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2 Website: or Copyright 2009 Edith Blondel-Hill MD All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the authors. Antimicrobial Susceptibility Testing Manual 4 th EDITION

3 Table of Contents Authors Editorial Contributors, Expert Advisor & Acknowledgements Preface Legend & User Guide Aerobic Gram Positive Cocci Section 1 Aerobic Gram Positive Bacilli Section 2 Aerobic Gram Negative Cocci Section 3 Aerobic Gram Negative Bacilli - Fermentative Section 4 Aerobic Gram Negative Bacilli - Non-fermentative Section 5 Fastidious Gram Negative Bacilli / Coccobacilli Section 6 Anaerobic Organisms Section 7 Appendices Section 4 Appendices 147 Appendix A - Drug Abbreviations Appendix B - Test Procedures Appendix 1 - Doubling Dilution Chart for Etest MIC 221 References 222 Index 250 Table of Contents: P age v i ii iii iv

4 SECTION 1 AEROBIC GRAM POSITIVE COCCI Pages 1-59 Abiotrophia spp. 1 Aerococcus spp. 3 Alloiococcus spp. 4 Enterococcus faecalis 5 Enterococcus faecium 8 Enterococcus spp. (Other) 11 Facklamia spp. 14 Gemella spp. 15 Globicatella spp. 16 Granulicatella spp. 17 Helcococcus spp. 18 Kocuria spp. / Kytococcus spp. 19 Lactococcus spp. 20 Leuconostoc spp. 21 Macrococcus spp. / Micrococcus spp. 22 Pediococcus spp. 23 Staphylococcus aureus 24 Staphylococcus intermedius / pseudintermedius 32 Staphylococcus lugdunensis 34 Staphylococcus saprophyticus 36 Staphylococcus schleiferi 38 Staphylococcus spp. (coagulase negative) 40 Streptococcus agalactiae 44 Streptococcus bovis group 47 Table of Contents: P age vi

5 Streptococcus Groups C and G 49 Streptococcus iniae 51 Streptococcus pneumoniae 52 Streptococcus pyogenes 57 Streptococcus spp. (other) 59 SECTION 2 AEROBIC GRAM POSITIVE BACILLI Pages Actinomycetes (Aerobic) 60 Arcanobacterium spp. 61 Bacillus cereus group 62 Bacillus spp. (other than B. cereus group) 64 Corynebacterium spp. 66 Coryneform Bacteria other than Corynebacterium spp 69 Erysipelothrix spp. 71 Gardnerella vaginalis 72 Listeria spp. 73 Rothia spp. 74 SECTION 3 AEROBIC GRAM NEGATIVE COCCI Pages Neisseria gonorrhoeae 75 Neisseria meningitidis 76 Neisseria spp. (other) 77 Table of Contents: P age vii

6 SECTION 4 AEROBIC GRAM NEGATIVE BACILLI - FERMENTATIVE Pages Aeromonas spp. 78 Cedecea spp. 80 Citrobacter spp. 82 Cronobacter sakazakii 84 Edwardsiella spp. 86 Enterobacter spp. 87 Escherichia spp. 89 Hafnia alvei. 91 Klebsiella spp. 93 Morganella spp. 95 Plesiomonas shigelloides 97 Proteus mirabilis 98 Proteus vulgaris complex 100 Providencia spp. 102 Salmonella spp. 104 Serratia spp. 107 Shigella spp. 109 Vibrio cholerae 110 Vibrio spp. 111 Yersinia spp. 112 Enterobacteriaceae (other) 114 Enterobacteriaceae Beta-lactam Resistance Detection Charts Beta-lactam Resistance Interpretation Chart Table of Contents: P age viii

7 Section 4 Appendix 147 Drug Abbreviations 147 SECTION 5 Test Procedures 147 AEROBIC GRAM NEGATIVE BACILLI NONFERMENTATIVE Pages Achromobacter spp. 148 Acidovorax spp. 150 Acinetobacter spp. 151 Alcaligenes spp. 154 Balneatrix spp. 156 Bordetella spp. 157 Brevundimonas spp. 158 Burkholderia spp. 159 Chryseobacterium spp. 161 Comamonas spp. 163 Delftia spp. 164 Empedobacter spp. 165 Methylobacterium spp. 166 Moraxella catarrhalis 167 Moraxella spp. (other than M. catarrhalis) 169 Myroides spp. 170 Ochrobactrum spp. 171 Oligella spp. 172 Pandoraea spp. 173 Table of Contents: P age ix

