108
Correspondence
Woodford, N., Johnson, A. P. & George, R. C. (1991). Detection of glycopeptide resistance in dinical isolates of Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 28, 483-6. Working Party of the British Society for Antimicrobial Chemotherapy (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy TJ, Suppl. D.
Activity of amoxycflHn/davulaiiic add against EscktricMa coB in who—correction J Antimicrob Chemother 1992; 30: 108
SmilhKUne Beecham Pharmaceuticals. Brockham Park, Betchworth. Surrey RH3 TAJ, UK References Aronoff, S. C , Jacobs, M. R-, Johenning, S. A Yamabe, S. (1984). Comparative activities of the 0-lactamase inhibitors YTR830, sodium davolanate, and sulbactam combined with amoxkallm or ampidllin. Antimicrobial Agents and Chemotherapy 26, 580-2. Cherubin, C. E., Eng, R. H. K., Smith, S. M. & Tan, E. N. (1991). An in-vitro and in-vivo comparison of the activity of /Mactamase inhibitor combinations with imipenem and cephatosporins against Escherichia coli producing TEM-1 or TEM-2 0-lactamase. Journal of Antimicrobial Chemotherapy 28, 61-70. Gisby, J. &. Beak, A. S. (1988). Comparative efficacies of amoxknllin-dflvulanic acid and amptcillmsulbactam against experimental Bacteroides fragilis-Escherichia coli mixed infections. Antimicrobial Agents and Chemotherapy 32, 1830-3. Reading, C. & Farmer, T. (1984). The inhibition of periplasmic ^-lactamase in E. coli by davulank acid and other 0-lactamase inhibitors. Postgraduate Medicine 1984, 163-8.
Classification of plasmM-encoded trimethopriin resistance genes
/ Antimicrob Chemother 1992; 30: 108-9 Sir, The classification of bacterial resistances genes is frequently a contentious issue and can lead to considerable confusion, as has become evident with the plasmid-encoded /Mactamases in Gram-negative bacteria. Plasmid-mediated trimethoprim resistance results from the production of an additional plasmid-encoded, trimethoprim-insuscepu'bte, dihydrofolate reductase (DHFR). At least IS such enzymes have been reported, and we are aware of further, potentially different, enzymes whose properties are being elucidated currently. It is important that general agreement is reached on the way in which these enzymes are to be designated and classified. The original demonstration that two different types of plasmid-encoded DHFR existed (types I and II), followed by the sub-
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 21, 2015
Sir, My attention has been drawn to a paper in the Journal by Cherubin and colleagues (Chcrubin et al., 1991) concerning the in-vitro and in-vivo activity of /Mactamase inhibitor compounds ('BICs') against strains of Escherichia coli producing TEM /7-lactamases. These workers have, in the course of their discussion, cited one of our papers on in-vivo activity (Gisby & Beak, 1988) but have drawn an incorrect conclusion from the data. In seeking to support their contention that the empirical use of BICs would be questionable in clinical sepsis involving large numbers of Gram-negative enteric bacteria, particularly E. coll, Cherubin and colleagues quote from our work, that 'a potent /Mactamaseproducing strain of E. coli reduced the activity of amoxycillin/clavulanic acid and ampidllin/ sulbactam in the abscess to the extent that an otherwise susceptible Bacteroides fragilis strain could survive...'. In this model, which incidentally was a subcutaneous abscess and not an abdominal one as quoted, we studied mixed infections caused by B. fragilis combined with strains of E. colt which produced either normal or elevated quantities of TEM /Mactamase. We found that treatment with amoxycillin/clavulanic acid, at doses designed to produce clinically-achievable concentrations in the infected animals, reduced counts of both aerobe and anaerobe to undetectable levels in all the mixed infections and thus prevented abscess formation. On the other hand, the efficacy of ampidllin/ sulbactam was markedly reduced in infections involving the more potent ^-lactamaseproducing strain of E. coli, reflecting the relatively poor /Mactamase inhibitory activity of sulbactam against TEM /Mactamase (Aronoff et al., 1984; Reading & Farmer, 1984). There was no evidence to suggest that the efficacy of amoxycillin/clavulanic acid was reduced
against either aerobe or anaerobe in the presence of the /J-lactamase-hyperproducing strain, and to infer otherwise from our data would be erroneous. ANGELA S. BEALE
Correspondence
cation will occur and that unnecessary confusion will be avoided. K. J. TOWNER' S. G. B. AMYES» H.-K. YOUNG* •Department of Microbiology A PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH; 'Department of Medical Microbiology, The Medical School. University of Edinburgh, Teriot Place. Edinburgh EH8 9AG; 'Department of Biological Sciences, University of Dundee. Dundee DD1 4HN, UK Reference Amyes, S. G. B., Towner, K. J. & Young, H.-K. (1992). Ckuification of plasmid-encoded dihydrofolate rcdoctaaes conferring trimetboprim resistance. Journal of Medical Microbiology 36, 1-3.
