Treating Complicated Skin & Diabetic Foot Infections: MRSA & the Role of Daptomycin Benjamin A. Lipsky, MD, FACP, FIDSA, FRCP Professor of Medicine, University of Washington Director, Primary Care Clinic & Antibiotic Research VA Puget Sound Health Care System, Seattle, Washington, USA
Overview of Discussion Skin and skin structure (soft tissue) infection Complicated (cssti); Acute bacterial skin/skin structure infection (ABSSSI) Diabetic foot infections (DFI) The importance of MRSA in cssti/dfi Approach to antibiotic therapy of cssti/dfi Empiric vs definitive therapy Oral vs parenteral agents The role of daptomycin Characteristics of the agent Data on treating diabetic foot infections
Which of the Following is Not a Complicated Skin and Soft Tissue Infections 1. Deep subcutaneous abscess 2. Infected ulcer 3. Cellulitis 4. Polymicrobial (GNR, anaerobic) infection 5. Infected burn wound
FDA Definitions: Skin & Skin Structure Infections Uncomplicated: minor, superficial simple abscess impetiginous lesions furuncles cellulitis Complicated: generally involve deeper soft tissues or require significant surgical intervention infected ulcers major abscesses burns underlying diseases superficial infection in sites where GNR or anaerobes likely pathogens US FDA Guidance for Industry (draft) July 1998
FDA: Acute Bacterial Skin and Skin Structure Infections (ABSSSI) Hallmark of infection definition is minimum surface area of redness, edema, and/or induration (i.e., 75 cm 2 of cellulitis) Patients with various complex infections, such as diabetic foot infection, should not be enrolled in ABSSSI trials Treatment regimens for these infections - Usually more complex than for ABSSSI trials - Interested sponsors must consult with FDA Draft guidance August 2010 fda.gov/drugs/guidancecomplianceregulatoryinformation/guidances/default.htm
Overview of cssti Point prevalence survey of antibiotic use in 20 European hospitals in 2006, SSTI 2 nd commonest indication for antibiotics Most common pathogens are aerobic GPC, Staph. aureus > β-hemolytic streptococci; polymicrobial in specific situations MRSA isolates more common past decade >10% of isolates in most European countries 59% of isolates in study in USA (2006) 30-50% reported in DFIs
Pathogens in SSTI in Hospitalized Diabetics Microbial Pathogen Foot (%) Other Gram-positive bacteria (n=2220) (n=810) Methicillin-sensi Staph aureus 13.2 16.4 Methicillin-resistant S. aureus 7.4 9.6 Coag.-negative staphylococci 5.5 5.9 Streptococcus spp. 7.2 4.3 Enterococcus spp. 2.1 2.2 Gram-negative bacteria Pseudomonas aeruginosa 1.4 2.0 Enterobacter spp. 0.4 0.5 Other 2.9 3.7 Anaerobic bacteria 0.5 0.6 Lipsky et al, Diabetologia 2010; 53:914
Which of These is Not a Risk Factor for MRSA Infection in cssti? 1. Recent course of antibiotic therapy 2. Recent or current hospitalization 3. Close contact (sports, military, prison) 4. Recent outpatient surgical procedure 5. Intensive, frequent outpatient care
Independent Risk Factors for MRSA in DFI 64% of S. aureus MRSA; 65% of MRSA hospital-acq Matched Case (n=21) Control (n=97) Study Characteristic OR (95% CI) P value Age (years) 1.02 (0.94 1.12) 0.61 Duration diabetes (yr) 1.02 (0.92 1.13) 0.74 HbA1c (%) 1.15 (0.78 1.71) 0.49 Size of ulcer (cm 2 ) 1.61 (1.22 2.12) 0.001 Osteomyelitis 18.51 (2.50 137.21) 0.004 Surgical procedure 0.53 (0.06 4.97) 0.53 Wang A-H, et al Diabetic Med 2010; 21:710
Adverse Outcomes With MRSA Infection Data from 8 Published Studies pts Effect No. Patients Eleftheriadou, et al. Drugs 2010; 70:1785
