Correspondence
'Institute of Microbiology. University of Ancona Medical School, 60131 Ancona. Italy; 'Department of Pathobiology. University of Washington. Seattle. Washington 98195. USA References Albritton. W. L. (1982). Infections due to Haemophilia species other than H. influenzae. Annual Review- of Microbiology 36, 199-216. Anderson, B., Albritton, W. L., Biddle, J. & Johnson, S. R. (1984). Common 0-lactamasespecifying plasmid in Haemophilia ducreyi and Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 25, 296-7. Brunton, J., Clare, D. 4 Meier, M. A. (1986a). Molecular epidemiology of antibiotic resistance plasmids of Haemophilia species and Neisseria gonorrhoeae. Reviews of Infectious Diseases 8, 713-24. Brunton, J., Meier, M., Ehrman, N.. Clare, D. & Almawy, R. (1986*). Origin of small 0-lactamasespecifying plasmids in Haemophilia species and Neisseria gonorrhoeae. Journal of Bacteriology 168, 374-9.
Brunton, J. L., Maclean, I., Ronald, A. R. & Albritton, W. L. (1979). Plasmid-mediated arapicillin resistance in Haemophilia ducreyi. Antimicrobial Agents and Chemotherapy 15, 294-9. Chen, S-T. & Clowes, R. C. (1987). Nuclcotide sequence comparisons of plasmids pHD131, pJBl, pFA3, and pFA7 and ^-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae. Journal of Bacteriology 169, 3124-30. de Graaff, J., Elwell, L. P. & Falkow, S. (1976). Molecular nature of two beta-lactamasespecifying plasmids isolated from Haemophilus influenzae type b. Journal of Bacteriology 126, 439-46. Green. M. J., Anderson, D. M., Norris, D. M. & Gubbinj, S. L. (1979). Antimicrobial resistance in Haemophilus species. New Zealand Medical Journal 90, 29. Groves, D. J. & Adeniyi-Jones, C. (1976). Ampicillin-resistant Haemophilus sp. Canadian Medical Association Journal 114, 407. Jones, R. N., Slepack, J. & Bigelow, J. (1976). Ampicillin-resistant Haemophilus paraphrophiha laryngo-epiglottitis. Journal of Clinical Microbiology 4, 405-7. Kauffman, C. A., Bergman, A. G. & Hertz, C. S. (1979). Antimicrobial resistance of Haemophilus species in patients with chronic bronchitis. American Review of Respiratory Diseases 120, 1382-5. KJlian, M. & Biberstein, E. L. (1984). Haemophilus. In Bergev's Manual of Systematic Bacteriology, Vol. I (Krieg, N. R. & Holt, J. G., Eds), p. 567. Williams & Wilkins, Baltimore, MD. Roberts, M. C. (1989). Gene transfer in the urogenital and respiratory tract. In Gene Transfer in the Environment (Levy, S. B. & Miller, R. V., Eds), pp. 347-75. McGraw-Hill, New York. Scheifele, D. W., Fussel, S. J. & Roberts, M. C. (1982). Characterization of ampiciUin-resistant Haemophilus parainfluenzae. Antimicrobial Agents and Chemotherapy 21, 734-9.
Artefactual resistance to metronidazole in anaerobes of the Bactermdes fragilis group / Amimicrob Chemolher 1992; 30: 553-554 Sir, The Anaerobe Reference Unit of the Public Health Laboratory Service receives isolates of anaerobic bacteria from laboratories situated throughout England and Wales. It is thus uniquely placed to monitor nationwide patterns and trends associated with anaerobic bacteria. The article by Cederbrant, Kahlmeter & Ljungh (1992) serves as a reminder of the importance of anaerobic conditions when testing susceptibilities to metronidazole. Despite its widespread use in prophylaxis and treatment of anaerobic infections for nearly 20
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 23, 2015
plasmid fragments and the N. gonorrhoeae probe and thus that the two plasmids were very closely related, if not identical. /}-Lactamase-producing H. haemolyticus and H. parahaenwlyticus, but not H. paraphrohaemolyticus have been reported previously (Bergman & Hertz, 1979; Green et al., 1979; Groves & Adeniyi-Jones, 1979). However, this is the first time that the location of the /Mactamase gene in a haemolytic /Mactamase producing Haemophilia sp. has been identified and shown to be on a 3-2 MDa /Mactamase plasmid. This plasmid is indistinguishable from the 3-2 MDa plasmid previously characterized in N. gonorrhoeae and is thus part of the family of small /Mactamase plasmids in the genus Neisseria and Haemophilia. As a result, it is tempting to speculate that the location of the /Mactamase gene in the previously-reported /Mactamaseproducing H. haemolyticus and H. parahaemolyticus strains may also have been on plasmids of the same family. Thus it would be interesting to screen commensal Haemophilia spp. to determine whether the small /Mactamase plasmids are present and whether these strains can act as reservoirs for this plasmid group. Acknowledgements. This work was supported in part by the CNR Target Project on Biotechnology and Bioinstrumentation grant 91.01251, to P.E.V. and by Public Health Service grant AI24136 to M.C.R. B. FACINELLI* R. TARSI' E. GIOVANETTT P. E. VARALDO* M. C. ROBERTS*