8 Pseudomonas aeruginosa 174 Beta-lactam Interpretation Chart for Carbapenem Resistance in Pseudomonas aeruginosa 176 Pseudomonas aeruginosa Beta-lactam Resistance Interpretation Chart 177 Pseudomonas spp. (other than P. aeruginosa) 178 Ralstonia spp. 180 Rhizobium spp. 181 Roseomonas spp. 182 Shewanella spp. 183 Sphingobacterium spp. 184 Sphingomonas spp. 185 Stenotrophomonas maltophilia 186 SECTION 6 FASTIDIOUS GRAM NEGATIVE BACILLI/COCCOBACILLI Pages Actinobacillus spp. 188 Aggregatibacter spp. 189 Bergeyella spp. 190 Brucella spp. 191 Campylobacter spp. 192 Capnocytophaga spp. 194 Capnocytophaga spp. (Canine) 195 Cardiobacterium hominis 196 CDC Group EF 4a and EF-4b 197 Dysgonomonas spp. 198 Table of Contents: P age x

9 Eikenella corrodens 199 Francisella spp. 200 Haemophilus influenzae 201 Haemophilus parainfluenzae 203 Haemophilus spp. (other) 205 Kingella spp. 206 Pasteurella spp. 207 Suttonella indologenes 209 Weeksella spp. 210 SECTION 7 ANAEROBES Pages Actinomyces spp. 211 Clostridium spp. 212 Lactobacillus spp. 213 Propionibacterium spp. 214 Anaerobic Gram Positive Bacilli - other 215 Anaerobic Gram Positive Cocci 216 Anaerobic Gram Negative Bacteria 218 Table of Contents: P age xi

10 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL REPORTING CHARTS AEROBIC GRAM POSITIVE COCCI Section 1 ORGANISM: Enterococcus faecalis CLINICAL: USUAL SUSCEPTIBILITY PATTERN: SUSCEPTIBILITY METHOD: E. faecalis is part of the normal flora of the human gastrointestinal and female genital tract. It may also be recovered from various environmental sources (soil, water, plants, animals). This organism is an important nosocomial pathogen associated with urinary tract infections (commonly in persons with structural abnormalities or following urologic manipulation), intra-abdominal and pelvic infections, endocarditis, and bacteremia. The role of E. faecalis in polymicrobial wound infections and respiratory infections is controversial and requires clinical evaluation, as well as review of Gram stain. E. faecalis is the most common of the enterococcal isolates, especially in the urinary tract. E. faecalis is usually susceptible to ampicillin, vancomycin, linezolid and chloramphenicol. Vancomycin tolerance has been described and linezolid resistance is increasing. This organism is resistant to cephalosporins, clindamycin, macrolides, TMP-SMX, fusidic acid and quinupristin/dalfopristin. (Note: some of these antibiotics may appear susceptible in vitro, but should not be used clinically.) Ciprofloxacin resistance is very common in urinary isolates. Gentamicin synergy resistance is common. Gentamicin is recommended in serious infections only as a synergistic agent if the gentamicin synergy is susceptible. Streptomycin may also be used for synergy if synergy susceptible. Other aminoglycosides should not be tested. Microbroth dilution. Additional tests include Kirby-Bauer or Etest method using Mueller-Hinton agar incubated in ambient air at 35 C for hours. (24 hours for Etest method) Note: For Etest use 0.5 McFarland suspension in broth. SUSCEPTIBILITY CSF/ BLOOD/ STERILE DEEP ENDOVASCULAR BODY REPORTING: BRAIN WOUND CATHETER SITE URINE OTHER COMMENTS Beta-lactamase If negative, do not report * Etest method Ampicillin * * Amoxicillin/clavulanate * * * * Report same as amp if S. aureus and/or anaerobes co-isolated Chloramphenicol rd line if amp, vanco and linezolid I/R Ciprofloxacin Do not report in patients < 18 y 3 rd line if amp/vanco/linezolid I/R Daptomycin Etest method Gentamicin Synergy * * * Report only if gent syn R Imipenem * * * * 2 nd line if amp and vanco R Linezolid Nitrofurantoin Piperacillin/tazobactam * * * * Quinupristin/ Dalfopristin * * * * * * Streptomycin Synergy nd line if gent syn R 2 2 nd line if amp I/R 3 rd line if amp, vanco and linezolid I/R Tetracycline Do not report in patients < 8 y Vancomycin 06 October 2009 Section 1: P age 4

