Journal of Antimicrobial Chemotherapy (2000) 45, 871-875
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Presence of ROB-1 ß-lactamase correlates with cefaclor resistance among recent isolates of Haemophilus influenzae
a Department of Medical Microbiology and b Faculty of Pharmacy, University of Manitoba; Departments of c Clinical Microbiology and d Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada
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Abstract |
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ß-Lactamase production in Canadian isolates of Haemophilus influenzae has remained relatively constant (25–35%) over the last decade despite increasing cefaclor resistance (MIC
32 mg/L). TEM (294/324, 90.7%) and ROB-1 (30/324, 9.3%) prevalence rates among 324 isolates of H. influenzae obtained from across Canada in 1997–1998 were similar (P > 0.05) to previously published reports. However, 66.7% (26/39) of cefaclor-resistant isolates were ROB-1-positive (P < 0.001) and the remaining four ROB-1-positive isolates were cefaclor-intermediate (MIC 16 mg/L). Susceptibilities to loracarbef (P < 0.001) and cefprozil were also reduced in the presence of ROB-1 while the activities of cefuroxime, cefotaxime, cefixime and imipenem were similar in both TEM- and ROB-1-positive solates. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Haemophilus influenzae is most frequently isolated from respiratory tract specimens. Two ß-lactamases, TEM-1 and ROB-1, have been identified in H. influenzae.1 Ampicillin resistance arising from TEM-1 in H. influenzae was first documented in 1975.1 ROB-1 was isolated in 1981 from an ampicillin-resistant isolate of H. influenzae and is considerably less prevalent than TEM-1.2,3 TEM-1 and ROB-1 share limited amino acid sequence homology but have similar substrate profiles and relative rates of ampicillin hydrolysis.1 Generally, any one isolate of H. influenzae produces only one of the two ß-lactamases,2,3 although rare isolates with both TEM-1 and ROB-1 have been reported.3 Recently published rates of ß-lactamase production in H. influenzae range from 24.0 to 31.3% in Canada4,5 and 34.2 to 36.1% in the USA.4,6 Doern and colleagues have suggested that the prevalence of ß-lactamase production among North American respiratory tract isolates of H. influenzae may have levelled off following dramatic increases during the 1980s and early 1990s.4
In Canada, rates of resistance to cefaclor (MIC 32 mg/L) among H. influenzae have increased from <3.2% in 19913 to 12% in 1997–1998.4,5 The prevalence of ß-lactamase production in the same isolates has remained relatively constant (24.0–31.3%) over the same period. Given the premiss that cefaclor is a substrate for TEM-1, albeit a significantly less ideal substrate than ampicillin,7 we attempted to determine if increasing cefaclor resistance may be the result of a change in ß-lactamase profile. To do this we identified ß-lactamase DNA sequences by PCR in 324 recent clinical isolates of H. influenzae collected from across Canada in 1997–98.5 The last similar study in Canada was conducted using isolates collected during 19913 and reported that 93.0% (146/157) of ß-lactamase-positive H. influenzae possessed a TEM ß-lactamase with the remaining 11 isolates (7%) having the ROB-1 ß-lactamase. As a follow-up to results found in the initial study, an additional 71 ß-lactamase-positive H. influenzae6 from the USA, with variable susceptibilities to cefaclor, were also tested for the presence of TEM and ROB-1 genes.
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Materials and methods |
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Bacterial isolates
A Canadian lower respiratory tract surveillance study conducted between September 1997 and November 1998 identified 324 (24.0%) unique clinical isolates of ß-lactamase-positive H. influenzae.5 Isolates had been identified previously as H. influenzae by standard methods and their antibiotic susceptibilities had been determined.5 A second collection of 20 cefaclor-susceptible (MIC 
8 mg/L), 26 cefaclor-intermediate (MIC 16 mg/L) and 25 cefaclorresistant (MIC 32 mg/L) ß-lactamase-positive strains of H. influenzae was obtained from authors of a recent USA national surveillance study for testing.6 ß-Lactamase production was confirmed in each isolate using nitrocefin-based disc testing (Cefinase; BBL Microbiology Systems, Cockeysville, MD, USA) according to the manufacturer's instructions.
