1. BSAC Standardised Disc Susceptibility Test User Group Day. Royal College of Physicians, London. 8 June 2007 Susceptibility testing of mucoid Pseudomonas and Burkholderia strains from patients with cystic fibrosis including evaluation of the BSAC standardised method. J.D. Perry Freeman Hospital Newcastle upon Tyne

2. Contents: Direct susceptibility testing of whole sputum from CF patients to detect resistant strains of P. aeruginosa. Direct susceptibility testing of whole sputum from CF patients to detect resistant strains of P. aeruginosa. Preliminary work to validate disc susceptibility testing with P. aeruginosa and B. cepacia from CF patients. Preliminary work to validate disc susceptibility testing with P. aeruginosa and B. cepacia from CF patients. MCBT testing of resistant strains of P. aeruginosa and B. cepacia from CF patients. MCBT testing of resistant strains of P. aeruginosa and B. cepacia from CF patients.

3. Direct susceptibility testing of Pseudomonas aeruginosa from sputa of patients with cystic fibrosis

4. Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing J. E. Foweraker*, C. R. Laughton, D. F. J. Brown and D. Bilton Department of Microbiology, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE, UK; Health Protection Agency, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge CB2 2QW, UK; Department of Chest Medicine, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE, UK Journal of Antimicrobial Chemotherapy (2005) 55, 921–927

5. Foweraker et al. 2005. Methods: One hundred and one sputa were cultured. Four colonies of each P.aeruginosa morphotype were suspended. One hundred and one sputa were cultured. Four colonies of each P.aeruginosa morphotype were suspended. Susceptibility to 12 agents by disc diffusion was tested individually or by pooling the four suspensions. Susceptibility to 12 agents by disc diffusion was tested individually or by pooling the four suspensions.

6. Foweraker et al. 2005. Results / Conclusions In some cases, all four colonies of a single morphotype had different antibiograms. In some cases, all four colonies of a single morphotype had different antibiograms. The susceptibility profiles of single isolates of P. aeruginosa correlated poorly with pooled cultures, with the pooled tests missing resistance. The susceptibility profiles of single isolates of P. aeruginosa correlated poorly with pooled cultures, with the pooled tests missing resistance. A range of susceptibility patterns is seen, even within a morphotype. Routine test results are not reproducible and underestimate resistance. A range of susceptibility patterns is seen, even within a morphotype. Routine test results are not reproducible and underestimate resistance.

7. Can antimicrobial resistance be detected more reliably by culture of whole sputum onto media containing antimicrobials ? Aim of the current investigation:

8. Routine culture: Sputum samples were homogenized 1:1 with Sputasol. Sputum samples were homogenized 1:1 with Sputasol. Routine culture: Routine culture: 10 µl aliquots of liquid sputum were plated onto: 10 µl aliquots of liquid sputum were plated onto: Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, Pseudomonas Selective agar & Burkholderia cepacia Selective agar. Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, Pseudomonas Selective agar & Burkholderia cepacia Selective agar.

9. Routine culture (continued): A 10 µl aliquot of liquid sputum was diluted by addition to 9.99 ml sterile water (1/1000). A 10 µl aliquot of liquid sputum was diluted by addition to 9.99 ml sterile water (1/1000). 10 µl of this diluted sample was also plated onto: 10 µl of this diluted sample was also plated onto: Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, Pseudomonas Selective agar & Burkholderia cepacia Selective agar. Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, Pseudomonas Selective agar & Burkholderia cepacia Selective agar.

10. Selective culture: A 10 µl aliquot of liquid sputum was inoculated onto 10 distinct Isosensitest agar plates incorporating the following antimicrobials: Amikacin (16 mg/L) Amikacin (16 mg/L) Gentamicin (4 mg/L) Gentamicin (4 mg/L) Tobramycin (4 mg/L) Tobramycin (4 mg/L) Aztreonam (8 mg/L) Aztreonam (8 mg/L) Ceftazidime (8 mg/L) Ceftazidime (8 mg/L) Meropenem (4 mg/L) Meropenem (4 mg/L) Temocillin (16 mg/l) Temocillin (16 mg/l) Ciprofloxacin (1 mg/L). Ciprofloxacin (1 mg/L). Piperacillin-tazobactam (16 mg/L) Piperacillin-tazobactam (16 mg/L) Ticarcillin-clavulanic acid (32 mg/L) Ticarcillin-clavulanic acid (32 mg/L)

