BSAC Standardised Disc Susceptibility Test User Group Day. Royal College of Physicians, London. 8 June 2007 Susceptibility testing of mucoid Pseudomonas.

BSAC Standardised Disc Susceptibility BSAC Standardised Disc Susceptibility Test User Group Day.Test User Group Day.

Royal College of Physicians, London.Royal College of Physicians, London.8 June 20078 June 2007

Susceptibility testing of mucoid Susceptibility testing of mucoid PseudomonasPseudomonas and and BurkholderiaBurkholderia strains from patients with cystic fibrosis strains from patients with cystic fibrosis

including evaluation of the BSAC standardised method.including evaluation of the BSAC standardised method.

J.D. PerryJ.D. PerryFreeman HospitalFreeman Hospital

Newcastle upon TyneNewcastle upon Tyne

Contents:Contents:• Direct susceptibility testing of whole sputum Direct susceptibility testing of whole sputum

from CF patients to detect resistant strains from CF patients to detect resistant strains of of P. aeruginosaP. aeruginosa..

• Preliminary work to validate disc Preliminary work to validate disc susceptibility testing with susceptibility testing with P. aeruginosaP. aeruginosa and and B. cepaciaB. cepacia from CF patients. from CF patients.

• MCBT testing of resistant strains of MCBT testing of resistant strains of P. P. aeruginosaaeruginosa and and B. cepaciaB. cepacia from CF patients. from CF patients.

Direct susceptibility testing of Direct susceptibility testing of Pseudomonas aeruginosaPseudomonas aeruginosa from from

sputa of patients with cystic sputa of patients with cystic fibrosisfibrosis

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

Foweraker Foweraker et alet al. 2005. . 2005. Methods:Methods:

• One hundred and one sputa were One hundred and one sputa were cultured. Four colonies of each cultured. Four colonies of each P.aeruginosaP.aeruginosa morphotype were morphotype were suspended. suspended.

•Susceptibility to 12 agents by Susceptibility to 12 agents by disc diffusion was tested disc diffusion was tested individually or by pooling the individually or by pooling the four suspensions. four suspensions.

Foweraker Foweraker et alet al. 2005. . 2005. Results / ConclusionsResults / Conclusions • In some cases, all four colonies of a single In some cases, all four colonies of a single

morphotype had different antibiograms. morphotype had different antibiograms.

• The susceptibility profiles of single isolates The susceptibility profiles of single isolates of of P. aeruginosaP. aeruginosa correlated poorly with correlated poorly with pooled cultures, with the pooled tests pooled cultures, with the pooled tests missing resistance.missing resistance.

• A range of susceptibility patterns is seen, A range of susceptibility patterns is seen, even within a morphotype. Routine test even within a morphotype. Routine test results are not reproducible and results are not reproducible and underestimate resistance. underestimate resistance.

Can antimicrobial resistance be Can antimicrobial resistance be detected more reliably by culture of detected more reliably by culture of

whole sputum onto media containing whole sputum onto media containing antimicrobials ?antimicrobials ?

Aim of the current Aim of the current investigation:investigation:

Routine culture:Routine culture:

• Sputum samples were homogenized Sputum samples were homogenized 1:1 with Sputasol.1:1 with Sputasol.

• Routine culture:Routine culture:

• 10 µl aliquots of liquid sputum were 10 µl aliquots of liquid sputum were plated onto:plated onto:

• Chocolate agar (+Bacitracin), Blood Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, agar, CLED agar, Isosensitest agar, Pseudomonas Selective agar & Pseudomonas Selective agar & Burkholderia cepaciaBurkholderia cepacia Selective agar. Selective agar.

Routine culture (continued):Routine culture (continued):

• A 10 µl aliquot of liquid sputum was diluted A 10 µl aliquot of liquid sputum was diluted by addition to 9.99 ml sterile water by addition to 9.99 ml sterile water (1/1000). (1/1000).