Selection of qutnolooe-fesiitaiit mutants of HaemopUbu imftumiat and Streptococcus J Antimkrob Chemother 1992; 30: 109-10 Sir, Recently, new fluorinated quinolones such as sparfloxacin and tosufloxacin have been developed with improved in-vitro activity against Gram-positive bacteria compared to earlier quinolones such as caprofloxacin (Cooper et al., 1990; Barry & Fuchs, 1991; Chin et al., 1991). It is suggested that such agents may be useful for the treatment of respiratory tract infections (Cooper et al., 1990; Simor, Fuller & Low, 1990). In this study five strains of Haemophiha inftuenzae (NCTC 10478, type e; NCTC 8466, type a; NCTC 8469, type c; and two type b recent clinical isolates) and five strains of Streptococcus pneumonlae (NCTC 7466, NCTC 7465 and three recent clinical isolates) were each exposed to 2 mg/L, and three, five and ten times their minimum inhibitory concentration (MIC) of sparfloxacin and ciprofloxacin. The liquid media for the preparation of the inocula was 'Iso-Sensitest' broth (Unipath) plus 1% supplement C (Difco) and the solid media for mutant selection and determination of susceptibility was 'Iso-Sensitest' agar supplemented with 5% defibrinated horse blood and 20 mg/L nicotinamide adenine dinudeotide (NAD; Sigma). All cultures were incubated at 37°C for 24 h (4856 h for mutant selection) in an atmosphere containing 5% carbon dioxide. Each strain had the typical susceptibility of
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 21, 2015
sequent recognition of types III and IV, was made entirely on the basis of biochemical properties. These distinctions have been supported by DNA sequence analysis which has revealed that there are considerable differences between the genes encoding these enzymes. Further justifiable sub-divisions within types I and II have been made on the basis of both sequence analysis and biochemical properties. Unfortunately, as with the plasmid-encoded /Mactamases, the recognition and designation of 'new' DHFR types is now resulting in increased confusion. In several cases, 'new' enzymes with ' slightly different biochemical properties have been shown by sequence analysis to be very similar to enzymes designated previously, while some enzymes allocated to sub-types on the basis of biochemical properties have been shown by sequence analysis to be completely unrelated to the suspected parent enzyme. While it seems foolhardy to change radically a system that has been in operation for over 15 years, clearly some rationalization is required. However, it is extremely difficult to alter classifications that already exist in the literature, and it seems logical, therefore, that a system should be operated which takes account of what has gone before. We therefore wish to draw the attention of your readers to our proposal (Amyes, Towner & Young, 1992) that all existing published DHFR types should remain as designated, including their subtypes, but that in future, no sub-types should be used and each newly discovered enzyme, should be allocated a new type only when it has been shown to be completely different from all others by both biochemical properties and sequence analysis. To aid in this process, we have established a register of all plasmid-encoded DHFRs so that the next available type number can be allocated to new enzymes. This will overcome the possibility of the same number being inadvertantly used by different investigators for different enzymes or genes. Gene probes for each of the existing recognized DHFRs can be supplied, so that investigators can ensure that they are not duplicating previous studies. In return, we would request that when an investigator is ready to confirm the identification of a new gene, that they contact one of us for the next available type number and donate a copy of the new gene, or the appropriate probe, for future distribution. By this method, although the type numbers will less and less reflect the biochemical properties, we hope that no dupli-
109
Correspondence
Woodford, N., Johnson, A. P. & George, R. C. (1991). Detection of glycopeptide resistance in dinical isolates of Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 28, 483-6. Working Party of the British Society for Antimicrobial Chemotherapy (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy TJ, Suppl. D.