Management of DFI In Turkey: Prospective Multicenter Study Turkish Assoc Clinical Microbiol & ID, DFI Working Group 96 patients enrolled, 10 centers, 2010-2011 Isolated bacteria were antibiotic-resistant in 43% Independent risk factors for antibiotic resistance Previous amputation (OR 7.2, p=0.02) Antibiotic Rx within 30 days (OR 3.8, p=0.03) Resistant bacteria infection independent risk factor for Treatment failure (OR 5.3, p=0.02) Higher cost of treatment ($2220 vs $1207) Ertugrul et al Eur J Clin Microboiol Infect Dis 2012; e-pub 22 Feb
Diabetic Foot Clinic as Source of MRSA Spread? 10 year analysis of MRSA acquisition & genotype trends Low potential for clinic-attributable incidence Rates comparable with regional & hospital Lagace-Wiens et al. Am J Infect Control 2009;37:587
Which Of These Antibiotics is Not FDA- Approved for Treating MRSA? Quinupristin-dalfopristin Tigeclycine Ceftaroline Imipenem-cilastatin Daptomycin Telavancin Linezolid Vancomycin
Susceptibility of MRSA from Infected DFU (n=21) Agent % Susceptible P value HA-MRSA CA-MRSA Gentamicin 4 (24) 0 (0) 0.11 Tetracycline 5 (29) 0 (0) 0.07 Clindamycin 3 (18) 6 (67) 0.01 Fusidate 15 (88) 9 (100) 0.28 Sulfamethoxazole 7 (41) 8 (89) 0.02 Rifampicin 11 (65) 9 (100) 0.04 Vancomycin/Teico 17 (100) 9 (100) NS Levofloxacin 4 (24) 6 (67) 0.03 Quinupristin-dalfo 15 (88) 5 (56) 0.06 Minocycline 7 (41) 7 (78) 0.08 Wang et al, J Med Microbiol 2010 (e-pub 2 July)
Anti-MRSA Agents for Soft Tissue/Bone Infxn Antibiotic Route Main benefits Main problems Clindamycin PO (IV) Good bone penetration Risk of C. difficile Linezolid PO (IV) Hi bioavai, tissue penet Heme SE limits duratn Resistance rare Neuropathy Fusidic acid PO (IV) Hi tissue penetration Only in combination Rifampicin PO (IV) Good tissue/bone Only in combination penetration Extensive interactions Doxycycline PO Once daily GI upset; photosensitiv Daptomycin IV Once daily Only available IV Resistance rare Myalgia, elev CPK Vancomycin IV Large experience Nephrotoxi; monitoring resistance concerns Teicoplanin IV nephrotoxic; QD Monitoring required Tigecycline IV For polymicrobial infx GI side effects;?mortality Thompson, Injury, Int J Care Injured 2011;42, S5: S7
Appropriateness of Initial Antibiotic Therapy by Severity Class: 189 Inpatients with SSTI Severity Total Antibiotic Treatment Administered** Class* no. pts Appropriate Over Under I 88 19 (22) 57 (65) 12 (14) II 56 20 (36) 14 (25) 22 (39) III 33 10 (30) 10 (30) 13 (39) IV 12 1 (8) 0 11 (92) All 189 50 (26) 81 (43) 58 (31) * Based on responsiveness, resp rate, O2 sat, temp, BP, pulse ** Treatment classification by comparison with UK guidance, N (%) Patients with MRSA infection more likely to receive inappropriate therapy (6/12, 50%) vs MSSA (3/20,15%) [Odds ratio 5.7] Marwick et al, J Antimicrob Chemother 2011; 66: 387
Current International MRSA SSTI Guidelines White, Infect Drug Resistance 2011:4 115
Daptomycin The first cyclic lipopetide antibiotic Approved for skin/soft tissue infections by US FDA (2003) and European Medicines Agency (2006) Active against gram-positive bacteria only: enterococci (including glycopeptide-resistant), staphylococci (including MRSA), streptococci and corynebacteria
Daptomycin: Unique Mechanism of Action Binds to bacterial cell membranes (not wall) Causes rapid depolarization of cell membrane potential with potassium efflux Inhibits synthesis of proteins, DNA & RNA Results in cell death without cell lysis Rapid, concentration dependent, bactericidal Silverman et al. Antimicrob Agents Chemother. 2003;47:2538
Comparative Daptomycin SSTI Studies: Design White, Infect Drug Resistance 2011:4 115
Comparative Daptomycin SSTI Studies: Outcome Results in all categories similar overall White, Infect Drug Resistance 2011:4 115