• 10 µl of this diluted sample was also plated 10 µl of this diluted sample was also plated onto: onto:

• Chocolate agar (+Bacitracin), Blood agar, Chocolate agar (+Bacitracin), Blood agar, CLED agar, Isosensitest agar, Pseudomonas CLED agar, Isosensitest agar, Pseudomonas Selective agar & Burkholderia cepacia Selective agar & Burkholderia cepacia Selective agar.Selective agar.

Selective culture:Selective culture:

A 10 µl aliquot of liquid sputum was A 10 µl aliquot of liquid sputum was inoculated onto 10 distinct Isosensitest agar inoculated onto 10 distinct Isosensitest agar plates incorporating the following plates incorporating the following antimicrobials: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 Piperacillin-tazobactam (16

mg/L) mg/L) • Ticarcillin-clavulanic acid Ticarcillin-clavulanic acid

(32 mg/L)(32 mg/L)

Summary of media usedSummary of media usedRoutine culture:Routine culture:

• 10 µl of Neat and diluted 10 µl of Neat and diluted homogenized sputa were homogenized sputa were inoculated onto:inoculated onto:

• Cholcolate-BacitracinCholcolate-Bacitracin• Blood agar.Blood agar.• CLED agar.CLED agar.• Isosensitest agar.Isosensitest agar.• PseudomonasPseudomonas selective agar. selective agar.• B. cepaciaB. cepacia selective agar. selective agar.

(Total = 10 culture plates).(Total = 10 culture plates).

‘‘Selective’ cultureSelective’ culture

• Culture of neat Culture of neat homogenized sputa on homogenized sputa on Isosensitest agar (x 10) Isosensitest agar (x 10) containing:containing:

• AmikacinAmikacin• GentamicinGentamicin• TobramycinTobramycin• AztreonamAztreonam• CeftazidimeCeftazidime• MeropenemMeropenem• Temocillin Temocillin • CiprofloxacinCiprofloxacin• Piperacillin-tazobactamPiperacillin-tazobactam• Ticarcillin-clavulanic acidTicarcillin-clavulanic acid

Interpretation of cultures:Interpretation of cultures:

• All plates were incubated for 72 hours and All plates were incubated for 72 hours and examined after 24, 48 and 72 hours.examined after 24, 48 and 72 hours.

• All colonial variants or ‘morphotypes’ of All colonial variants or ‘morphotypes’ of Gram-negative bacteria were sub-cultured Gram-negative bacteria were sub-cultured onto a blood agar plate to obtain pure onto a blood agar plate to obtain pure cultures. These subcultures were used for cultures. These subcultures were used for MIC testing, identification and storage in MIC testing, identification and storage in glycerol for potential further studies.glycerol for potential further studies.

Identification:Identification:• P. aeruginosaP. aeruginosa was identified by inoculation of all was identified by inoculation of all

morphotypes onto PC agar, cetrimide agar and blood morphotypes onto PC agar, cetrimide agar and blood agar to test for growth at 42°C.agar to test for growth at 42°C.

• PC agar contains: PC agar contains: • 30 mg/L 30 mg/L 9-chloro-9-[4-(diethylamino)phenyl]-9,10-9-chloro-9-[4-(diethylamino)phenyl]-9,10-

dihydro-10-phenylacridine hydrochloride (C-390) and 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 Growth on this agar is diagnostic for P. aeruginosaP. aeruginosa with 100 % specificitywith 100 % specificity11..

1 1 J Clin Microbiol. 1988 Sep;26(9):1910-2. J Clin Microbiol. 1988 Sep;26(9):1910-2.

Identification:Identification:• For this study:For this study:

• Growth on cetrimide + growth on PC agar Growth on cetrimide + growth on PC agar + growth at 42°C = + growth at 42°C = P. aeruginosaP. aeruginosa..

• All other strains were identified by API 20 All other strains were identified by API 20 NE.NE.

Susceptibility testing:Susceptibility testing:

• All morphotypes (from any medium) All morphotypes (from any medium) were referred for MIC testing against were referred for MIC testing against 10 antibiotics. 10 antibiotics.

