate; R, resistant. The antibiotics tested were ampicillin (Amp2, 2 μg; Amp, 10 μg); chloramphenicol (Chl), 30 μg; ceftiaxone (Cro), 30 μg; trimethoprim-sulfamethoxazole (Sxt), 25 μg; amoxicillin-clavulanic acid (Amc), 25 μg; cefaclor (Cec), 30 μg; ciprofloxacin (Cip), 5 μg; moxifloxacin (Mxf), 5 μg; and clarithromycin (Clr), 15 μg.
Intermediate values with the 2-μg and 10-μg ampicillin disks.
Studies in both the United States (5) and Canada (11) have shown a significant decline in the prevalence of β-lactamase-producing H. influenzae in recent years. In our current study, the rate of ampicillin-resistant strains from 1990 to 1999 was 21.1%, while only 16.4% of the strains from 2000 to 2006 showed resistance to ampicillin. Although the previous studies were mostly focused on respiratory isolates, the decreasing trend of β-lactamase-positive ampicillin-resistant strains holds true even for invasive isolates. Also, both BLNAR and β-lactamase-positive amoxicillin-clavulanic acid-resistant (BLPACR) strains were rarely found in the earlier studies (5, 11). However, in this study, we found two β-lactamase-positive isolates that were also resistant to amoxicillin-clavulanate (BLPACR), and it is possible that both had mutations affecting their penicillin binding proteins (PBPs). There were also 13 β-lactamase-negative isolates that were ampicillin intermediate. An additional six β-lactamase-negative isolates were identified as being potential BLNAR strains because they showed intermediate susceptibility to the 2-μg ampicillin disk, which has been proposed for detection of these strains (7). Currently, we are examining these potential BLNAR and BLPACR strains for mutations in their PBPs to correlate their in vitro susceptibility behavior to ampicillin and amoxicillin-clavulanate antibiotics with genetic characterization of their PBP3s.
No resistance to ceftriaxone, ciprofloxacin, or moxifloxacin was observed in this study. Therefore, it appears that third-generation cephalosporins and the newer fluoroquinolones are the most suitable choices of antibiotics for treatment of serious systemic H. influenzae infections.
In summary, our data suggest a change in the distribution of serotypes involved in invasive H. influenzae disease in the postvaccination era, with Hib being replaced by either non-type b serotypes or NT strains. Over the period of this study, the results indicate a decline in the prevalence of β-lactamase-positive ampicillin-resistant isolates and an increase in the number of isolates showing resistance to trimethoprim-sulfamethoxazole. It is possible that Canadian isolates of H. influenzae may have evolved a non-β-lactamase-mediated mechanism to show a stepwise increase in resistance to ampicillin, as the number of β-lactamase-negative ampicillin-intermediate isolates appears to have increased over the years. Finally, even though our study did not include a systematic collection of strains, it provides a snapshot analysis of the overall trend in the antibiotic susceptibility patterns of H. influenzae in Canada over the last 17 years.
Acknowledgments
We thank the directors and staff of the provincial public health laboratories, St. Boniface General Hospital, and Winnipeg Health Science Centre for providing the isolates for this study. We also thank Kathryn Bernard of the National Microbiology Laboratory (NML) for making isolates from her laboratory available for this study and Dennis Law and Jianwei Zhou for confirming the serotypes of the isolates by PCR.
R. S. W. Tsang receives grants from Health Canada's Genomics Research and Development Fund for research on vaccine-preventable bacterial diseases.
Footnotes
Published ahead of print on 28 January 2008.
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