European Cubicin Outcomes Registry and Experience (EU-CORE SM ) Retrospective, non-interventional registry; collects information on patients treated with daptomycin In 2010 3621 patients enrolled; 277 had DFI 4% had concomitant osteo or bacteremia 193 had + cultures; most common pathogen Staph aureus (118, 69% MRSA) 39% had previous antibiotics; failure most common reason to change therapy to dapto Cogo et al, Abstract L1-1492, 51 st ICAAC, Chicago 17 Sept 2011
European Cubicin Outcomes Registry & Experience (EU-CORE SM ) Cogo et al, Abstract L1-1492, 51 st ICAAC, Chicago 17 Sept 2011
European Cubicin Outcomes Registry and Experience (EU-CORE SM ) Antibiotic therapy Median duration dapto therapy: outpatient 6 days, inpatient 12 days, ICU 8 days 159 (57%) received combination therapy Surgical interventions documented for 160 Outcomes Clinical success in 86% CPK levels & adverse events possibly related to dapto reported in 4 Cogo et al, Abstract L1-1492, 51 st ICAAC, Chicago 17 Sept 2011
European Cubicin Outcomes Registry & Experience (EU-CORE SM ) Cogo et al, Abstract L1-1492, 51 st ICAAC, Chicago 17 Sept 2011
Susceptibility of DFI Isolates to Daptomycin All patients with DFI, Nîmes U hospital, 2006-7 Among 200 strains, dapto active against 99.5% Streptococcus sp. (100%) Enterococcus sp. (100%) Coagulase-negative Staphylococcus (100%) MS-Staphylococcus aureus (100%) 1 MR-S. aureus strain was not covered (%) MIC s MIC (mg/l) Sotto et al, Pathologie Biologie 2010;58:73
Daptomycin Soft Tissue/Bone Penetration in DFI Measured interstitial fluid concentrations in 9 pts Serial sampling after infusion of 6 mg/kg at steady state from 0-8 hours, & 8-16 hours Compared free AUC in plasma to tissues for Healthy & inflamed subcutaneous tissue Metatarsal bone Key findings Free daptomycin in plasma equilibrates completely with soft tissues & bone w/n 3 hours Inflammation did not affect concentration Traunmuller et al, J Antimcrob Chemother 2010;65:1252
Daptomycin Penetration (6 mg/kg IV) in DFI MIC 90s<0.5 mg/l (N=5) (N=4) Traunmuller et al, J Antimcrob Chemother 2010;65:1252
Daptomycin for Treating Diabetic Foot Infections Subset analysis of cssti: 103 clinically evaluable DFI patients 47 got daptomycin, 56 vancomycin or semi-synthetic penicillin Lipsky BA, Stoutenburg U. J Antimicrob Chemother 2005;55:240
Daptomycin for Treating Diabetic Foot Infections (MSSA) Similar clinical and microbiological efficacy and safety Lipsky BA, Stoutenburg U. J Antimicrob Chemother 2005;55:240
Retrospective Study Daptomycin for DFI: Multicenter, multi-year, observational registry (CORE) Cubicin Outcomes Registry & Experience (CORE) - Standardized case report form & protocol - Collected demographic & clinical data - US medical institutions, 2005-2006 Of 781 patients with cssti, ~10% had DFI - Dose: 39% 4 mg/kg; 36% 6 mg/kg; 6% >6 mg/kg - Duration: median 14 days; 22 with osteomyelitis - Prior antibiotics before switch to dapto in 71% - Half had concomitant antibiotic agents Johnson et al. J Wound Care 2009;18:396
Retrospective Study Daptomycin for DFI: Multicenter, multi-year, observational registry (CORE) 60/66 (91%) clinically successful outcomes Johnson et al. J Wound Care 2009;18:396
IDSA/IWGDF Guidelines on Treating MRSA Mild Infection Moderate/ Severe Infection IWGDF Lipsky et al, Clin Inf Dis 2012;54;e132; Lipsky et al, Diab Met Res Rev 2012;28 Suppl 1:234
High Dose (>6 mg/kg) Daptomycin: Review of the Literature Approved: 4 mg/kg for csssi; 6 mg/kg for BSIs, RIE Found 2 prospective trials, 3 retrospective reviews, case reports and in vitro simulations All documented safety & tolerability, even when administered for a prolonged duration Additional in vitro benefits included suppression of emergence of daptomycin resistance & increased rapidity of bactericidal activity Might consider in selected difficult infections Wu et al. Int J Antimicrob Ag 2011;38:192