• MIC testing was performed using MIC testing was performed using agar dilution in Isosensitest agar with agar dilution in Isosensitest agar with a final inoculum of 10 000 cfu/spot.a final inoculum of 10 000 cfu/spot.

• MIC’s were recorded after both 24 MIC’s were recorded after both 24 and 48 hours of incubation at 37°C.and 48 hours of incubation at 37°C.

Susceptibility testing:Susceptibility testing:

Ranges of antibiotics Ranges of antibiotics for MIC testing:for MIC testing:

• AMIK AMIK (64 - 2 mg/L) (64 - 2 mg/L)

• GENT GENT (16 - 0.5 mg/L)(16 - 0.5 mg/L)

• TOBRA TOBRA (16 - 0.5 (16 - 0.5 mg/L)mg/L)

• ATM ATM (32 - 1 mg/L)(32 - 1 mg/L)

• CAZ CAZ (32 - 1 mg/L)(32 - 1 mg/L)

• CIPRO CIPRO (4 - 0.125 mg/L)(4 - 0.125 mg/L)• TEM TEM (64 - 2 mg/l)(64 - 2 mg/l)• TIM TIM (128 - 4 mg/L) (128 - 4 mg/L) • PIPTAZO PIPTAZO (64 - 2 mg/L)(64 - 2 mg/L)• MERO MERO (16 - 0.5 mg/L)(16 - 0.5 mg/L)

Results:Results:

• From 45 sputum From 45 sputum samples, 705 samples, 705 bacterial bacterial morphotypes were morphotypes were referred for referred for identification and identification and susceptibility susceptibility testing:testing:

Identification of 705 bacterial Identification of 705 bacterial isolates:isolates:

Pseudomonas aeruginosaPseudomonas aeruginosa6464

55

Alcaligenes xylosoxidansAlcaligenes xylosoxidans 2727

Pseudomonas fluorescensPseudomonas fluorescens 2222

Stenotrophomonas Stenotrophomonas maltophiliamaltophilia 88

Bulkholderia cenocepacia Bulkholderia cenocepacia 11

Moraxella speciesMoraxella species 11

Sphingobacterium Sphingobacterium spiritovorum spiritovorum 11

Results:Results:

• 43 / 45 samples yielded 43 / 45 samples yielded P. aeruginosaP. aeruginosa (one sample: (one sample: B. cenocepaciaB. cenocepacia only. only.

one sample: one sample: A. xylosoxidansA. xylosoxidans only). only).

• An average of three morphotypes was An average of three morphotypes was tested from routine media (range 1 – tested from routine media (range 1 – 5).5).

Table 1: Number of specimens containing strains Table 1: Number of specimens containing strains of of P. aeruginosaP. aeruginosa resistant to antimicrobials resistant to antimicrobials

AMIKAMIK ATMATM CAZCAZ CIPROCIPRO GENTGENT MEROMERO TAZOTAZO TIMTIM TEMTEMTOBRTOBR

AA

TotalTotal 3131 2626 2929 3030 3535 3030 3333 3535 2929 1313

Routine Routine methodmethod

No. detected: No. detected: 1313 2323 1818 2424 2020 2121 2020 2323 2626 77

% detected:% detected: 4242 8888 6262 8080 5757 7070 6161 6666 9090 5454

Selective Selective methodmethod

No. detected: No. detected: 3131 2020 2626 2727 3535 2929 3333 3535 2525 1313

% detected:% detected: 100100 7777 9090 9090 100100 9797 100100101000 8686 100100

