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 University of Iowa Libraries/Serials Acquisitions on May 30, 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
110
Table. Susceptibility of two (trains of S. pneumoniae and three typical mutants to quinolones at 105 cfu
Strain
SPAR
cn»
TOS
—_
06 1-25
3-5 160 06 90 40
06
60 — 20 0-75
MIC (mg/L)*
0-5 10 06
1-25
0-7 0-8 07
from MIC determination using doubling dilutions. •Decimal dilutions.
the species to each agent (Table) (Cooper et al., 1990; Simor et al., 1990; Barry & Fuchs, 1991; Chin et al., 1991). Mutant selection was performed three times, and only for two strains of S. pneumonia* (NCTC 7465 and a clinical isolate) could mutants be selected with decreased susceptibility to quinolones. Increased MICs of the three quinolones were seen for the mutant S. pneumoniae (Table), but no cross resistance to erythromycin or penicillin was seen (data not shown). The frequency of mutation to resistance of all strains was ~ 10~ 7 -10~\ suggesting a mutation in a single gene. All the mutant 5. pneumoniae would be resistant to the recommended breakpoint concentration of ciprofloxacin (1 mg/L) for respiratory tract infections. The suggested breakpoint concentration of sparfloxacin, 2 mg/L (C. Leperlier, Rhone Poulenc D P Q would inhibit all the selected mutants, although the MIC of sparfloxacin for some mutants was only one doubling dilution below that concentration. This study has shown that spontaneous quinolone resistant mutants of S. pneumoniae can arise after one exposure to quinolones, and that if such strains arise during quinolone therapy the decreased susceptibility of such mutants may cause therapeutic failure. Clinical failures of quinolones have been previously reported for pneumococci (Lee et al., 1991). In addition this study also suggests that whilst resistant pneumococci are unlikely to be selected with sparfloxacin, strains previously exposed to ciprofloxacin will be less susceptible to the action of new fluoroquinolones. L. J. V. PIDDOCK YU FANG JIN Antimicrobial Agents Research Group, Department of Infection, University of Birmingham, Birmingham BIS 2TT, UK
References Barry, A. L. & Fuchs, P. C. (1991). In vitro activities of sparfloxacin, tosufloxacin, ciprofloxacin and fleroxacin. Antimicrobial Agents and Chemotherapy 35, 955-60. Chin, N.-X., Gu, J.-W., Zhang, Y.-X. & Neu, H. (1991). In vitro activity of sparfloxacin. Antimicrobial Agents and Chemotherapy 35, 567-71. Cooper, M. A., Andrews, J. M., Ashby, J. P., Matthews, R. S. & Wise, R. (1990). In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent Journal of Antimicrobial Chemotherapy 26, 667-76. Lee, B. L., Padula, A. M., Kimbrough, R. C , Jones, S. R., Chaisson, R. E., Mills, J. & Sandy, M. A. (1991). Infectious complication* with respiratory pathogens despite ciprofloxacin therapy. New England Journal of Medicine 325, 520. Simor, A. E., Fuller, S. A. & Low, D. E. (1990). Comparative in vitro activity of sparfloxacin, (CI978; AT-4140) and other antimicrobial agents against staphylococci, enterococci and respiratory tract pathogens. Antimicrobial Agents and Chemotherapy 34, 2283-6.
Faecal carriage of antUriotic-resfatarrt enterococci in hospitalized «"•! Don-bospttalin imUvMnab J Antimicrob Chemother 1992; 30: 110-2
Sir, Enterococci have emerged as important nosocomial pathogens. Among the few antibiotics useful in treating serious enterococcal infections are certain /Mactams (such as ampiciUin or amoxycillin), aminogrycosides (nicfa as gentamicin), and glycopeptides. However, enterococci resistant to ampiciUin (MIC > 16 mg/L) or highly resistant to gentamicin (MIC > 500 mg/L) are now prevalent in many
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 30, 2015
NCTC 7465 (M4) M4/C1 Clinical isolate (M5) M5/C1 M5/C3
Selecting concentration of aprofloxaan* (mg/L)
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 University of Iowa Libraries/Serials Acquisitions on May 30, 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
110
Table. Susceptibility of two (trains of S. pneumoniae and three typical mutants to quinolones at 105 cfu
Strain
SPAR
cn»
TOS
—_
06 1-25
3-5 160 06 90 40
06
60 — 20 0-75
MIC (mg/L)*
0-5 10 06
1-25
0-7 0-8 07
from MIC determination using doubling dilutions. •Decimal dilutions.