553
554
Correspondence fragilis NCTC 9343. Bonafide resistant strains should be referred to the Anaerobe Reference Unit for further study. J. S. BRAZIER VALERIE HALL B. I. DUERDEN PHLS Anaerobe Reference Unit, University Hospital of Wales. Heath Park. Cardiff CF4 4XW. UK References Cederbrant, G., Kahlmeter, G. & Ljungb, A. (1992). Proposed mechanism for metronidazole resistance in Helicobacter pylori. Journal of Antimicrobial Chemotherapy 29, 115-20. Edwards, D. I. (1979). Mechanism of antimicrobial action of metronidazole. Journal of Antimicrobial Chemotherapy 5, 499-502. Sebald, M., Reysset, G. & Breuil, J. (1990). What's new in 5-nitroimidazole resistance in the Bacteroides fragilis group? In Clinical and Molecular Aspects of Anaerobes (Bordello, S. P., Ed.), pp. 217-25. Wrightson Biomedkal Publications. The activity of ceflxinte against 715 urinary isolates of Enterobacteriaceae isolated from general practice and out-patients in twenty centres across the British Isles J Antimicrob Chemother 1992; 30: 554-556 Sir, Cefixime is an orally available cephem structurally related to some third generation cephalosporins and showing similar antibacterial activity (Kamimura el al., 1984; Bauernfeind, 1985). It is stable to a wide range of chromosomal and plasmid coded /?-lactamases and, hence, is active against Enterobacteriaceae resistant to ampitillin, cephalexin and cefaclor (Sanders, 1989). Therefore cefixime is likely to be of value in the therapy of some urinary tract infections in an out-patient setting. The objective of our study was to establish the in-vitro activity of cefixime against more than 700 Gram-negative urinary pathogens isolated in twenty centres across the United Kingdom with an emphasis on isolates from general practice and out-patients. Clinical laboratories were asked to collect consecutive and clinically significant nonrepeat isolates from general practice and outpatients. Each laboratory contributed 10 Escherichia coli and 50 other urinary tract pathogens (excluding Pseudomonas spp. and Enterococcus faecalis). A total of 715 strains were collected and sent to Southmead Hospital, Bristol for testing. MICs of cefixime,
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 23, 2015
years, resistance to this agent is still unusual, although plasmid-mcdiatcd resistance to the nitroimidazoles has been reported (Sebald, Reysset & Breuil, 1990). Recently an upward trend in the number of referrals of putative resistance to mctronidazolc among the Bacteroides fragilis group became apparent. In the space of less than one month, five supposedly metronidazole resistant strains of B. fragilis were submitted to the Unit for confirmation. Four laboratories, (two isolates were from one laboratory) reported isolates which showed greatly reduced sensitivity to a 5 fig metronidazole disc. The strains were isolates from a hip abscess, a blood culture, a wound swab (site unspecified), and two from high vaginal swabs. Upon subculture in the Anaerobe Reference Unit under controlled anaerobic conditions, all five strains proved to be fully susceptible on disc testing to a 5 m metronidazole disc. Four of the strains were identified as B. fragilis, (sensu stricto) and one as B. distasonis. Further examination using the E Test (AB Biodisk Sweden) showed MICs of metronidazole ranging from 0-38 to O75mg/L—well within therapeutic levels. The most probable explanation for this apparent resistance found by the submitting laboratories was sub-optimal anaerobiosis. The 'B. fragilis group' are very aero tolerant and will grow in the presence of small amounts of oxygen. Metronidazole only becomes active when it is reduced via its nitro group accepting electrons from a low-redox-potential electron transport enzyme pyruvate:ferredoxin oxidoreductase (Edwards, 1979). If conditions are not sufficiently reduced ( < 430 mV) the drug will not become reduced to its active form. The end result is bacterial growth with either no zone or a very reduced zone to the metronidazole disc. Some credence was given to this explanation in a telephone conversation with the laboratory that submitted two 'resistant' isolates, who reported having reliability problems with their anaerobic chamber. As the submitting laboratories were widespread in their geographical location (London and the north of England) it is unlikely that a single factor (i.e. a common batch of faulty anaerobic gas mixture or metronidazole discs) contributed to these reports of false resistance. It is more likely that a series of local factors leading to incomplete anaerobiosis was responsible. Diagnostic laboratories identifying possible resistance to metronidazole with the B. fragilis group are advised to check this result by using a control strain of known sensitivity such as B.