Example: 14 Yr old CF pateint: LTAExample: 14 Yr old CF pateint: LTA

AMIKAMIK ATATMMCACAZZ CIPROCIPRO GENTGENT MERMEROO TAZTAZOO TIMTIM TETEMM TOBRATOBRA

Met ®Met ® P.aeruginosaP.aeruginosa <2 (4)<2 (4) <2<2 <2<2 0.25 (0.5)0.25 (0.5) 11 0.5 (2)0.5 (2) <2<2 <4 (64)<4 (64) 2 (4)2 (4) <0.5<0.5L ®L ® P.aeruginosaP.aeruginosa <2<2 <2<2 <2<2 0.5 (1)0.5 (1) 11 0.50.5 <2<2 <4<4 4 (8)4 (8) <0.5<0.5muc ®muc ® P.aeruginosaP.aeruginosa <2<2 <2<2 <2<2 0.25 (0.5)0.25 (0.5) 0.5 (1)0.5 (1) 0.50.5 <2<2 <4<4 44 <0.5<0.5

L (Amik)L (Amik) P.aeruginosaP.aeruginosa 6464

L (Caz)L (Caz) P.aeruginosaP.aeruginosa 3232

L (Cip)L (Cip) P.aeruginosaP.aeruginosa 22

gr (gent)gr (gent) P.aeruginosaP.aeruginosa 1616

L (Mero)L (Mero) P.aeruginosaP.aeruginosa 88 ( (1616))

M (Tazo)M (Tazo) P.aeruginosaP.aeruginosa >64>64

L (Tim)L (Tim) P.aeruginosaP.aeruginosa >>128128

S (Tob)S (Tob) P.aeruginosaP.aeruginosa 1616

L (Atm)L (Atm) P.fluorescensP.fluorescens 3232

S (Caz)S (Caz) P.fluorescensP.fluorescens 3232 --

L (Gent)L (Gent) P.fluorescensP.fluorescens 44

L (Tazo)L (Tazo) S. S. spiritovorum spiritovorum 3232

M (Tim)M (Tim) P.fluorescensP.fluorescens >128>128

L (Tem)L (Tem) P.fluorescensP.fluorescens >64>64

tiny tiny (Tob)(Tob) P.fluorescensP.fluorescens 8 (16)8 (16)

Table 2: Positive predictive value of growth Table 2: Positive predictive value of growth on selective agars to predict antimicrobial on selective agars to predict antimicrobial resistance by MIC testing.resistance by MIC testing.

AMIKAMIK ATMATM CAZCAZ CIPROCIPRO GENTGENT MEROMERO TAZOTAZO TIMTIM TEMTEM TOBRATOBRA

PPV (%)PPV (%) 9292 100100 9393 9494 8888 9393 9696 9595 9696 8989

Conclusions:Conclusions:

• Growth on selective media containing Growth on selective media containing breakpoint concentrations of antimicrobials breakpoint concentrations of antimicrobials can be used to detect antimicrobial can be used to detect antimicrobial resistance in resistance in P. aeruginosaP. aeruginosa (PPV: 88 – 100 %). (PPV: 88 – 100 %).

• For most antimicrobials, the use of selective For most antimicrobials, the use of selective media facilitates detection of more media facilitates detection of more antimicrobial resistance when compared with antimicrobial resistance when compared with routine methods that involve selection of routine methods that involve selection of morphotypes for susceptibility testing.morphotypes for susceptibility testing.

Disc susceptibility testing of Disc susceptibility testing of Pseudomonas aeruginosaPseudomonas aeruginosa from from

sputa of patients with cystic sputa of patients with cystic fibrosisfibrosis

Disc susceptibility testing was Disc susceptibility testing was performed by BSAC method:performed by BSAC method:

• 38 strains of 38 strains of P. aeruginosaP. aeruginosa were tested. Each strain was isolated were tested. Each strain was isolated from a distinct patient with CF.from a distinct patient with CF.

• 0.5 McFarland suspension of strains cultured overnight on blood 0.5 McFarland suspension of strains cultured overnight on blood agar.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 Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A).agar (PO O779A).

• Incubation for 24 h at 37°C (48 h only if required) to obtain semi-Incubation for 24 h at 37°C (48 h only if required) to obtain semi-confluent growth.confluent growth.

• Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the same 0.5 McFarland using the BSAC method using the same 0.5 McFarland suspensions.suspensions.