Combination Therapy with Daptomycin Diminishing susceptibility of S. aureus to daptomycin during treatment of complicated infections w/ clinical failure described Combination of daptomycin with rifampicin May be effective for foreign-body infections Data for IE is somewhat contradictory Combinations of daptomycin with beta-lactams, TMP-SMX, or clarithromycin Provides potential strategy for MDR S. aureus Value supported by the findings of in vitro & in vivo studies but not yet assessed clinically Nadrah, Chemother Res Pract 2011; 2011: 619321
Potential Problems with Daptomycin Adverse effects CPK elevation & myopathy; dose/duration related; (6.7% at 6 mg/kg for bacteremia); monitor Eosinophilic pneumonia (.43/10,000 pts; warning) Emergence of Resistance Noted after approval, with clinical failures Often associated with reduced susceptibility to vancomycin, esp with prior vanco Rx No differences in AEs or resistance in metaanalysis (4 studies) comparing daptomycin to vancomycin or β-lactam agents Bliziotis et al, Ann Phamacother 2010;44:97
When Would You Consider Using Daptomycin for Treating cssti/dfi? All patients with likely gram-positive infection Patients with MRSA pneumonia Patients with a mixed (gram +/-) infection Patients with a mild DFI Selected patients with likely or proven infection with MRSA
When to Consider Using Daptomycin in Treating Diabetic Foot Infections Empirical therapy For infections likely to be caused by MRSA Where rapidly -cidal activity is important If other anti-mrsa agents are not appropriate Combine with anti-gnr agent, when needed Definitive therapy MRSA isolated, especially if hi MIC to vanco Contraindications to other anti-mrsa agents Clinical failure of other antibiotic agents OPAT approach appropriate
Conclusions Complicated SSTI & DFI are common; morbid Commonest cause GPC, especially S. aureus Prevalence of MRSA infection has increased, and is associated with worse outcomes Several po/iv anti-mrsa agents now available Daptomycin Excellent activity against cssti/dfi strains Good penetration to inflamed soft tissue/bone Evidence of efficacy in retrospective, prospective and registry studies
Thank You
EXTRA SLIDES
Antibiotic Effectiveness vs MRSA for csstis 14 studies on 6 antibiotics with 28 treatment arms (n = 1840) Agent Pooled Success Cred Intervals 95% Vancomycin 74.7% 64.1% 83.5% Dalbavancin 87.7% 74.6% 95.4% Linezolid 84.4% 76.6% 90.6% Telavancin 83.5% 73.6% 90.8% Daptomycin 78.1% 54.6% 93.2% Tigecycline 70.4% 48.0% 87.6% Comparisons between others vs vancomycin Dalbavancin (+13.1%; CrI 95% : 1.0% 23.8% Linezolid (+ 9.7%; CrI 95% : 4.4% 15.8%) Telavancin (+ 8.8%; CrI 95% : 1.5% 16.7%) Logman et al, Current Med Res Opin 2010;26:1565
When to Consider Daptomycin Doses 6 mg/kg Associated sepsis syndrome Suspected/confirmed bacteremia or endocarditis Diabetic foot infections Suspected osteomyelitis or septic arthritis Intravenous drug user Burn injury a Glycopeptide intermediate-resistant MRSA Creatinine clearance <30 ml/minute b ------------------------------------------------------------------------------------------------ a Consider doses of 10 12 mg/kg; b Increase dosing interval to q 48 hours White, Infect Drug Resistance 2011:4 115
Preventing MRSA Infection by Hand Hygiene procurement hand cleanser associated with MRSA bacteremia Per 10,000 bed days Stone et al, BMJ 2012;344:e300
MRSA in cssti Risk factors for MRSA as cause of infection Recent exposure to antibiotics Hospitalization or intensive outpatient care Close contact (sports, military, prison) US FDA approved agents for MRSA: Vancomycin -Tygecycline (Tygicil) Linezolid (Zyvox) -Telavancin (Vibativ) Daptomycin (Cubicin) - Ceftaroline (Teflaro) Other agents used: Teicoplainin, Clindamycin, Doxycycline, TMP/SMX, Fosfomycin, Ceftobiprole (several new agents in pipeline)
