Correspondence Table 1. Quinolone resistance mutations in GyrA and ParC of ciprofloxacin-resistant clinical isolates of A. baumannii
A. baumannii. Furthermore, it is also possible that other mutations at other locations within gyrA, parC, or in other genes may also contribute to the modulation of the MIC level since these mutations did not entirely explain resistance.
Amino acid change
Strains 8 9 52 55 60 71 72 78 85
Ciprofloxacin MIC (mg/L)
GyrA
ParC
4 64 8 32 64 32 32 8 4
Gly-81 ! Cys Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu Ser-83 ! Leu
no mutation Gly-78 ! Cys no mutation no mutation Glu-84 ! Lys no mutation no mutation no mutation no mutation
Acknowledgements
81 conferred a ciprofloxacin-resistance level of 4 mg/L where glycine had been substituted by cysteine with no mutation concurrent at codon Ser-83. This mutation has already been described in Escherichia coli.5 The GyrA mutations do not explain why isolates with the same mutation have different MICs of ciprofloxacin. This variation in MICs could be explained by a mutation in the parC gene. In A. baumannii, topoisomerase IV is a target of quinolones and mutation at residues Ser-80 and Glu-84 of ParC contribute to decreased fluoroquinolone susceptibility.3 In the present study, only a change at residue Glu-84 was observed. This study showed that a double mutation in both gyrA and parC genes is needed to acquire a high-level resistance to ciprofloxacin and this is similar to previously published data.2,3 In contrast, in E. coli a double mutation affecting Ser-83 of GyrA and Ser-80 of ParC leads only to a moderate level of resistance to ciprofloxacin, whereas three or four mutations in both gyrA and parC genes are required to obtain high-level resistance. This difference is probably because of the decreased permeability of A. baumannii to quinolones.6 Moreover, no parC mutation has been found without the presence of mutation in the gyrA gene, suggesting that parC could be a secondary target for quinolones. In A. baumannii, restriction fragment length polymorphism was used to detect mutations at codon 83 in gyrA or at codon 80 in parC.2,3 The presence or absence of mutations in these genes at these particular codons can be determined by the digestion of PCR products with HinfI. HinfI restriction of the PCR products of gyrA from isolate 8 (MIC = 4 mg/L) and a ciprofloxacin-susceptible strain yielded two fragments of 291 bp and 52 bp, indicating that the HinfI site was intact. For the rest of the isolates the restriction failed to produce restriction fragments, indicating the loss of the HinfI site at the codons for amino acids Asp-82 – Ser-83, and suggesting that mutation either at codon Ser-82 or Ser-83 had occurred. HinfI restriction of PCR products of parC produced two DNA fragments of 144 bp and 53 bp, respectively, in all isolates, indicating that the HinfI restriction site at the codons for amino acids Asp-79 –Ser-80 was intact, and suggesting the absence of mutation at codon Ser-80. In conclusion, we showed that mutations in the amino acids corresponding to Gly-81 and Gly-78 in GyrA and ParC, respectively, contribute to decreased ciprofloxacin susceptibility in
We gratefully acknowledge the University of Edinburgh for supporting this work.
References 1. Acar, J. F., O’Brien, T. F., Goldstein, F. W. et al. (1993). The epidemiology of bacterial resistance to quinolones. Drugs 45, Suppl. 3, 24–8. 2. Vila, J., Ruiz, J., Goni, P. et al. (1995). Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 39, 1201– 3. 3. Vila, J., Ruiz, J., Goni, P. et al. (1997). Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 39, 757– 62. 4. British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1– 50. 5. Yoshida, H., Bogaki, M., Nakamura, M. et al. (1990). Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrobial Agents and Chemotherapy 34, 1271–2. 6. Sato, K. & Nakae, T. (1991). Outer membrane permeability of Acinetobacter calcoaceticus and its implication in antibiotic resistance. Journal of Antimicrobial Chemotherapy 28, 35–45.
Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkh382 Advance Access publication 28 July 2004
Susceptibility of capsular Staphylococcus aureus strains to some antibiotics, triclosan and cationic biocides Paul Seaman1, Martin Day1*, Dietmar Ochs3
A. Denver Russell2 and
1
Cardiff School of Biosciences and 2Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3TL, UK; 3 Ciba Spezialita¨tenchemie Grenzach GmbH, Postfach 1266, D-79630 Grenzach-Wyhlen, Germany Keywords: S. aureus, capsule polysaccharides, antimicrobial susceptibility *Corresponding author. Tel: +44-29-20875768; Fax: +44-29-20-874305; E-mail:
[email protected] Sir, Approximately 90% of Staphylococcus aureus isolates produce one of 11 serotypes of capsular polysaccharides. Serotypes CP5 and CP8 account for 25% and 50%, respectively, of isolates recovered from humans, offering support for their
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Correspondence Table 1. Antibiotic susceptibility of the three S. aureus capsule strains alongside the control strain NCTC 6571 for which the antibiogram is known Antibiotic susceptibility Strain
PEN
OXA
VAN
MUP
CHL
TET
ERY
GEN
FD
AMP
CEC
CRO
RIF
TEC
CP5 CP8 CP6571
R R R S
S S S S
S S S S
S S S S
S S S S
S S S S
S S R S
S S S S
S S S S
R R R S
R R R S
S S S S
S S S S
S S S S
R, resistant; S, susceptible; PEN, penicillin; OXA, oxacillin; VAN, vancomycin; MUP, mupirocin; CHL, chloramphenicol; TET, tetracycline; ERY, erythromycin; GEN, gentamicin; FD, fusidic acid; AMP, ampicillin; CEC, cefaclor; CRO, ceftriaxone; RIF, rifampicin; TEC, teicoplanin.