Disc susceptibility testing was Disc susceptibility testing was performed by BSAC method:performed by BSAC method:• Controls:Controls:

• With each batch of disc susceptibility tests With each batch of disc susceptibility tests and agar dilution tests we included:and agar dilution tests we included:

• Pseudomonas aeruginosaPseudomonas aeruginosa NCTC 10662 NCTC 10662• Escherichia coliEscherichia coli NCTC 10418 NCTC 10418

This was performed to ensure that zones of This was performed to ensure that zones of inhibition fell within published acceptable inhibition fell within published acceptable limits and MIC values were within one limits and MIC values were within one dilution of published acceptable values.dilution of published acceptable values.

Results:Results:

• 36 / 38 strains of 36 / 38 strains of P. aeruginosaP. aeruginosa grew grew well within 24 h and produced an ideal well within 24 h and produced an ideal semi-confluent inoculum.semi-confluent inoculum.

• 1 strain generated a light growth 1 strain generated a light growth within the ‘acceptable range’.within the ‘acceptable range’.

• One strain produced no growth on One strain produced no growth on repeated disc susceptibility testing.repeated disc susceptibility testing.

• (Control strains produced acceptable (Control strains produced acceptable results)results)

Amikacin 10 µg disc with P.aeruginosa

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MIC (mg/L)

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R or I by disc (6) = Resistant by MIC

Sensitive by disc (31):

Sensitive : 17 Intermediate: 6Resistant : 8

Sensitive Intermediate Resistant

≤ 8 mg/L 16 mg/L ≥ 32 mg/L≥ 19 mm 16-18 mm ≤ 15 mm

Resistant by disc (8) = Resistant by MIC


Sensitive : 18 Resistant : 10

Gentamicin 10 µg disc with P.aeruginosa

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MIC (mg/L)

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Sensitive Resistant

≤ 4 mg/L ≥ 8 mg/L≥ 18 mm ≤ 17 mm




Tobramycin 10 µg disc with P.aeruginosa

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MIC (mg/L)

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Sensitive Resistant

≤ 4 mg/L ≥ 8 mg/L≥ 20 mm ≤ 19 mm




Ciprofloxacin 5 µg disc with P.aeruginosa

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≤ 0.5 mg/L 1 mg/L ≥ 2 mg/L≥ 30 mm 20-29 mm ≤ 19 mm



Sensitive : 23Resistant : 6

Aztreonam 30 µg disc with P.aeruginosa

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MIC (mg/L)

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SensitiveResistant

≤ 8 mg/L ≥ 16 mg/L≥ 23 mm ≤ 22 mm

Resistant by disc (10) = 9 Resistant by MIC (1 S)



Ceftazidime 30 µg disc with P.aeruginosa

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MIC (mg/L)

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SensitiveResistant

≤ 8 mg/L ≥ 16 mg/L≥ 24 mm ≤ 23 mm

Resistant by disc (11) = R or I by MIC


Sensitive : 19 Intermediate: 3 (MIC = 4)Resistant : 0

Meropenem 10 µg disc with P.aeruginosa

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0 5 10 15 20 25 30 35

MIC (mg/L)

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≤ 2 mg/L 4-8 mg/L ≥ 16 mg/L≥ 27 mm 22-26 mm ≤ 21 mm



Sensitive : 21 Resistant : 8 (MIC 16-32)

Piperacillin / tazobactam 75 / 10 µg disc with P.aeruginosa

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0 50 100 150 200 250 300

MIC (mg/L)

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)Sensitive Resistant

≤ 16 mg/L ≥ 32 mg/L≥ 22 mm ≤ 21 mm

Resistant by disc (17) Resistant by MIC: 16 R 1S

Sensitive by disc : 19

Sensitive : 18Resistant : 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/LE. coli NCTC 10418 MIC = 4 mg/L

Temocillin 30 µg disc with P.aeruginosa

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Disc susceptibility testing of Disc susceptibility testing of Burkholderia cepaciaBurkholderia cepacia complex complex

using BSAC criteria for using BSAC criteria for interpretation of interpretation of PseudomonasPseudomonas

spp.spp.