Importance of MRSA/MDROs in DFIs Methicillin-resistant Staph aureus (MRSA) Inpatient & community settings; 20-50% of isolates MRSA isolation associated with clinical infection, &: longer healing time higher amputation rate higher mortality Extended-spectrum β-lactamase resistance (ESBL) In 5%-15% of DFIs in some reports Associated with more severe infection, worse outcome Mantey et al, Comun Dis Public Health 2000;3:288 Dang et al, Diabetic Med 2003;20: 159 Tentolouris et al, Clin Microbiol Infect Dis 2006;12:186 Varaiya et al. Indian J Pathol Microbiol. 2008;51:370 Gadepalli et al Diabetes Care 2006;29:1727
Microbiology of Diabetic Foot Infections Infection type Microorganisms Cellulitis Staphylococcus aureus, β-hemolytic streptococci (usually group A) Ulcer w/o antibiotic Rx S. aureus, β-hemolytic streptococci (esp grpup B) Ulcer with antibiotic Rx S. aureus, MRSA, coag staph or chronic wound streptococci, enterococci (often polymicrobial) Enterobacteriaceae, Pseudomonas aeruginosa Necrotizing fascitis or Anaerobes (GPC>GNR), Nonmyonecrosis (often non- fermenting GNR, aerobic polymicrobial) GPC Blanes-Mompo, Rev Esp Quimioter 2011;24: 233
High Dose (>6 mg/kg) Daptomycin: Review of the Literature FDA approved in single daily IV doses In 2003 at 4 mg/kg for csssis in 2006 at 6 mg/kg for S. aureus BSIs, R IE Mechanism of action distinct from any currently available antibiotics; conc-dependent kiling Exhibits generally linear pharmacokinetics at doses of 4 12 mg/kg every 24 h Effective against most gram-positive pathogens, including MRSA, MRSE, GISA, VRSA, VRE Wu et al. Int J Antimicrob Ag 2011;38:192
Susceptibility of DFI Isolates to Daptomycin Sotto et al, Pathologie Biologie 2010;58:73
Daptomycin Soft Tissue/Bone Penetration in DFI Key findings Free daptomycin in plasma equilibrates completely with soft tissues & bone w/n 3h Inflammation did not affect concentrations Comparisons with other studies in diabetic foot No significant differences between diabetic & control patients for: fosfomycin, moxifloxacin, linezolid {need further data for this slide} Traunmuller et al, J Antimcrob Chemother 2010;65:1252
Daptomycin for Infections in Diabetic Patients Cubicin Outcomes Registry & Experience (CORE) 60 patients with skin/soft tissue infections Clinical success rate of 83% Other recent studies of daptomycin vs vancomycin or semi-synthetic penicillins: cure rates 70%-80% European CORE: EUROCORE Patients with bacteremia or endocarditis due to GPC- success in 76.8% & 72%, respectively Daptomycin vs vancomycin for MRSA or SSpenicillin for MSSA: no significant differences in diabetic patients with bacteremia or endocarditis Montejo, Enferm Infecc Microbiol Clínica 2012;30, Suppl 1:54
Management of DFI In Turkey: Prospective Multicenter Study Turkish Association of Clinical Microbiology & Infectious Diseases, DFI Working Group 96 pts enrolled from 10 medical centers 2010-2011 95% PEDIS 2/3 (mild/moderate); 62% osteomyelitis Microbiological assessment in 86 115 causative bacteria from 71 patients Most frequently isolated: Ps aeruginosa (18.3%) Bacteria antibiotic-resistant in 43% with + culture Antibiotic costs ($US) $2220 for patients with antibiotic-resistant strains $1160 for patients with antibiotic susceptible strains Ertugrul et al Eur J Clin Microboiol Infect Dis 2012; e-pub 22 Feb
Daptomycin vs Glycopeptides for cssti: RCT 134 pts in multicenter trial; 66% S. aureus D/C due to AEs: 9.8% vs. 3.1% for daptomycin Microbiological success: 98.2% dapto vs. 90.7% Duration Rx w/ dapto (75% required 4 10 days of therapy vs 62% for comparators) Quist, Int J Antimicrob Ag 2012; 39:90