11 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL REPORTING CHARTS AEROBIC GRAM POSITIVE COCCI Section 1 Enterococcus faecalis (cont d) SPECIAL CONSIDERATIONS: Betalactamase: Ampicillin: Ciprofloxacin: Daptomycin: Gentamicin Synergy: Imipenem: The presence of beta-lactamase is thought to be very rare but may require a much higher inoculum to be detected. If beta-lactamase positive consult microbiologist. Beta-lactamase positive strains should be considered resistant to penicillin, amoxicillin and piperacillin. Note: Enterococcal isolates susceptible to penicillin are predictably susceptible to ampicillin, amoxicillin +/-clavulanate, piperacillin +/- tazobactam Enterococcal isolates susceptible to ampicillin are predictably susceptible to amoxicillin +/- clavulanate and piperacillin+/- tazobactam, but not necessarily to penicillin or imipenem. CSF / Brain isolates: Perform ampicillin Etest report MIC value. For susceptible isolates with MIC >2 µg/ml, add comment: In serious infections, pharmacodynamic evaluation should be considered for optimal dosing of ampicillin. In patients < 18 years old, ciprofloxacin may be reported on urine specimens at physician request only, add comment: Ciprofloxacin susceptibility reported at physician s request. Daptomycin is inactivated by surfactant in the lungs. This antibiotic should not be used for the treatment of respiratory infections. For blood isolates where daptomycin is reported, add comment: Daptomycin is inactivated by lung surfactant and should not be used for respiratory infections CSF / Brain / Blood or Sterile body site / Endovascular catheter if pure/predominant: If gentamicin synergy Sensitive, add comment: Gentamicin synergy screen SENSITIVE. In serious infections, combination therapy with gentamicin should be considered. If gentamicin synergy Resistant, add comment Gentamicin synergy screen RESISTANT. Therapy with gentamicin for synergy is NOT indicated. Imipenem is the only carbapenem with activity against E. faecalis. Ertapenem and meropenem are NOT active against enterococci. Imipenem should be reported if patient receiving imipenem therapy or has a polymicrobial infections especially with co-isolation of anaerobes and/or Pseudomonas aeruginosa. If ampicilin is resistant, imipenem can reliably be reported as resistant. Neither penicillin nor ampicillin susceptibility can predict imipenem. This agent must be tested separately. Note: Alterations in penicillin binding proteins may result in resistance to imipenem +/- penicillin but not ampicillin. There are no CLSI breakpoints for imipenem but EUCAST sets the susceptible breakpoint at 4µg/mL. However, if penicillin is resistant it seems prudent to report imipenem as susceptible only if the MIC is 2µg/mL. See Interpretation. For sterile body sites where imipenem is not reported, add comment: Ampicillin should not be used to predict imipenem susceptibility. Contact laboratory immediately if imipenem susceptibility testing is required. If imipenem reported, add comment: Ertapenem and meropenem have no activity against Enterococcus spp. 06 October 2009 Section 1: P age 5