Disc susceptibility testing was Disc susceptibility testing was performed by BSAC method:performed by BSAC method:• 0.5 McFarland suspension of strains cultured 0.5 McFarland suspension of strains cultured

overnight on blood agar.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 Dilution spread with a swab onto pre-poured Oxoid Isosensitest agar (PO O779A).Oxoid Isosensitest agar (PO O779A).

• Incubation for 24 h at 37°C (48 h only if required) Incubation for 24 h at 37°C (48 h only if required) to obtain semi-confluent growth.to obtain semi-confluent growth.

• Agar dilution MIC’s were performed in Isosensitest Agar dilution MIC’s were performed in Isosensitest agar (Oxoid) using the BSAC method using the agar (Oxoid) using the BSAC method using the same 0.5 McFarland suspensions.same 0.5 McFarland suspensions.

40 strains of Burkholderia cepacia complex used for disc susceptibility testing :

LMG 16654 Burkholderia cenocepacia LMG 13010 Burkholderia multivorans







LMG 18832 Burkholderia cenocepacia

LMG 18863 Burkholderia cenocepacia

40 strains of Burkholderia cepacia complex (cont…)

LMG 14086 Burkholderia stabilis CEP 0092 Burkholderia cepacia complex




LMG 10929 Burkholderia vietnamiensis CEP 0686 Burkholderia cepacia complex




LMG 1222 Burkholderia cepacia CEP 0945 Burkholderia cepacia complex




Results:Results:

• 35 / 40 strains of 35 / 40 strains of B. cepaciaB. cepacia complex complex grew well within 24 h and produced grew well within 24 h and produced an ideal semi-confluent inoculum.an ideal semi-confluent inoculum.

• 5 strains generated a light growth 5 strains generated a light growth within the ‘acceptable range’.within the ‘acceptable range’.

• One strain produced an unacceptable One strain produced an unacceptable lawn (too light) which required lawn (too light) which required incubation for 48 h.incubation for 48 h.

Resistant by disc = Resistant by MIC



Ciprofloxacin 5 µg disc with B. cepacia complex

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MIC (mg/L)

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)Sensitive IntermediateResistant

≤ 0.5 mg/L 1 mg/L ≥ 2 mg/L

≥ 30 mm 20-29 mm ≤ 19 mm




Aztreonam 30 µg disc with B. cepacia complex

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Sensitive Resistant

≤ 8 mg/L ≥ 16 mg/L≥ 23 mm ≤ 22 mm




Ceftazidime 30 µg disc with B. cepacia complex

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Sensitive Resistant

≤ 8 mg/L ≥ 16 mg/L≥ 24 mm ≤ 23 mm

R or I by disc (6) = R or I by MIC



Meropenem 10 µg disc with B. cepacia complex

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MIC (mg/L)

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≤ 2 mg/L 4-8 mg/L ≥ 16 mg/L≥ 27 mm 22-26 mm ≤ 21 mm

No resistance by disc



Piperacillin / tazobactam 75 / 10 µg disc with B. cepacia complex

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Sensitive Resistant

≤ 16 mg/L ≥ 32 mg/L≥ 22 mm ≤ 21 mm




Temocillin 30 µg disc with B. cepacia complex

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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/LE. coli NCTC 10418 MIC = 4 mg/L

MULTIPLE ANTIBIOTIC MULTIPLE ANTIBIOTIC SYNERGY TESTING AGAINST SYNERGY TESTING AGAINST

P. AERUGINOSAP. AERUGINOSA AND AND B. CEPACIAB. CEPACIA COMPLEX COMPLEX

STRAINS FROM CYSTIC STRAINS FROM CYSTIC FIBROSIS PATIENTS.FIBROSIS PATIENTS.

Principles:Principles:

•Inoculation of 10Inoculation of 106 6 cfu/ml test cfu/ml test bacteria into Isosensitest bacteria into Isosensitest broth with 78 distinct broth with 78 distinct antimicrobial combinations.antimicrobial combinations.