pathogenic significance.1 The importance and relevance of these capsule types is confirmed by the development of a conjugate vaccine, StaphVAX that includes type 5 and 8 capsule polysaccharides.2 CP5 and CP8 serotypes are considered as microcapsules because they are much smaller than those produced by the mucoid serotypes 1 and 2. The microcapsules of CP5 and CP8 are extracellular, uronic acid-containing polysaccharides that are too small to be visualized by negative stains such as India ink. These two capsules are very similar and differ only in the position of O-acetyl groups and the linkages between the amino sugars. The function of CP5 and CP8 in S. aureus virulence has been investigated in great depth, especially with regard to their role in impeding phagocytosis.1 However, few authors have reported upon the potential for a capsule polysaccharide to present a permeability barrier to antimicrobial agents. Gram-positive bacteria possess a cell wall that is usually permeable and does not limit the incursion of antimicrobials. However, resistance through reduced penetration has been shown to occur, for example vancomycin-intermediate resistant S. aureus (VISA) strains produce a distinctly thickened cell wall.3 Furthermore, in 1984 Kolawole4 discussed the effect of a mucoid capsule upon disinfectant and antiseptic susceptibility in S. aureus. He concluded that the thick capsule, as associated with serotypes CP1 and CP2, does provide a permeability barrier to common biocides but fell short of testing the more common and clinically important serotypes. We wish
to report how CP5 and CP8 microcapsules affect the susceptibility of S. aureus to several antibiotics and three widely used biocides: triclosan, chlorhexidine gluconate and cetylpyridinium chloride. A range of 14 antibiotic discs were purchased from Oxoid (Basingstoke, UK) and used to analyse S. aureus Reynolds and two isogenic capsule mutants. Antibiotic susceptibility was established as per the BSAC standardized disc susceptibility testing methodology5 (Table 1). The MICs of nine of these clinically relevant antibiotics were elucidated by Etest strips (AB Biodisk, Sweden) according to the manufacturer’s recommendations. MICs for the three biocides were calculated using Iso-Sensitest agar (Oxoid, Basingstoke, UK), multipoint inoculator (Denley; Mast Diagnostics, Bootle, UK) and incubation in air at 378C for 18 –20 h, in accordance with BSAC guidelines.6 Triclosan (Irgasan DP300) was a gift from Ciba Speciality Chemicals; chlorhexidine gluconate and cetylpyridinium chloride were purchased from ICN Biomedicals Inc. (Ohio, USA). Of the three S. aureus Reynolds strains, one was wild-type, expressing a serotype CP5 capsule, one a mutant expressing CP8 and the third a second mutant, lacking a capsule (CP –).7 The acapsular Reynolds strain was constructed by replacing the serotype-specific capsule genes cap5HIJK on the bacterial chromosome with an erm(B) gene, conferring erythromycin resistance. S. aureus NCTC 6571 (Oxford) was included alongside the capsule strains as a control. The MICs were defined as the lowest concentration
Table 2. MICs for S. aureus Reynolds expressing either capsule serotype CP5, CP8 or CP– (acapsular) MIC (mg/L) Strain
b-Lactamase
PENa
OXAa
VANa
MUPa
CHLa
TETa
ERYa
GENa
FDa
Tricb
CHXb
CPCb
CP5 CP8 CP – 6571
+ + +
16 16 16 0.023
0.75 0.75 0.75 0.125
1.0 1.0 1.5 1.0
0.125 0.125 0.125 0.19
6 6 4 4
0.28 0.38 0.5 0.064
0.38 0.38 >256 0.19
0.25 0.25 0.19 0.19
0.064 0.064 0.064 0.094
0.13 0.13 0.13 0.13
2.0 2.0 2.0 2.0
2.0 2.0 2.0 2.0
a
Calculated by Etest (AB Biodisk, Sweden). Calculated in accordance with BSAC guidelines. PEN, penicillin; OXA, oxacillin; VAN, vancomycin; MUP, mupirocin; CHL, chloramphenicol; TET, tetracycline; ERY, erythromycin; GEN, gentamicin; FD, fusidic acid; Tric, triclosan; CHX, chlorhexidine gluconate; CPC, cetylpyridinium chloride. b
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Correspondence of antimicrobial with which there was no visible growth of the organism and are shown in Table 2, along with the Etest data. In addition, all strains were investigated for b-lactamase production by nitrocefin stick (Oxoid, Basingstoke, UK; Table 2). The antibiogram for NCTC 6571, as deduced by antibiotic disc susceptibility testing, was as expected. No difference in antibiotic susceptibility was observed between the capsule strains other than for erythromycin, resistance to which was found in CP–. This was as expected due to the construction of the CP– strain by disruption of the serotype-specific capsule genes with an erm(B) gene. The nitrocefin test implies that penicillin and ampicillin resistance is conferred by b-lactamase production in S. aureus Reynolds. MICs for NCTC 6571 were within plus or minus one two-fold dilution of the expected MIC.6 The capsule strains all demonstrated MICs for each antimicrobial within plus or minus one two-fold dilution of each other. The only exception to this was—once again—erythromycin, confirming the results of the susceptibility disc test. From this we deduced that there is no significant difference between the antibiotic and biocide susceptibilities of the strains investigated herein. Consequently, we conclude that the capsule polysaccharide serotypes CP5 and CP8 do not present a permeability barrier to the antibiotics used in this investigation, or to triclosan, chlorhexidine gluconate or cetylpyridinium chloride. These results indicate that whereas S. aureus capsule polysaccharides are implicated in virulence, they are not involved in conferring reduced antibiotic or biocide susceptibility. Clinical implications are pertinent through the choice of clinically relevant antibiotics and biocides regularly used in the hospital setting.
