BSAC Standardised Disc Susceptibility Test User Group Day' Royal College of Physicians, London' 8 Ju

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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

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Contents

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

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Direct susceptibility testing of Pseudomonas
aeruginosa from sputa of patients with cystic
fibrosis
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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, Addenbrookes
Hospital, Cambridge CB2 2QW, UK Department of
Chest Medicine, Papworth Hospital, Papworth
Everard, Cambridge CB3 8RE, UK Journal of
Antimicrobial Chemotherapy (2005) 55, 921927
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Foweraker et al. 2005. Methods

• 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.

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Foweraker et al. 2005. Results / Conclusions

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

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Can antimicrobial resistance be detected more
reliably by culture of whole sputum onto media
containing antimicrobials ?

• Aim of the current investigation

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Routine culture

• Sputum samples were homogenized 11 with
  Sputasol.
• Routine culture
• 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.

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• Routine culture (continued)
• 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
• Chocolate agar (Bacitracin), Blood agar, CLED
  agar, Isosensitest agar, Pseudomonas Selective
  agar Burkholderia cepacia Selective agar.

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Selective cultureA 10 µl aliquot of liquid
sputum was inoculated onto 10 distinct
Isosensitest agar plates incorporating the
following antimicrobials

• Amikacin (16 mg/L)
• Gentamicin (4 mg/L)
• Tobramycin (4 mg/L)
• Aztreonam (8 mg/L)
• Ceftazidime (8 mg/L)

• Meropenem (4 mg/L)
• Temocillin (16 mg/l)
• Ciprofloxacin (1 mg/L).
• Piperacillin-tazobactam (16 mg/L)
• Ticarcillin-clavulanic acid (32 mg/L)

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Summary of media used

• Routine culture
• 10 µl of Neat and diluted homogenized sputa were
  inoculated onto
• Cholcolate-Bacitracin
• Blood agar.
• CLED agar.
• Isosensitest agar.
• Pseudomonas selective agar.
• B. cepacia selective agar.
• (Total 10 culture plates).

• Selective culture
• Culture of neat homogenized sputa on Isosensitest
  agar (x 10) containing
• Amikacin
• Gentamicin
• Tobramycin
• Aztreonam
• Ceftazidime
• Meropenem
• Temocillin
• Ciprofloxacin
• Piperacillin-tazobactam
• Ticarcillin-clavulanic acid

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Interpretation of cultures

• 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.

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Identification

• P. aeruginosa was identified by inoculation of
  all morphotypes onto PC agar, cetrimide agar and
  blood agar to test for growth at 42C.
• PC agar contains
• 30 mg/L 9-chloro-9-4-(diethylamino)phenyl-9,10-d
  ihydro-10-phenylacridine hydrochloride (C-390)
  and
• 30 mg/L 1,10-phenantholine.
• Growth on this agar is diagnostic for P.
  aeruginosa with 100 specificity1.
• 1 J Clin Microbiol. 1988 Sep26(9)1910-2.

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Identification

• For this study
• Growth on cetrimide growth on PC agar growth
  at 42C P. aeruginosa.
• All other strains were identified by API 20 NE.

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Susceptibility testing

• 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.
• MICs were recorded after both 24 and 48 hours of
  incubation at 37C.

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Susceptibility testing

• Ranges of antibiotics for MIC testing
• AMIK (64 - 2 mg/L)
• GENT (16 - 0.5 mg/L)
• TOBRA (16 - 0.5 mg/L)
• ATM (32 - 1 mg/L)
• CAZ (32 - 1 mg/L)

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

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Results

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

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Results

• 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).

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Conclusions

• 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.

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Disc susceptibility testing of Pseudomonas
aeruginosa from sputa of patients with cystic
fibrosis
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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.
• 0.5 McFarland suspension of strains cultured
  overnight on blood agar.
• 1/100 dilution of suspension in sterile water.
• Dilution spread with a swab onto pre-poured Oxoid
  Isosensitest agar (PO O779A).
• Incubation for 24 h at 37C (48 h only if
  required) to obtain semi-confluent growth.
• Agar dilution MICs were performed in
  Isosensitest agar (Oxoid) using the BSAC method
  using the same 0.5 McFarland suspensions.

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Disc susceptibility testing was performed by BSAC
method

• Controls
• With each batch of disc susceptibility tests and
  agar dilution tests we included
• Pseudomonas aeruginosa NCTC 10662
• 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.

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Results

• 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.
• One strain produced no growth on repeated disc
  susceptibility testing.
• (Control strains produced acceptable results)

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Sensitive Intermediate Resistant 8 mg/L 16
mg/L 32 mg/L 19 mm 16-18 mm 15 mm
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Sensitive Resistant 4 mg/L 8 mg/L 18 mm
17 mm
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Sensitive Resistant 4 mg/L 8 mg/L 20 mm
19 mm
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Sensitive Intermediate Resistant 0.5 mg/L 1
mg/L 2 mg/L 30 mm 20-29 mm 19 mm
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Sensitive Resistant 8 mg/L 16 mg/L 23
mm 22 mm
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Sensitive Resistant 8 mg/L 16 mg/L 24
mm 23 mm
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Sensitive Intermediate Resistant 2 mg/L
4-8 mg/L 16 mg/L 27 mm 22-26 mm
21 mm
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Sensitive Resistant 16 mg/L 32 mg/L 22
mm 21 mm
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S 8 mg/L (Unconfirmed) R gt 8 mg/L
(Unconfirmed) S 20 mm (Unconfirmed) R 19
mm (Unconfirmed) P. aeruginosa NCTC 10662 MIC
gt 64 mg/L E. coli NCTC 10418 MIC 4 mg/L
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Disc susceptibility testing of Burkholderia
cepacia complex using BSAC criteria for
interpretation of Pseudomonas spp.
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Disc susceptibility testing was performed by BSAC
method

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

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40 strains of Burkholderia cepacia complex used
for disc susceptibility testing
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40 strains of Burkholderia cepacia complex
(cont)
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Results

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

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Sensitive Intermediate Resistant 0.5 mg/L
1 mg/L 2 mg/L 30 mm 20-29 mm
19 mm
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Sensitive Resistant 8 mg/L 16 mg/L 23
mm 22 mm
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Sensitive Resistant 8 mg/L 16 mg/L 24
mm 23 mm
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Sensitive Intermediate Resistant 2 mg/L 4-8
mg/L 16 mg/L 27 mm 22-26 mm 21 mm
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Sensitive Resistant 16 mg/L 32 mg/L 22
mm 21 mm
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S 8 mg/L (Unconfirmed) R gt 8 mg/L
(Unconfirmed) S 20 mm (Unconfirmed) R 19
mm (Unconfirmed) P. aeruginosa NCTC 10662 MIC
gt 64 mg/L E. coli NCTC 10418 MIC 4 mg/L
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MULTIPLE ANTIBIOTIC SYNERGY TESTING AGAINST P.
AERUGINOSA AND B. CEPACIA COMPLEX STRAINS FROM
CYSTIC FIBROSIS PATIENTS.
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Principles

• Inoculation of 106 cfu/ml test bacteria into
  Isosensitest broth with 78 distinct antimicrobial
  combinations.
• Incubation for 48 h at 37C.

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Antimicrobial combinations tested

• Tests performed in microtitre wells.
• Final volume 100µl.

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

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Work performed by

• Larissa Laine, Susan Hughes,
• Audrey Nicholson John Perry.
• Microbiology Department
• Freeman Hospital
• Newcastle upon Tyne