12 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL REPORTING CHARTS AEROBIC GRAM POSITIVE COCCI Section 1 Enterococcus faecalis (cont d) SPECIAL CONSIDERATIONS: INTERPRETATION: Linezolid: Piperacillin/ tazobactam: Quinupristin/ Dalfopristin: Streptomycin Synergy: Tetracycline: Vancomycin: If linezolid susceptibility reported, confirm all resistant isolates with second method. If confirmed R, consult microbiologist. May be reported in polymicrobial infection with co-isolation of anaerobes and/or Pseudomonas aeruginosa, on physician request only. Report same as ampicillin. E. faecalis is intrinsically resistant to this antibiotic. Consult microbiologist if susceptible. CSF / Brain / Blood or Sterile body site / Endovascular catheter if pure/predominant: If streptomycin synergy Sensitive, add comment: Streptomycin synergy screen SENSITIVE. In serious infections, combination therapy with streptomycin should be considered. Doxycycline and minocycline may have better activity than tetracycline, and may be reported on physician request. Isolates that are susceptible to tetracycline are predictably susceptible to doxycycline and minocycline. If tetracycline I/R test doxycycline and/or minocycline separately. If MIC 4 µg/ml, confirm by Etest method. If MIC 4 µg/ml by Etest, send to reference laboratory for detection of Van genes. Add comment: This isolate exhibits an elevated MIC to Vancomycin and has been sent to reference laboratory for detection of Van genes. Report Vancomycin I/R isolates to Infection Control and/or Public Health. For Etest, report actual MIC result. For interpretation (S, I, or R) report according to the nearest higher doubling dilution (Appendix 1). Use CLSI interpretive document for Enterococcus spp. For imipenem : If MIC 2µg/mL S If MIC 4 µg/ml I If MIC 8µg/mL R Add comment: Susceptibility testing of this organism to imipenem is not standardized. Results are probable but not definite. 06 October 2009 Section 1: P age 6

13 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL REPORTING CHARTS AEROBIC GRAM POSITIVE BACILLI Section 2 ORGANISM: CLINICAL: USUAL SUSCEPTIBILITY PATTERN: SUSCEPTIBILITY METHOD: SUSCEPTIBILITY REPORTING: Bacillus cereus group - B. anthracis - B. cereus - B. mycoides - B. thuringiensis These organisms are widely distributed in nature, especially in soil environments. Their spores may contaminate dried foods (spices, tea, flour, powders). B. anthracis - is an obligate pathogen of animals and humans and is the cause of anthrax which may cause infections in different forms (cutaneous, intestinal and inhalational). Any request for isolation of this organism must be referred to a Level III Laboratory. B. cereus is an opportunistic pathogen in immunocompromised patients (cancer, alcoholism, preterm neonates, intravenous drug users) and has been associated with bacteremia, septicemia, meningitis, brain abscess, endocarditis, pneumonia, lung abscess, and osteomyelitis. It may also cause wound or ocular infections in otherwise healthy patients, especially following burns, surgery or trauma (usually following soil exposure). Neonates are prone to infection with this organism, especially umbilical stump and ventilator associated respiratory tract infections. B. cereus is also associated with food-borne illnesses (two enterotoxigenic food poisoning syndromes associated with B. cereus: a diarrheal type and a vomiting type). B. thuringiensis - has been associated with wound, burn and ocular infections. B. cereus and B. thuringiensis produce various chromosomal beta-lactamases including two penicillinases and a broad spectrum inducible metallo beta-lactamase resulting in resistance to penicillins, cephalosporins (especially 3 rd generation cephalosporins) and beta-lactamase inhibitor combinations. Induction of the metalloenzyme may result in elevated MICs to carbapenems. Expression of the beta-lactamases is slow and not well detected by nitrocefin. These organisms are usually susceptible to quinolones, aminoglycosides, tetracyclines, clindamycin, chloramphenicol, and rifampin (the latter should not be used alone). They have variable susceptibility to macrolides and are usually resistant to TMP-SMX. Rare resistance to vancomycin has been reported. Clindamycin plus gentamicin may be best therapy for ocular infections (antibiotic penetration). B. anthracis is usually susceptible to penicillin, ciprofloxacin and doxycycline. It is resistant to cephalosporins. Susceptibility testing for B. anthracis must be performed in a Level III laboratory. Refer to CLSI guidelines for Potential Agents of Bioterrorism. Microbroth dilution or Etest method using Mueller-Hinton agar incubated in ambient air at 35 o C for hours. Note: 1) Broth microdilution may be superior for Vancomycin.. 2) Kirby-Bauer may be substituted for Etest method, except when testing vancomycin. CSF/ STERILE BLOOD BRAIN BODY SITE EYES + OTHER COMMENTS Chloramphenicol Ciprofloxacin * * * * Do not report in patients < 18 y Clindamycin * * Gentamicin Imipenem 2 2 nd line if cipro I/R Penicillin R R R R R Report all isolates as R Tetracycline Do not report in patients < 8 y Vancomycin + See Note 16 September 2009 Section 2: P age 62