Acknowledgements This work was supported by Ciba Speciality Chemicals Inc. We are grateful to Dr Jean Lee of the Channing Laboratory, Harvard University, Boston, MA, USA, who supplied the S. aureus Reynolds strains.
References 1. O’Riordan, K. & Lee, J. C. (2004). Staphylococcus aureus capsular polysaccharides. Clinical Microbiology Reviews 17, 218–34. 2. Fattom, A. I., Horwith, G., Fuller, S. et al. (2004). Development of StaphVAXe, a polysaccharide conjugate vaccine against S. aureus infection: from the lab bench to phase III clinical trials. Vaccine 22, 880–7. 3. Lambert, P. A. (2002). Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. Journal of Applied Microbiology 92, 46S –54S. 4. Kolawole, D. O. (1984). Resistance mechanisms of mucoidgrown Staphylococcus aureus to the antibacterial action of some disinfectants and antiseptics. FEMS Microbiology Letters 25, 205–9. 5. Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43– 57. 6. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16. 7. Nilsson, I. M., Lee, J. C., Bremell, T. et al. (1997). The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis. Infection and Immunity 65, 4216– 21.
Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkh394 Advance Access publication 4 August 2004
BSAC Respiratory Resistance Surveillance Programme (2002– 2003): comparative susceptibility of Streptococcus pneumoniae, cultured from patients in Great Britain and Ireland with communityacquired lower respiratory tract infection, to gemifloxacin David Felmingham1*, Tillotson2 1
Jemma Shackcloth1 and Glenn
GR Micro Ltd, London, UK; Waltham, MA, USA
2
Oscient Pharmaceuticals,
Keywords: S. pneumoniae, RTIs, antibacterials, resistance *Corresponding author. Tel: +44-207-3887320; Fax: +44-207-3887324; E-mail:
[email protected]
Sir, Streptococcus pneumoniae is an important pathogen of the respiratory tract where it may be responsible for a wide range of infections including community-acquired pneumonia, acute exacerbations of chronic bronchitis and obstructive airways disease, sinusitis and otitis media.1 Therapeutic choice for infections which are likely to include S. pneumoniae among the causative bacteria is increasingly complicated by resistance to a range of otherwise suitable antimicrobials with the prevalence of resistance reaching alarming proportions in the Far East region, the USA and some countries within Europe.2 Global, national and local surveillance programmes are essential aids in defining rates and the evolution of antimicrobial resistance.3 The BSAC Respiratory Resistance Surveillance Programme began in 1999 and exists to provide long-term, large-scale antimicrobial susceptibility surveillance of the major pathogens causing community-acquired lower respiratory tract infections in Great Britain and Ireland, including S. pneumoniae.3 Gemifloxacin is a novel, fluorinated, naphthyridone antibacterial characterized by excellent activity against Gram-negative species typical of most fluoroquinolones, whilst possessing enhanced potency against Gram-positive bacterial pathogens, most notably S. pneumoniae, which exceeds that of other currently licensed members of this class of antimicrobials.4 During the 2002 –2003 ‘cold season’, as part of the BSAC Resistance Surveillance Programme, the susceptibility of 772 isolates of S. pneumoniae to various antimicrobials, including gemifloxacin, was determined using BSAC procedures and interpretation of results.3 The results are presented in Table 1. Non-susceptibility to penicillin (intermediate, MIC 0.12 – _ 2 mg/L, 0.3%) was 1 mg/L, 8.3% + high-level resistance, MIC > found in a total of 8.6% of the isolates of S. pneumoniae. Erythromycin resistance was found in 10% of the isolates of S. pneumoniae with 4.7% resistant to clindamycin suggesting
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