•Incubation for 48 h at 37°C.Incubation for 48 h at 37°C.

1 2 3 4 5 6 7 8 9 10 11 12A TIM CAZ CIP TAZ AK MER AZT TOB CHL MIN FOS SXTB TIM/CAZ CAZ/TOB CIP/TOB TAZ/SXT AK/TIM MER/CHL AZT/MER TOB/AZT CHL/TAZ MIN/MER FOS/MER SXT/AKC TIM/CIP CAZ/CIP CIP/AZT TAZ/AK AK/CAZ MER/TAZ AZT/CHL TOB/MER CHL/SXT MIN/AZT FOS/AZT SXT/TIMD TIM/MIN CAZ/MIN CIP/MIN TAZ/TIM AK/CIP MER/SXT AZT/TAZ TOB/CHL CHL/AK MIN/TOB FOS/TOB SXT/CAZE TIM/FOS CAZ/FOS CIP/FOS TAZ/CAZ AK/MIN MER/AK AZT/SXT TOB/TAZ CHL/TIM MIN/CHL FOS/CHL SXT/CIPF x x x TAZ/CIP AK/FOS MER/TIM AZT/AK TOB/SXT CHL/CAZ MIN/FOS FOS/TAZ SXT/MING x x x x x MER/CAZ AZT/TIM TOB/AK CHL/CIP MIN/TAZ Sterility SXT/FOSH x x x x x MER/CIP AZ/CAZ TOB/TIM x x Sterility Growth

Antimicrobial combinations Antimicrobial combinations tested:tested:

Tests performed in microtitre wells. Tests performed in microtitre wells.

Final volume: 100µl.Final volume: 100µl.

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

Synergy testing (continued…)Synergy testing (continued…)

• Clear or ‘no growth’ wells are Clear or ‘no growth’ wells are subcultured (50 µl) onto blood agar.subcultured (50 µl) onto blood agar.

• Colony counts are then performed Colony counts are then performed after 48 h incubation.after 48 h incubation.

• Antibiotics are assessed as Antibiotics are assessed as bacteriostatic or bactericidal.bacteriostatic or bactericidal.

• Antimicrobial combinations that Antimicrobial combinations that result in complete kill are result in complete kill are recommended for therapy.recommended for therapy.

Best 10 antimicrobial combinations for kill of P.aeruginosa (no. of strains = 42) from cases of CF.

Antibiotic combination % of strains killed

Piperacillin-tazobactam/Ciprofloxacin 50Tobramycin/Piperacillin-tazobactam 40

Amikacin/Ceftazidime 38Ceftazidime/Tobramycin 36

Ceftazidime/Ciprofloxacin 36Tobramycin/Aztreonam 36Tobramycin/Timentin 36

Piperacillin-tazobactam 31Piperacillin-tazobactam/Amikacin 31

Fosfomycin/Tobramycin 31

Best 10 antimicrobial combinations for kill of B. cepacia (no. of strains = 36) from cases of CF.

Antibiotic combination % of strains killed

Piperacillin-tazobactam/Amikacin 33Ceftazidime/Tobramycin 28

Minocycline/Piperacillin-tazobactam 28Ceftazidime/Ciprofloxacin 25

Amikacin/Ceftazidime 25Tobramycin/Piperacillin-tazobactam 25Piperacillin-tazobactam/Ceftazidime 22

Aztreonam/Ceftazidime 22Timentin/Ceftazidime 19

Chloramphenicol/Piperacillin-tazobactam 19

Work performed by:Work performed by:

Larissa Laine, Susan Hughes, Larissa Laine, Susan Hughes,

Audrey Nicholson & John Perry.Audrey Nicholson & John Perry.

Microbiology DepartmentMicrobiology Department

Freeman HospitalFreeman Hospital

Newcastle upon TyneNewcastle upon Tyne

BSAC Standardised Disc Susceptibility Test User Group Day. Royal College of Physicians, London. 8 June 2007 Susceptibility testing of mucoid Pseudomonas.

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