11. Summary of media used Routine culture: 10 µl of Neat and diluted homogenized sputa were inoculated onto: 10 µl of Neat and diluted homogenized sputa were inoculated onto: Cholcolate-Bacitracin Cholcolate-Bacitracin Blood agar. Blood agar. CLED agar. CLED agar. Isosensitest agar. Isosensitest agar. Pseudomonas selective agar. Pseudomonas selective agar. B. cepacia selective agar. B. cepacia selective agar. (Total = 10 culture plates). ‘Selective’ culture Culture of neat homogenized sputa on Isosensitest agar (x 10) containing: Culture of neat homogenized sputa on Isosensitest agar (x 10) containing: Amikacin Amikacin Gentamicin Gentamicin Tobramycin Tobramycin Aztreonam Aztreonam Ceftazidime Ceftazidime Meropenem Meropenem Temocillin Temocillin Ciprofloxacin Ciprofloxacin Piperacillin-tazobactam Piperacillin-tazobactam Ticarcillin-clavulanic acid Ticarcillin-clavulanic acid

12. Interpretation of cultures: All plates were incubated for 72 hours and examined after 24, 48 and 72 hours. All plates were incubated for 72 hours and examined after 24, 48 and 72 hours. All colonial variants or ‘morphotypes’ of Gram-negative bacteria were sub-cultured onto a blood agar plate to obtain pure cultures. These subcultures were used for MIC testing, identification and storage in glycerol for potential further studies. All colonial variants or ‘morphotypes’ of Gram-negative bacteria were sub-cultured onto a blood agar plate to obtain pure cultures. These subcultures were used for MIC testing, identification and storage in glycerol for potential further studies.

13. Identification: P. aeruginosa was identified by inoculation of all morphotypes onto PC agar, cetrimide agar and blood agar to test for growth at 42°C. P. aeruginosa was identified by inoculation of all morphotypes onto PC agar, cetrimide agar and blood agar to test for growth at 42°C. PC agar contains: PC agar contains: 30 mg/L 9-chloro-9-[4-(diethylamino)phenyl]-9,10- dihydro-10-phenylacridine hydrochloride (C-390) and 30 mg/L 9-chloro-9-[4-(diethylamino)phenyl]-9,10- dihydro-10-phenylacridine hydrochloride (C-390) and 30 mg/L 1,10-phenantholine. 30 mg/L 1,10-phenantholine. Growth on this agar is diagnostic for P. aeruginosa with 100 % specificity 1. Growth on this agar is diagnostic for P. aeruginosa with 100 % specificity 1. 1 J Clin Microbiol. 1988 Sep;26(9):1910-2.

14. Identification: For this study: For this study: Growth on cetrimide + growth on PC agar + growth at 42°C = P. aeruginosa. Growth on cetrimide + growth on PC agar + growth at 42°C = P. aeruginosa. All other strains were identified by API 20 NE. All other strains were identified by API 20 NE.

15. Susceptibility testing: All morphotypes (from any medium) were referred for MIC testing against 10 antibiotics. All morphotypes (from any medium) were referred for MIC testing against 10 antibiotics. MIC testing was performed using agar dilution in Isosensitest agar with a final inoculum of 10 000 cfu/spot. MIC testing was performed using agar dilution in Isosensitest agar with a final inoculum of 10 000 cfu/spot. MIC’s were recorded after both 24 and 48 hours of incubation at 37°C. MIC’s were recorded after both 24 and 48 hours of incubation at 37°C.

16. Susceptibility testing: Ranges of antibiotics for MIC testing: AMIK (64 - 2 mg/L) AMIK (64 - 2 mg/L) GENT (16 - 0.5 mg/L) GENT (16 - 0.5 mg/L) TOBRA (16 - 0.5 mg/L) TOBRA (16 - 0.5 mg/L) ATM (32 - 1 mg/L) ATM (32 - 1 mg/L) CAZ (32 - 1 mg/L) CAZ (32 - 1 mg/L) CIPRO (4 - 0.125 mg/L) CIPRO (4 - 0.125 mg/L) TEM (64 - 2 mg/l) TEM (64 - 2 mg/l) TIM (128 - 4 mg/L) TIM (128 - 4 mg/L) PIPTAZO (64 - 2 mg/L) PIPTAZO (64 - 2 mg/L) MERO (16 - 0.5 mg/L) MERO (16 - 0.5 mg/L)

17. Results: From 45 sputum samples, 705 bacterial morphotypes were referred for identification and susceptibility testing: From 45 sputum samples, 705 bacterial morphotypes were referred for identification and susceptibility testing: Identification of 705 bacterial isolates: Pseudomonas aeruginosa 645 Alcaligenes xylosoxidans 27 Pseudomonas fluorescens 22 Stenotrophomonas maltophilia 8 Bulkholderia cenocepacia 1 Moraxella species 1 Sphingobacterium spiritovorum 1

18. Results: 43 / 45 samples yielded P. aeruginosa (one sample: B. cenocepacia only. 43 / 45 samples yielded P. aeruginosa (one sample: B. cenocepacia only. one sample: A. xylosoxidans only). An average of three morphotypes was tested from routine media (range 1 – 5). An average of three morphotypes was tested from routine media (range 1 – 5).