'Institute of Microbiology. University of Ancona Medical School, 60131 Ancona. Italy; 'Department of Pathobiology. University of Washington. Seattle. Washington 98195. USA References Albritton. W. L. (1982). Infections due to Haemophilia species other than H. influenzae. Annual Review- of Microbiology 36, 199-216. Anderson, B., Albritton, W. L., Biddle, J. & Johnson, S. R. (1984). Common 0-lactamasespecifying plasmid in Haemophilia ducreyi and Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 25, 296-7. Brunton, J., Clare, D. 4 Meier, M. A. (1986a). Molecular epidemiology of antibiotic resistance plasmids of Haemophilia species and Neisseria gonorrhoeae. Reviews of Infectious Diseases 8, 713-24. Brunton, J., Meier, M., Ehrman, N.. Clare, D. & Almawy, R. (1986*). Origin of small 0-lactamasespecifying plasmids in Haemophilia species and Neisseria gonorrhoeae. Journal of Bacteriology 168, 374-9.
Brunton, J. L., Maclean, I., Ronald, A. R. & Albritton, W. L. (1979). Plasmid-mediated arapicillin resistance in Haemophilia ducreyi. Antimicrobial Agents and Chemotherapy 15, 294-9. Chen, S-T. & Clowes, R. C. (1987). Nuclcotide sequence comparisons of plasmids pHD131, pJBl, pFA3, and pFA7 and ^-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae. Journal of Bacteriology 169, 3124-30. de Graaff, J., Elwell, L. P. & Falkow, S. (1976). Molecular nature of two beta-lactamasespecifying plasmids isolated from Haemophilus influenzae type b. Journal of Bacteriology 126, 439-46. Green. M. J., Anderson, D. M., Norris, D. M. & Gubbinj, S. L. (1979). Antimicrobial resistance in Haemophilus species. New Zealand Medical Journal 90, 29. Groves, D. J. & Adeniyi-Jones, C. (1976). Ampicillin-resistant Haemophilus sp. Canadian Medical Association Journal 114, 407. Jones, R. N., Slepack, J. & Bigelow, J. (1976). Ampicillin-resistant Haemophilus paraphrophiha laryngo-epiglottitis. Journal of Clinical Microbiology 4, 405-7. Kauffman, C. A., Bergman, A. G. & Hertz, C. S. (1979). Antimicrobial resistance of Haemophilus species in patients with chronic bronchitis. American Review of Respiratory Diseases 120, 1382-5. KJlian, M. & Biberstein, E. L. (1984). Haemophilus. In Bergev's Manual of Systematic Bacteriology, Vol. I (Krieg, N. R. & Holt, J. G., Eds), p. 567. Williams & Wilkins, Baltimore, MD. Roberts, M. C. (1989). Gene transfer in the urogenital and respiratory tract. In Gene Transfer in the Environment (Levy, S. B. & Miller, R. V., Eds), pp. 347-75. McGraw-Hill, New York. Scheifele, D. W., Fussel, S. J. & Roberts, M. C. (1982). Characterization of ampiciUin-resistant Haemophilus parainfluenzae. Antimicrobial Agents and Chemotherapy 21, 734-9.
Artefactual resistance to metronidazole in anaerobes of the Bactermdes fragilis group / Amimicrob Chemolher 1992; 30: 553-554 Sir, The Anaerobe Reference Unit of the Public Health Laboratory Service receives isolates of anaerobic bacteria from laboratories situated throughout England and Wales. It is thus uniquely placed to monitor nationwide patterns and trends associated with anaerobic bacteria. The article by Cederbrant, Kahlmeter & Ljungh (1992) serves as a reminder of the importance of anaerobic conditions when testing susceptibilities to metronidazole. Despite its widespread use in prophylaxis and treatment of anaerobic infections for nearly 20
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 23, 2015
plasmid fragments and the N. gonorrhoeae probe and thus that the two plasmids were very closely related, if not identical. /}-Lactamase-producing H. haemolyticus and H. parahaenwlyticus, but not H. paraphrohaemolyticus have been reported previously (Bergman & Hertz, 1979; Green et al., 1979; Groves & Adeniyi-Jones, 1979). However, this is the first time that the location of the /Mactamase gene in a haemolytic /Mactamase producing Haemophilia sp. has been identified and shown to be on a 3-2 MDa /Mactamase plasmid. This plasmid is indistinguishable from the 3-2 MDa plasmid previously characterized in N. gonorrhoeae and is thus part of the family of small /Mactamase plasmids in the genus Neisseria and Haemophilia. As a result, it is tempting to speculate that the location of the /Mactamase gene in the previously-reported /Mactamaseproducing H. haemolyticus and H. parahaemolyticus strains may also have been on plasmids of the same family. Thus it would be interesting to screen commensal Haemophilia spp. to determine whether the small /Mactamase plasmids are present and whether these strains can act as reservoirs for this plasmid group. Acknowledgements. This work was supported in part by the CNR Target Project on Biotechnology and Bioinstrumentation grant 91.01251, to P.E.V. and by Public Health Service grant AI24136 to M.C.R. B. FACINELLI* R. TARSI' E. GIOVANETTT P. E. VARALDO* M. C. ROBERTS*