Daptomycin: EUROCORE Registry, Spain 2006-10 Retrospective European post-marketing non-comparative database 726 patients : 66% M, 49% >65 yo, 70% comorbid. Administered in outpatient setting in 20% >50% received a dose of 6mg/kg/day In 80% daptomycin was a rescue therapy Median treatment duration was 14 days Infections treated: bacteremia (33%), skin & soft tissue (31%), endocarditis (14%), prosthetic materials (11%), osteoarticular (6%) Pathogens: S. aureus (41%; 14% MRSA), CoN (35%), enterococci (12%), streptococci (3%). Cercenado E, Enferm Infecc Microbiol Clin 2012;30 Suppl 1:3
Daptomycin: EUROCORE Registry, Spain 2006-10 Clinical success rate: overall 78.4% 82% when used as 1 st line therapy 78% when used as rescue therapy Efficacy lower in patients with renal failure At the end of therapy 8.7% of patients had creatinine clearance In 25 patients creatine kinase values >10 times higher than normal values Cercenado E, Enferm Infecc Microbiol Clin 2012;30 Suppl 1:3
Evidence of Efficacy of Daptomycin in Complicated Skin/Soft Tissue Infections Surgical Site infections: CORE 2007 population 118 of 962 patients had SSI; 104 (11%) evaluable for efficacy analysis Most common pathogens: S. aureus (59%), of which 25 (24% of all cases) MRSA Overall success (cure + improved) 91% VRE infection lower cure rate (63%) Outpatient parenteral antibiotic therapy (OPAT) CORE 2005 170 with cssti: success rate 95% EU-CORE 2006 & 2008: 34 pts, 89% success White B, Seaton R, Infect Drug Resist 2011;4:115
Daptomycin vs Glycopeptide Therapy: Retrospective Case-Control Analysis Patients with SSTIs in glycopeptide group longer mean duration of antibiotic therapy (18.2 days vs. 14.6 days; P=0.009) longer mean length of hospital stay (28.2 days vs. 19.6 days; P=0.01) Longer mean duration therapy also noted with bloodstream infxns (25.6 v 18 days; p=0.004) Food clinical efficacy of daptomycin associated with amore rapid resolution of clinical syndrome & reduced length of hospitalization Falcone et al, Int J Antimicrob Agents 2012;39:64
Proposed Antibiotic Algorithm for MRSA csstis White, Infect Drug Resistance 2011:4 115
Antibiotic Resistance for Anti-S. aureus Agents Methicillin Clindamycin Vancomycin Associated with acquisition & expression of the mec gene Cross resistance w/ macrolides (inducible expression mutated erm gene) Mediated by cell-wall thickening or acquisition of vana gene Treatment failure associated with MICs 1 µg/ml to 2 µg/ml Linezolid Resistance is most often with target site mutation at G2576; most often in coag-negative staphylococci & enterococci Daptomycin Resistance associated with point mutations in a host of genes, leading to altered membrane surface charge, phospholipid asymmetry, drug binding. Associated with prior vancomycin exposure, but clinical significance of this observation remains to be determined
Properties of New Antibiotics for DFI Agents with Efficacy for cssti ± DFI Agent MRSA VRG+ Gm- Ana Route Freq Notes Linezolid* + + ± IV/po q12 Tox/cost Ertapenem* ± 1 + IV QD No Ps.aer Moxifloxacin + 1 + IV/po QD FQ Daptomycin + + ± IV QD Cidal Tigecycline + + + 1,2 + IV q12 ESBLs Dalbavancin + ± IV QW Not aprvd Ceftobiprole + + ± IV q12 * FDA-approved for DFI; 1 No Ps aerug; 2 Including ESBLs ±Ps,ESBL Telavancin Omar et at, Int J + Antimicrob + Ag 2008:31:411 ± IV QD Cidal
MRSA in Infected & Uninfected Diabetic Foot Ulcers in Diabetic Foot Clinic 84 consecutive patients with 91 foot ulcers Infection diagnosed by IWGDF consensus criteria Culture result Infected (n=59) Uninfected (n=32) S. aureus 18 17 MSSA 7 (39%) 7 (88%) MRSA 11 (61%) 1 (13%)* [* p=.03] Gram negs 57 31 Anaerobes 8 5 MRSA colonization not associated with usual risks Tentolouris et al, Clin Microbiol Infect 2006;12:186