Activity of amoxycflHn/davulaiiic add against EscktricMa coB in who—correction J Antimicrob Chemother 1992; 30: 108
SmilhKUne Beecham Pharmaceuticals. Brockham Park, Betchworth. Surrey RH3 TAJ, UK References Aronoff, S. C , Jacobs, M. R-, Johenning, S. A Yamabe, S. (1984). Comparative activities of the 0-lactamase inhibitors YTR830, sodium davolanate, and sulbactam combined with amoxkallm or ampidllin. Antimicrobial Agents and Chemotherapy 26, 580-2. Cherubin, C. E., Eng, R. H. K., Smith, S. M. & Tan, E. N. (1991). An in-vitro and in-vivo comparison of the activity of /Mactamase inhibitor combinations with imipenem and cephatosporins against Escherichia coli producing TEM-1 or TEM-2 0-lactamase. Journal of Antimicrobial Chemotherapy 28, 61-70. Gisby, J. &. Beak, A. S. (1988). Comparative efficacies of amoxknllin-dflvulanic acid and amptcillmsulbactam against experimental Bacteroides fragilis-Escherichia coli mixed infections. Antimicrobial Agents and Chemotherapy 32, 1830-3. Reading, C. & Farmer, T. (1984). The inhibition of periplasmic ^-lactamase in E. coli by davulank acid and other 0-lactamase inhibitors. Postgraduate Medicine 1984, 163-8.
Classification of plasmM-encoded trimethopriin resistance genes
/ Antimicrob Chemother 1992; 30: 108-9 Sir, The classification of bacterial resistances genes is frequently a contentious issue and can lead to considerable confusion, as has become evident with the plasmid-encoded /Mactamases in Gram-negative bacteria. Plasmid-mediated trimethoprim resistance results from the production of an additional plasmid-encoded, trimethoprim-insuscepu'bte, dihydrofolate reductase (DHFR). At least IS such enzymes have been reported, and we are aware of further, potentially different, enzymes whose properties are being elucidated currently. It is important that general agreement is reached on the way in which these enzymes are to be designated and classified. The original demonstration that two different types of plasmid-encoded DHFR existed (types I and II), followed by the sub-
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 21, 2015
Sir, My attention has been drawn to a paper in the Journal by Cherubin and colleagues (Chcrubin et al., 1991) concerning the in-vitro and in-vivo activity of /Mactamase inhibitor compounds ('BICs') against strains of Escherichia coli producing TEM /7-lactamases. These workers have, in the course of their discussion, cited one of our papers on in-vivo activity (Gisby & Beak, 1988) but have drawn an incorrect conclusion from the data. In seeking to support their contention that the empirical use of BICs would be questionable in clinical sepsis involving large numbers of Gram-negative enteric bacteria, particularly E. coll, Cherubin and colleagues quote from our work, that 'a potent /Mactamaseproducing strain of E. coli reduced the activity of amoxycillin/clavulanic acid and ampidllin/ sulbactam in the abscess to the extent that an otherwise susceptible Bacteroides fragilis strain could survive...'. In this model, which incidentally was a subcutaneous abscess and not an abdominal one as quoted, we studied mixed infections caused by B. fragilis combined with strains of E. colt which produced either normal or elevated quantities of TEM /Mactamase. We found that treatment with amoxycillin/clavulanic acid, at doses designed to produce clinically-achievable concentrations in the infected animals, reduced counts of both aerobe and anaerobe to undetectable levels in all the mixed infections and thus prevented abscess formation. On the other hand, the efficacy of ampidllin/ sulbactam was markedly reduced in infections involving the more potent ^-lactamaseproducing strain of E. coli, reflecting the relatively poor /Mactamase inhibitory activity of sulbactam against TEM /Mactamase (Aronoff et al., 1984; Reading & Farmer, 1984). There was no evidence to suggest that the efficacy of amoxycillin/clavulanic acid was reduced
against either aerobe or anaerobe in the presence of the /J-lactamase-hyperproducing strain, and to infer otherwise from our data would be erroneous. ANGELA S. BEALE
Correspondence
cation will occur and that unnecessary confusion will be avoided. K. J. TOWNER' S. G. B. AMYES» H.-K. YOUNG* •Department of Microbiology A PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH; 'Department of Medical Microbiology, The Medical School. University of Edinburgh, Teriot Place. Edinburgh EH8 9AG; 'Department of Biological Sciences, University of Dundee. Dundee DD1 4HN, UK Reference Amyes, S. G. B., Towner, K. J. & Young, H.-K. (1992). Ckuification of plasmid-encoded dihydrofolate rcdoctaaes conferring trimetboprim resistance. Journal of Medical Microbiology 36, 1-3.