553
554
Correspondence fragilis NCTC 9343. Bonafide resistant strains should be referred to the Anaerobe Reference Unit for further study. J. S. BRAZIER VALERIE HALL B. I. DUERDEN PHLS Anaerobe Reference Unit, University Hospital of Wales. Heath Park. Cardiff CF4 4XW. UK References Cederbrant, G., Kahlmeter, G. & Ljungb, A. (1992). Proposed mechanism for metronidazole resistance in Helicobacter pylori. Journal of Antimicrobial Chemotherapy 29, 115-20. Edwards, D. I. (1979). Mechanism of antimicrobial action of metronidazole. Journal of Antimicrobial Chemotherapy 5, 499-502. Sebald, M., Reysset, G. & Breuil, J. (1990). What's new in 5-nitroimidazole resistance in the Bacteroides fragilis group? In Clinical and Molecular Aspects of Anaerobes (Bordello, S. P., Ed.), pp. 217-25. Wrightson Biomedkal Publications. The activity of ceflxinte against 715 urinary isolates of Enterobacteriaceae isolated from general practice and out-patients in twenty centres across the British Isles J Antimicrob Chemother 1992; 30: 554-556 Sir, Cefixime is an orally available cephem structurally related to some third generation cephalosporins and showing similar antibacterial activity (Kamimura el al., 1984; Bauernfeind, 1985). It is stable to a wide range of chromosomal and plasmid coded /?-lactamases and, hence, is active against Enterobacteriaceae resistant to ampitillin, cephalexin and cefaclor (Sanders, 1989). Therefore cefixime is likely to be of value in the therapy of some urinary tract infections in an out-patient setting. The objective of our study was to establish the in-vitro activity of cefixime against more than 700 Gram-negative urinary pathogens isolated in twenty centres across the United Kingdom with an emphasis on isolates from general practice and out-patients. Clinical laboratories were asked to collect consecutive and clinically significant nonrepeat isolates from general practice and outpatients. Each laboratory contributed 10 Escherichia coli and 50 other urinary tract pathogens (excluding Pseudomonas spp. and Enterococcus faecalis). A total of 715 strains were collected and sent to Southmead Hospital, Bristol for testing. MICs of cefixime,
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 23, 2015
years, resistance to this agent is still unusual, although plasmid-mcdiatcd resistance to the nitroimidazoles has been reported (Sebald, Reysset & Breuil, 1990). Recently an upward trend in the number of referrals of putative resistance to mctronidazolc among the Bacteroides fragilis group became apparent. In the space of less than one month, five supposedly metronidazole resistant strains of B. fragilis were submitted to the Unit for confirmation. Four laboratories, (two isolates were from one laboratory) reported isolates which showed greatly reduced sensitivity to a 5 fig metronidazole disc. The strains were isolates from a hip abscess, a blood culture, a wound swab (site unspecified), and two from high vaginal swabs. Upon subculture in the Anaerobe Reference Unit under controlled anaerobic conditions, all five strains proved to be fully susceptible on disc testing to a 5 m metronidazole disc. Four of the strains were identified as B. fragilis, (sensu stricto) and one as B. distasonis. Further examination using the E Test (AB Biodisk Sweden) showed MICs of metronidazole ranging from 0-38 to O75mg/L—well within therapeutic levels. The most probable explanation for this apparent resistance found by the submitting laboratories was sub-optimal anaerobiosis. The 'B. fragilis group' are very aero tolerant and will grow in the presence of small amounts of oxygen. Metronidazole only becomes active when it is reduced via its nitro group accepting electrons from a low-redox-potential electron transport enzyme pyruvate:ferredoxin oxidoreductase (Edwards, 1979). If conditions are not sufficiently reduced ( < 430 mV) the drug will not become reduced to its active form. The end result is bacterial growth with either no zone or a very reduced zone to the metronidazole disc. Some credence was given to this explanation in a telephone conversation with the laboratory that submitted two 'resistant' isolates, who reported having reliability problems with their anaerobic chamber. As the submitting laboratories were widespread in their geographical location (London and the north of England) it is unlikely that a single factor (i.e. a common batch of faulty anaerobic gas mixture or metronidazole discs) contributed to these reports of false resistance. It is more likely that a series of local factors leading to incomplete anaerobiosis was responsible. Diagnostic laboratories identifying possible resistance to metronidazole with the B. fragilis group are advised to check this result by using a control strain of known sensitivity such as B.