19. Table 1: Number of specimens containing strains of P. aeruginosa resistant to antimicrobials AMIKATMCAZCIPROGENTMEROTAZOTIMTEMTOBRA Total31262930353033352913 Routine method No. detected: 1323182420212023267 % detected: 42886280577061669054 Selective method No. detected: 31202627352933352513 % detected: 1007790901009710010086100

20. Example: 14 Yr old CF pateint: LTA Example: 14 Yr old CF pateint: LTA AMIKATMCAZCIPROGENTMEROTAZOTIMTEMTOBRA Met ® P.aeruginosa <2 (4) <2<2 0.25 (0.5) 1 0.5 (2) <2 <4 (64) 2 (4) <0.5 L ® P.aeruginosa<2<2<2 0.5 (1) 10.5<2<4 4 (8) <0.5 muc ® P.aeruginosa<2<2<2 0.25 (0.5) 0.5 (1) 0.5<2<44<0.5 L (Amik) P.aeruginosa64 L (Caz) P.aeruginosa32 L (Cip) P.aeruginosa2 gr (gent) P.aeruginosa16 L (Mero) P.aeruginosa 8 ( 16 ) M (Tazo) P.aeruginosa>64 L (Tim) P.aeruginosa > 128 S (Tob) P.aeruginosa16 L (Atm) P.fluorescens32 S (Caz) P.fluorescens32- L (Gent) P.fluorescens4 L (Tazo) S. spiritovorum 32 M (Tim) P.fluorescens>128 L (Tem) P.fluorescens>64 tiny (Tob) P.fluorescens 8 (16)

21. Table 2: Positive predictive value of growth on selective agars to predict antimicrobial resistance by MIC testing. AMIKATMCAZCIPROGENTMEROTAZOTIMTEMTOBRA PPV (%) 921009394889396959689

22. Conclusions: Growth on selective media containing breakpoint concentrations of antimicrobials can be used to detect antimicrobial resistance in P. aeruginosa (PPV: 88 – 100 %). Growth on selective media containing breakpoint concentrations of antimicrobials can be used to detect antimicrobial resistance in P. aeruginosa (PPV: 88 – 100 %). For most antimicrobials, the use of selective media facilitates detection of more antimicrobial resistance when compared with routine methods that involve selection of morphotypes for susceptibility testing. For most antimicrobials, the use of selective media facilitates detection of more antimicrobial resistance when compared with routine methods that involve selection of morphotypes for susceptibility testing.

23. Disc susceptibility testing of Pseudomonas aeruginosa from sputa of patients with cystic fibrosis

24. Disc susceptibility testing was performed by BSAC method: 38 strains of P. aeruginosa were tested. Each strain was isolated from a distinct patient with CF. 38 strains of P. aeruginosa were tested. Each strain was isolated from a distinct patient with CF. 0.5 McFarland suspension of strains cultured overnight on blood agar. 0.5 McFarland suspension of strains cultured overnight on blood agar. 1/100 dilution of suspension in sterile water. 1/100 dilution of suspension in sterile water. Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A). Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A). Incubation for 24 h at 37°C (48 h only if required) to obtain semi- confluent growth. Incubation for 24 h at 37°C (48 h only if required) to obtain semi- confluent growth. Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the same 0.5 McFarland suspensions. Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the same 0.5 McFarland suspensions.

25. Disc susceptibility testing was performed by BSAC method: Controls: Controls: With each batch of disc susceptibility tests and agar dilution tests we included: With each batch of disc susceptibility tests and agar dilution tests we included: Pseudomonas aeruginosa NCTC 10662 Pseudomonas aeruginosa NCTC 10662 Escherichia coli NCTC 10418 Escherichia coli NCTC 10418 This was performed to ensure that zones of inhibition fell within published acceptable limits and MIC values were within one dilution of published acceptable values.