ß-Lactamase identification by PCR
TEM and ROB-1 ß-lactamase sequences were identified in all isolates of H. influenzae by PCR amplification.3 TEM-specific primers were chosen from the published sequence of pBR322.8 The primers 5'-TGGGTGCACGAGTGGGTTAC-3' and 5'-TTATCCGCCTCCATCCAGTC-3' amplified a TEM internal sequence 525 bp in length. Thermocycling conditions for the TEM primer pair were 94°C for 5 min, 30 cycles of 94°C for 2 min, 57°C for 1 min and 72°C for 2 min, followed by a final elongation step of 10 min at 72°C. Digestion of the 525 bp amplicon with DraI (Boehringer–Mannheim, Montreal, Quebec, Canada) into 97 and 428 bp fragments confirmed amplicon identity. ROB-1-specific primers and cycling conditions were identical to those previously described by Scriver and colleagues.3 ROB-1 amplicon identity was also confirmed with DraI digestion as previously described.3
DNA template was obtained for PCR by heating a suspension of bacteria to 94°C for 5 min.3 Each 50 µL PCR reaction contained 15 mM Mg2+, 1.25 mM total dNTPs, 25 pmol of each sense and antisense primers, 2.5 units of Taq DNA polymerase (Pharmacia, Baie d'Urfe, Quebec, Canada) and double-distilled sterile water in addition to DNA template. H. influenzae ATCC 49247 was used as a negative control organism for both TEM and ROB-1 PCR assays. H. influenzae clinical isolates 103 and 546 were used as TEM-positive and ROB-1-positive control organisms, respectively.3
Statistical analysis
Statistical analyses used the nQuery advisor program (Statistical Solutions, San Francisco, USA) and assumed an eventual one-sample 
2 test (normal approximation). TEM- and ROB-1-positive isolates of H. influenzae were assumed to be equally susceptible to each antibiotic tested. P values of <0.05 were considered significant.
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Results |
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Of the 324 Canadian isolates tested, 294 (90.7%) were PCR-positive for TEM and 30 (9.3%) PCR-positive for ROB-1. The MICs of these isolates are presented in Table 1
. TEM- and ROB-1-positive isolates in this collection were 98.0% susceptible to amoxycillin–clavulanate, cefuroxime, cefotaxime, cefixime and imipenem regardless of the ß-lactamase present (Table I).9 All isolates of ß- lactamase-positive H. influenzae were resistant to ampicillin (Table I).9 Rates of cefaclor and loracarbef resistance were significantly (P < 0.001) higher in ROB-1-positive isolates than in TEM-positive isolates (Table I). None of the 30 ROB-1-positive isolates tested was susceptible to cefaclor or loracarbef (Table I). Cefprozil-resistant isolates were all ROB-1-positive (Table I). Cefuroxime resistance was only demonstrated among TEM-positive, cefaclor-resistant isolates (Table I).
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Of the 71 H. influenzae isolates tested from the USA, all 20 cefaclor-susceptible isolates and all 26 cefaclor-intermediate isolates were TEM-positive (Table II
). Of the 25 cefaclor-resistant isolates tested 17 (68.0%) were ROB- 1-positive and 8 (32.0%) were TEM-positive (Table II). The MICs of these isolates are presented in Table II. All 71 isolates were resistant to ampicillin and susceptible to cefotaxime, cefixime and imipenem regardless of the ß- lactamase present (Table II). Two ß-lactamase-positive amoxycillin/clavulanate-resistant (BLPACR) isolates were identified, one being TEM-positive and cefaclor-intermediate and the other TEM-positive and cefaclor-resistant (Table II). Again, cefuroxime resistance was only demonstrated among TEM-positive, cefaclor-resistant isolates (Table II). At least 25% (range 25–52.9%) of cefaclor-resistant H. influenzae isolates were cross-resistant to cefprozil and loracarbef regardless of the ß-lactamase present (Table II). As demonstrated by the Canadian isolates, cefaclor and loracarbef resistance rates were significantly (P < 0.001) higher in ROB-1-positive isolates than in TEM-positive isolates; none of the ROB-1-positive isolates tested was susceptible to cefaclor or loracarbef (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A 1991 Canadian H. influenzae antibiotic resistance surveillance study reported 93.0% (146/157) of isolates as TEM-positive and the remaining 7.0% (11/157) as ROB-1-positive.3 In other previous studies the prevalence of ROB-1 ß-lactamase has been reported to be 8.0% and 2.8% in hospitalized and community isolates, respectively.2,10 The present study, in which TEM was identified in 90.7% of ß-lactamase-positive H. influenzae isolates and ROB-1 in the remaining 9.3% of isolates indicates that the proportion of TEM- and ROB-1 ß-lactamases in H. influenzae has not changed significantly (P < 0.05) in Canada during the 1990s.
The aforementioned 1991 Canadian surveillance study also reported the absence of a correlation between antibiotic susceptibilities, including cefaclor, and the presence of TEM or ROB-1 ß-lactamases.3 Of the 479 ß-lactamase-positive isolates tested, 96.8% were susceptible to cefaclor.3 In contrast, in the present study, which was conducted with 1997–98 ß-lactamase-positive H. influenzae isolates also collected from across Canada, 66.7% (26/39) of cefaclor-resistant isolates had ROB-1 and 33.3% (13/39) had TEM. For the other four ROB-1-positive isolates cefaclor MICs were also higher (16 mg/L). A similar trend was observed among the 71 isolates6 tested from the USA with all cefaclor-susceptible and -intermediate isolates being TEM-positive and 68.0% (17/25) of the cefaclor-resistant isolates being ROB-1-positive.