the species to each agent (Table) (Cooper et al., 1990; Simor et al., 1990; Barry & Fuchs, 1991; Chin et al., 1991). Mutant selection was performed three times, and only for two strains of S. pneumonia* (NCTC 7465 and a clinical isolate) could mutants be selected with decreased susceptibility to quinolones. Increased MICs of the three quinolones were seen for the mutant S. pneumoniae (Table), but no cross resistance to erythromycin or penicillin was seen (data not shown). The frequency of mutation to resistance of all strains was ~ 10~ 7 -10~\ suggesting a mutation in a single gene. All the mutant 5. pneumoniae would be resistant to the recommended breakpoint concentration of ciprofloxacin (1 mg/L) for respiratory tract infections. The suggested breakpoint concentration of sparfloxacin, 2 mg/L (C. Leperlier, Rhone Poulenc D P Q would inhibit all the selected mutants, although the MIC of sparfloxacin for some mutants was only one doubling dilution below that concentration. This study has shown that spontaneous quinolone resistant mutants of S. pneumoniae can arise after one exposure to quinolones, and that if such strains arise during quinolone therapy the decreased susceptibility of such mutants may cause therapeutic failure. Clinical failures of quinolones have been previously reported for pneumococci (Lee et al., 1991). In addition this study also suggests that whilst resistant pneumococci are unlikely to be selected with sparfloxacin, strains previously exposed to ciprofloxacin will be less susceptible to the action of new fluoroquinolones. L. J. V. PIDDOCK YU FANG JIN Antimicrobial Agents Research Group, Department of Infection, University of Birmingham, Birmingham BIS 2TT, UK
References Barry, A. L. & Fuchs, P. C. (1991). In vitro activities of sparfloxacin, tosufloxacin, ciprofloxacin and fleroxacin. Antimicrobial Agents and Chemotherapy 35, 955-60. Chin, N.-X., Gu, J.-W., Zhang, Y.-X. & Neu, H. (1991). In vitro activity of sparfloxacin. Antimicrobial Agents and Chemotherapy 35, 567-71. Cooper, M. A., Andrews, J. M., Ashby, J. P., Matthews, R. S. & Wise, R. (1990). In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent Journal of Antimicrobial Chemotherapy 26, 667-76. Lee, B. L., Padula, A. M., Kimbrough, R. C , Jones, S. R., Chaisson, R. E., Mills, J. & Sandy, M. A. (1991). Infectious complication* with respiratory pathogens despite ciprofloxacin therapy. New England Journal of Medicine 325, 520. Simor, A. E., Fuller, S. A. & Low, D. E. (1990). Comparative in vitro activity of sparfloxacin, (CI978; AT-4140) and other antimicrobial agents against staphylococci, enterococci and respiratory tract pathogens. Antimicrobial Agents and Chemotherapy 34, 2283-6.
Faecal carriage of antUriotic-resfatarrt enterococci in hospitalized «"•! Don-bospttalin imUvMnab J Antimicrob Chemother 1992; 30: 110-2
Sir, Enterococci have emerged as important nosocomial pathogens. Among the few antibiotics useful in treating serious enterococcal infections are certain /Mactams (such as ampiciUin or amoxycillin), aminogrycosides (nicfa as gentamicin), and glycopeptides. However, enterococci resistant to ampiciUin (MIC > 16 mg/L) or highly resistant to gentamicin (MIC > 500 mg/L) are now prevalent in many
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 30, 2015
NCTC 7465 (M4) M4/C1 Clinical isolate (M5) M5/C1 M5/C3
Selecting concentration of aprofloxaan* (mg/L)