Selection of qutnolooe-fesiitaiit mutants of HaemopUbu imftumiat and Streptococcus J Antimkrob Chemother 1992; 30: 109-10 Sir, Recently, new fluorinated quinolones such as sparfloxacin and tosufloxacin have been developed with improved in-vitro activity against Gram-positive bacteria compared to earlier quinolones such as caprofloxacin (Cooper et al., 1990; Barry & Fuchs, 1991; Chin et al., 1991). It is suggested that such agents may be useful for the treatment of respiratory tract infections (Cooper et al., 1990; Simor, Fuller & Low, 1990). In this study five strains of Haemophiha inftuenzae (NCTC 10478, type e; NCTC 8466, type a; NCTC 8469, type c; and two type b recent clinical isolates) and five strains of Streptococcus pneumonlae (NCTC 7466, NCTC 7465 and three recent clinical isolates) were each exposed to 2 mg/L, and three, five and ten times their minimum inhibitory concentration (MIC) of sparfloxacin and ciprofloxacin. The liquid media for the preparation of the inocula was 'Iso-Sensitest' broth (Unipath) plus 1% supplement C (Difco) and the solid media for mutant selection and determination of susceptibility was 'Iso-Sensitest' agar supplemented with 5% defibrinated horse blood and 20 mg/L nicotinamide adenine dinudeotide (NAD; Sigma). All cultures were incubated at 37°C for 24 h (4856 h for mutant selection) in an atmosphere containing 5% carbon dioxide. Each strain had the typical susceptibility of
Downloaded from http://jac.oxfordjournals.org/ at Carleton University on June 21, 2015
sequent recognition of types III and IV, was made entirely on the basis of biochemical properties. These distinctions have been supported by DNA sequence analysis which has revealed that there are considerable differences between the genes encoding these enzymes. Further justifiable sub-divisions within types I and II have been made on the basis of both sequence analysis and biochemical properties. Unfortunately, as with the plasmid-encoded /Mactamases, the recognition and designation of 'new' DHFR types is now resulting in increased confusion. In several cases, 'new' enzymes with ' slightly different biochemical properties have been shown by sequence analysis to be very similar to enzymes designated previously, while some enzymes allocated to sub-types on the basis of biochemical properties have been shown by sequence analysis to be completely unrelated to the suspected parent enzyme. While it seems foolhardy to change radically a system that has been in operation for over 15 years, clearly some rationalization is required. However, it is extremely difficult to alter classifications that already exist in the literature, and it seems logical, therefore, that a system should be operated which takes account of what has gone before. We therefore wish to draw the attention of your readers to our proposal (Amyes, Towner & Young, 1992) that all existing published DHFR types should remain as designated, including their subtypes, but that in future, no sub-types should be used and each newly discovered enzyme, should be allocated a new type only when it has been shown to be completely different from all others by both biochemical properties and sequence analysis. To aid in this process, we have established a register of all plasmid-encoded DHFRs so that the next available type number can be allocated to new enzymes. This will overcome the possibility of the same number being inadvertantly used by different investigators for different enzymes or genes. Gene probes for each of the existing recognized DHFRs can be supplied, so that investigators can ensure that they are not duplicating previous studies. In return, we would request that when an investigator is ready to confirm the identification of a new gene, that they contact one of us for the next available type number and donate a copy of the new gene, or the appropriate probe, for future distribution. By this method, although the type numbers will less and less reflect the biochemical properties, we hope that no dupli-
109