A recent Canadian antibiotic surveillance study found that 12.0% of H. influenzae isolates were resistant to cefaclor.5 Results presented in this study have demonstrated that increasing cefaclor resistance in Canada appears not to be the result of changing ß-lactamase profiles among clinical isolates of H. influenzae. Therefore, consideration must be given to mutation of TEM and ROB-1 ß-lactamases as the most probable reason underlying increases in cefaclor resistance. As previously demonstrated in Escherichia coli and Klebsiella pneumoniae, one to four amino acid substitutions in the TEM-1 enzyme or its promoter region can result in hyperproduction of ß-lactamase or a change in substrate specificity.1 Similarly, the TEM-1 ß-lactamase in some isolates of H. influenzae may have acquired mutations that make cefaclor a more favourable substrate for this enzyme.1 Compared with the TEM ß-lactamase, ROB-1 is largely unstudied and little is known of its activity against cefaclor. Mutations in the ROB-1 enzyme have not been described previously, but may help to explain the high prevalence of ROB-1 among the cefaclor-resistant isolates of H. influenzae studied here. The data may also suggest that ROB-1 may be a more potent hydrolyser of cefaclor than TEM. It is also possible that another ß-lactamase gene, distinct from TEM and ROB-1, may have been introduced into H. influenzae and co-exists compatibly with TEM or ROB-1 to confer resistance.
In conclusion, the ß-lactamase profile of H. influenzae in Canada appears not to have changed between 1991 and 1997–98, but ß-lactamase-positive cefaclor-resistant H. influenzae harboured ROB-1 ß-lactamase more commonly (P < 0.001) than TEM. The significant increases in cefaclor resistance appear to have occurred in the absence of a change in ß-lactamase profile, suggesting that mutation and/or changes in the expression of ß-lactamase genes and/or alterations in penicillin-binding proteins may be responsible.
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Acknowledgments |
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The authors gratefully acknowledge the financial support of Lilly Research Laboratories, Indianapolis, IN, USA. This work was presented in part at the Twenty-First International Congress of Chemotherapy, Birmingham, UK, 4–7 July 1999.
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Notes |
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* Correspondence address. Department of Clinical Microbiology, Health Sciences Centre, MS673, 820 Sherbrook Street, Winnipeg, Manitoba R3A 1R9, Canada. Tel: +1-204-787-4683; Fax: +1-204-787-4699; E-mail: jkarlowsky{at}hsc.mb.ca
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References |
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1 . Bush, K. (1989). Classification of ß-lactamases: groups 1, 2a, 2b, and 2b'. Antimicrobial Agents and Chemotherapy 33, 264–70.
2 . Daum, R. S., Murphey-Corb, M., Shapira, E. & Dipp, S. (1988). Epidemiology of rob ß-lactamase among ampicillin-resistant Haemophilus influenzae isolates in the United States. Journal of Infectious Diseases 157, 450–5.[Web of Science][Medline]
3
.
Scriver, S. R., Walmsley, S. L., Kau, C. L., Hoban, D. J., Brunton, J., McGeer, A. et al. (1994). Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Canadian Haemophilus Study Group. Antimicrobial Agents and Chemotherapy 38, 1678–80.
4
.
Doern, G. V., Jones, R. N., Pfaller, M. A. & Kugler, K. (1999). Haemophilus influenzae and Moraxella catarrhalis from patients with community-acquired respiratory tract infections: antimicrobial susceptibility patterns from the SENTRY Antimicrobial Surveillance Program (United States and Canada, 1997). Antimicrobial Agents and Chemotherapy 43, 385–9.
5 . Hoban, D. J., Zhanel, G. G. & Karlowsky, J. A. (1999). In vitro activity of the novel ketolide HMR 3647 and comparative oral antibiotics against Canadian respiratory tract isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Diagnostic Microbiology and Infectious Disease 35, 37–44.[Web of Science][Medline]
6 . Jones R. N., Jacobs, M. R., Washington, J. A. & Pfaller, M. A. (1997). A 1994–95 survey of Haemophilus influenzae susceptibility to ten orally administered agents. A 187 clinical laboratory center sample in the United States. Diagnostic Microbiology and Infectious Diseases 27, 75–83.[Web of Science][Medline]
7
.
Wise, R., Andrews, J. M., Ashby, J. P. & Thornber, D. (1990). The in-vitro activity of cefpodoxime: a comparison with other oral cephalosporins. Journal of Antimicrobial Chemotherapy 25, 541–50.
8
.
Sutcliffe, J. G. (1978). Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proceedings of the National Academy of Sciences, USA 75, 3737–41.
9 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement M100-S9. NCCLS, Wayne, PA.
10 . Walmsley, S. L., Fuller, S., Juteau, J. M., Simor, A. E. & Low, D. E. (1990). Prevalence of ROB-1 ß-lactamase among community isolates of Haemophilus influenzae. In Program and Abstracts of the Nintieth Annual Meeting of the American Society for Microbiology, Anaheim, CA, 1990. Abstract A-74, p. 13. American Society for Microbiology, Washington, DC.
Received 8 October 1999; returned 29 November 1999; revised 22 December 1999; accepted 17 January 2000
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