26. Results: 36 / 38 strains of P. aeruginosa grew well within 24 h and produced an ideal semi- confluent inoculum. 36 / 38 strains of P. aeruginosa grew well within 24 h and produced an ideal semi- confluent inoculum. 1 strain generated a light growth within the ‘acceptable range’. 1 strain generated a light growth within the ‘acceptable range’. One strain produced no growth on repeated disc susceptibility testing. One strain produced no growth on repeated disc susceptibility testing. (Control strains produced acceptable results) (Control strains produced acceptable results)

27. R or I by disc (6) = Resistant by MIC Sensitive by disc (31): Sensitive : 17 Intermediate: 6 Resistant : 8 SensitiveIntermediateResistant ≤ 8 mg/L16 mg/L ≥ 32 mg/L ≥ 19 mm 16-18 mm ≤ 15 mm

28. Resistant by disc (8) = Resistant by MIC Sensitive by disc (28): Sensitive : 18 Resistant : 10 SensitiveResistant ≤ 4 mg/L ≥ 8 mg/L ≥ 18 mm ≤ 17 mm

29. Resistant by disc (1) = Resistant by MIC Sensitive by disc (35): Sensitive : 34 Resistant : 1 SensitiveResistant ≤ 4 mg/L ≥ 8 mg/L ≥ 20 mm ≤ 19 mm

30. Resistant by disc (8) = Resistant by MIC Sensitive by disc (17): Sensitive : 7 Intermediate: 9 Resistant : 1 SensitiveIntermediateResistant ≤ 0.5 mg/L1 mg/L ≥ 2 mg/L ≥ 30 mm 20-29 mm ≤ 19 mm

31. Resistant by disc (8) = Resistant by MIC Sensitive by disc (29): Sensitive : 23 Resistant : 6 SensitiveResistant ≤ 8 mg/L ≥ 16 mg/L ≥ 23 mm ≤ 22 mm

32. Resistant by disc (10) = 9 Resistant by MIC (1 S) Sensitive by disc (27): Sensitive : 21 Resistant : 6 SensitiveResistant ≤ 8 mg/L ≥ 16 mg/L ≥ 24 mm ≤ 23 mm

33. Resistant by disc (11) = R or I by MIC Sensitive by disc (22): Sensitive : 19 Intermediate: 3 (MIC = 4) Resistant : 0 Sensitive IntermediateResistant ≤ 2 mg/L 4-8 mg/L≥ 16 mg/L ≥ 27 mm 22-26 mm ≤ 21 mm

34. Resistant by disc (7) = Resistant by MIC Sensitive by disc (29): Sensitive : 21 Resistant : 8 (MIC 16-32) SensitiveResistant ≤ 16 mg/L ≥ 32 mg/L ≥ 22 mm ≤ 21 mm

35. Resistant by disc (17) Resistant by MIC: 16 R 1S Sensitive by disc : 19 Sensitive : 18 Resistant : 1 S ≤ 8 mg/L (Unconfirmed) R > 8 mg/L (Unconfirmed) S ≥ 20 mm (Unconfirmed) R ≤ 19 mm (Unconfirmed) P. aeruginosa NCTC 10662 MIC = > 64 mg/L E. coli NCTC 10418 MIC = 4 mg/L

36. Disc susceptibility testing of Burkholderia cepacia complex using BSAC criteria for interpretation of Pseudomonas spp.

37. Disc susceptibility testing was performed by BSAC method: 0.5 McFarland suspension of strains cultured overnight on blood agar. 0.5 McFarland suspension of strains cultured overnight on blood agar. 1/100 dilution of suspension in sterile water. 1/100 dilution of suspension in sterile water. Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A). Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A). Incubation for 24 h at 37°C (48 h only if required) to obtain semi-confluent growth. Incubation for 24 h at 37°C (48 h only if required) to obtain semi-confluent growth. Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the same 0.5 McFarland suspensions. Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the same 0.5 McFarland suspensions.

38. 40 strains of Burkholderia cepacia complex used for disc susceptibility testing : LMG 16654Burkholderia cenocepaciaLMG 13010Burkholderia multivorans LMG 16656Burkholderia cenocepaciaLMG 16665Burkholderia multivorans LMG 16659Burkholderia cenocepaciaLMG 17588Burkholderia multivorans LMG 18826Burkholderia cenocepaciaLMG 18822Burkholderia multivorans LMG 18827Burkholderia cenocepaciaLMG 18823Burkholderia multivorans LMG 18829Burkholderia cenocepaciaLMG 18824Burkholderia multivorans LMG 18830Burkholderia cenocepaciaLMG 18825Burkholderia multivorans LMG 18832Burkholderia cenocepacia LMG 18863Burkholderia cenocepacia