14 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL REPORTING CHARTS AEROBIC GRAM POSITIVE BACILLI Section 2 Bacillus cereus group (cont d) SPECIAL CONSIDERATIONS: INTERPRETATION: NOTE: Eye Specimens: Deep: Perform susceptibility test if: - vitreous fluid - corneal ulcer - contact lens related infections - keratitis - endophthalmitis - ophthalmology clinic/ward - injury/surgery - history of failure of therapy Superficial: Susceptibility testing of superficial eye specimens not routinely performed. Add comment: Susceptibility testing of topical antibiotics is not standardized and is not routinely performed on superficial eye specimens. Note: Susceptibility testing may be selectively performed on superficial eye specimens based on Gram stain and clinical history, after consultation with microbiologist. Other Specimens: Susceptibility testing is recommended if organism isolated from sterile body site. For other sites, or if isolated with other organisms, clinical correlation and correlation with Gram stain is required. On blood culture/sterile body sites/deep eye isolates, add comment: In serious infections, combination therapy with gentamicin should be considered. Clindamycin: Clindamycin may be reported on blood culture isolates on physician request. It is NOT recommended for treatment of endocarditis. Imipenem: Vancomycin: Induction of the metalloenzyme may result in heteroresistance to carbapenems If reporting imipenem as sensitive, add comment: Penicillin and cephalosporin resistance in this organism is mediated by an inducible beta-lactamase that can also affect carbapenems. Carbapenems should be used with caution Alternatively, check for inducible resistance with a double disc (DD) test: NOTE: This is investigational test only Place a 10 µg penicillin disc 15 mm away from edge of a 10 µg imipenem disc on standard BAP or Mueller-Hinton agar plate. Incubate for hours in ambient air at 35 0 C. If flattening of imipenem zone adjacent to penicillin disc: Report imipenem R. Add comment: This isolate is presumed to be resistant to carbapenems based on the detection of inducible resistance in vitro If no flattening of imipenem zone adjacent to penicillin disc: Report imipenem S These organisms should be susceptible to vancomycin. E-test has occasionally been associated with false resistance. If R confirm with broth microdilution. Use CLSI interpretive document for Bacillus species (not B. anthracis). If using Kirby Bauer method, use CLSI interpretative document for Staphylococcus spp. Note: If using Kirby Bauer method, add comment: Susceptibility testing for this organism was performed by an unstandardized method. Results are probable but not definite. 16 September 2009 Section 2: P age 63

15 ANTIMICROBIAL SUSCEPTIBILITY TESTING MANUAL ENTEROBACTERIACEAE BETA-LACTAM RESISTANCE DETECTION CHARTS 1 µg/ml Comment 1 ETP 2µg/mL Chart 6 8 µg/ml ESBL discs µg/ml CTX or CRO or CAZ Chart 5 Chart 4 Detection of Beta Lactam Resistance if ESBL Screen Positive E. coli Klebsiella spp Proteus mirabilis Salmonella spp Shigella spp FOX < 2 µg/ml 2 µg/ml CZ 8 µg/ml Chart 2 ESBL screen positive* 16 µg/ml ESBL discs Cefepime ESBL disc + - CTX or CRO or CAZ < 2 µg/ml 2 µg/ml Consult Microbiologist Chart 2 CTX or CRO or CAZ Chart 3 Chart 2 <2 µg/ml 2 µg/ml Chart 2 CTX or CRO or CAZ 2 µg/ml YES Other 4µg/mL *ESBL screen POSITIVE: Cefpodoxime 8 µg/ml (for P. mirabilis, cefpodoxime 2 µg/ml) or ESBL screen on automated system Note: Reporting of resistance to 3 rd generation cephalosporins should not be delayed until confirmatory tests are completed. NO 8 µg/ml CZ FOX 8µg/mL Chart 5 Chart 4 O NOT COPY) 16 µg/ml Chart 3