39. 40 strains of Burkholderia cepacia complex (cont…) LMG 14086Burkholderia stabilisCEP 0092Burkholderia cepacia complex LMG 14294Burkholderia stabilisCEP 0408Burkholderia cepacia complex LMG 16660Burkholderia stabilisCEP 0591Burkholderia cepacia complex LMG 18888Burkholderia stabilisCEP 0615Burkholderia cepacia complex LMG 10929Burkholderia vietnamiensisCEP 0686Burkholderia cepacia complex LMG 16232Burkholderia vietnamiensisCEP 0769Burkholderia cepacia complex LMG 18835Burkholderia vietnamiensisCEP 0786Burkholderia cepacia complex LMG 18836Burkholderia vietnamiensisCEP 0891Burkholderia cepacia complex LMG 1222Burkholderia cepaciaCEP 0945Burkholderia cepacia complex LMG 17997Burkholderia cepaciaCEP 1012Burkholderia cepacia complex LMG 18821Burkholderia cepaciaCEP 1190Burkholderia cepacia complex LMG 2162Burkholderia cepaciaCEP 1235Burkholderia cepacia complex

40. Results: 35 / 40 strains of B. cepacia complex grew well within 24 h and produced an ideal semi-confluent inoculum. 35 / 40 strains of B. cepacia complex grew well within 24 h and produced an ideal semi-confluent inoculum. 5 strains generated a light growth within the ‘acceptable range’. 5 strains generated a light growth within the ‘acceptable range’. One strain produced an unacceptable lawn (too light) which required incubation for 48 h. One strain produced an unacceptable lawn (too light) which required incubation for 48 h.

41. Resistant by disc = Resistant by MIC Sensitive by disc (31): Sensitive : 17 Intermediate: 6 Resistant : 8 Sensitive IntermediateResistant ≤ 0.5 mg/L 1 mg/L ≥ 2 mg/L ≥ 30 mm 20-29 mm ≤ 19 mm

42. Resistant by disc (7) = Resistant by MIC Sensitive by disc (33): Sensitive : 31 Resistant : 2 SensitiveResistant ≤ 8 mg/L ≥ 16 mg/L ≥ 23 mm ≤ 22 mm

43. Resistant by disc (4) = Resistant by MIC Sensitive by disc (36): Sensitive : 34 Resistant : 2 SensitiveResistant ≤ 8 mg/L ≥ 16 mg/L ≥ 24 mm ≤ 23 mm

44. R or I by disc (6) = R or I by MIC Sensitive by disc (34): Sensitive : 32 Resistant : 2 SensitiveIntermediateResistant ≤ 2 mg/L4-8 mg/L ≥ 16 mg/L ≥ 27 mm 22-26 mm ≤ 21 mm

45. No resistance by disc Sensitive by disc (40): Sensitive : 36 Resistant : 4 SensitiveResistant ≤ 16 mg/L ≥ 32 mg/L ≥ 22 mm ≤ 21 mm

46. Resistant by disc (8) = Resistant by MIC Sensitive by disc (32): Sensitive : 30 Resistant : 2 S ≤ 8 mg/L (Unconfirmed) R > 8 mg/L (Unconfirmed) S ≥ 20 mm (Unconfirmed) R ≤ 19 mm (Unconfirmed) P. aeruginosa NCTC 10662 MIC = > 64 mg/L E. coli NCTC 10418 MIC = 4 mg/L

47. MULTIPLE ANTIBIOTIC SYNERGY TESTING AGAINST P. AERUGINOSA AND B. CEPACIA COMPLEX STRAINS FROM CYSTIC FIBROSIS PATIENTS.

48. Principles: Inoculation of 10 6 cfu/ml test bacteria into Isosensitest broth with 78 distinct antimicrobial combinations. Inoculation of 10 6 cfu/ml test bacteria into Isosensitest broth with 78 distinct antimicrobial combinations. Incubation for 48 h at 37°C. Incubation for 48 h at 37°C.

49. Antimicrobial combinations tested: Tests performed in microtitre wells. Final volume: 100µl.

50. Microtitre wells interpreted as : ‘growth’ or ‘no growth’ by spectrophotometry.

51. Synergy testing (continued…) Clear or ‘no growth’ wells are subcultured (50 µl) onto blood agar. Clear or ‘no growth’ wells are subcultured (50 µl) onto blood agar. Colony counts are then performed after 48 h incubation. Colony counts are then performed after 48 h incubation. Antibiotics are assessed as bacteriostatic or bactericidal. Antibiotics are assessed as bacteriostatic or bactericidal. Antimicrobial combinations that result in complete kill are recommended for therapy. Antimicrobial combinations that result in complete kill are recommended for therapy.

54. Work performed by: Larissa Laine, Susan Hughes, Audrey Nicholson & John Perry. Microbiology Department Freeman Hospital Newcastle upon Tyne