Transcript A new approach to the selective isolation of Salmonella

BRITISH SOCIETY FOR MICROBIAL
TECHNOLOGY
SCIENTIFIC WORKSHOP
Enterics – a bog standard approach?
An Appraisal of Enteric Culture Media
27 November 2009
J.D. Perry
Freeman Hospital
Newcastle upon Tyne
In enteric microbiology, culture
media may be used for isolation of:
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Salmonella species.
Shigella species.
E. coli O157
Campylobacter species
Vibrio cholerae.
Yersinia enterocolitica
Clostridium difficile
Conventional media commonly used in
clinical microbiology for
Salmonella/Shigella isolation.
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Deoxycholate citrate agar
XLD agar
Salmonella-Shigella medium.
Hektoen Enteric agar.
MLCB agar (Salmonella only)
Advantages of conventional media:
• Relatively inexpensive and effective.
• May allow for isolation of other pathogens e.g..
Shigella.
Disadvantages of conventional media:
• Often highly non-specific and labour intensive.
Examples of Chromogenic agars
for isolation of Salmonella.
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Rambach medium (Chromagar Co.)
SMID agar (bioMérieux)
CSE Agar (PPR Diagnostics)
Compass Salmonella agar (Biokar Diagnostics).
Salmonella Chromogenic agar (Oxoid).
Chromagar Salmonella (Chromagar Co.)
ABC medium (Lab M).
Rambach agar
(Chromagar Co.)
Substrates used:
5-Bromo-4-chloro-3-indolyl-ß-D-galactoside
(X-Gal):
E. coli, Klebsiella, Enterobacter, Citrobacter.
Propylene glycol: Salmonella
[Rambach, A. 1990, Appl. Environ. Microbiol. 56:301-303].
SM-ID agar
(bioMérieux)
Substrates used:
5-Bromo-4-chloro-3-indolyl-ß-D-galactoside
(X-Gal):
E. coli, Klebsiella, Enterobacter, Citrobacter.
Sodium Glucuronate: Salmonella.
[Poupart, M.C., et al. ECCMID 1991, abstr. 1254].
5-bromo-6-chloro-3-indolyl-octanoate
(magenta-caprylate)
O-octanoic acid
Cl
Br
N
H
Esterase production by Salmonella
using magenta-caprylate.
A range of
chromogenic media
is available for
detection of
Salmonella spp
based on esterase
detection.
ASAP (AES CHEMUNEX) for
isolation of Salmonella from stools.
Salmonella species
producing C8
esterase
‘Coliform’
producing
ß-glucosidase.
Further examples of Salmonella
detection using an esterase substrate.
SM-ID 2 for Salmonella
BBL™ CHROMagar™ Salmonella
ABC medium (Lab M).
3,4-cyclohexenoesculetin -ß-D-galactoside:
E. coli, Klebsiella, Enterobacter, Citrobacter.
5-bromo-4-chloro-3-indolyl-alpha-Dgalactoside:
Salmonella (green colonies).
[J. Clin. Microbiol. 1999; 37: 766-768].
Salmonella napoli isolated from a
stool sample on ABC medium.
Salmonella
E. coli
(α and ß GAL +)
(α-GAL +)
Advantages of chromogenic media:
• Highly specific for target pathogens and
therefore less labour intensive.
Disadvantages of chromogenic media:
• Relatively expensive.
• Do not usually allow for isolation of more than
one target pathogen.
• May be less sensitive than conventional media.
Comparative evaluation of four chromogenic media
with Hektoen enteric agar using 916 stool specimens
from three hospitals.
[Perez et al. 2003. JCM 41:1130-1134.]
ABC
medium
COMPASS
agar
CHROMagar
Salmonella
SM-ID
Hektoen
No. of Salmonella
57
60
57
55
63
No. of false
positives
17
108
130
83
215
Evaluation and Implementation of a Chromogenic Agar
Medium for Salmonella Detection in Stool in Routine
Laboratory Diagnostics.
[Journal of Clinical Microbiology, February 2009, p. 456-458, Vol. 47, No. 2]
Sensitivities and specificities of the tested media calculated from culture results after 48 h
and including enrichment
No. of Salmonella.
Sensitivity (%)
No. of falsepositives
Specificity (%)
SS
23
71.9
277
78.8
XLD
31
96.9
261
80
Hektoen
27
84.4
245
81.3
BBL
28
87.5
56
95.7
SM-ID 2
25
78.1
51
96.1
Medium
Q: How good are chromogenic
media at inhibiting the growth of
commensal bacteria ?
A: Most media for Salmonella
allow the growth of most
Enterobacteriaceae.
Selectivity of various Salmonella agars
No. of strains Hektoen XLD
Salmonella spp.
63
95.2
98.4
Total non-Salmonellae
372
70.4
90.9
Total ß-gal producers
235
73.6
89.8
Total E.coli
108
45.4
78.7
ABC Chromagar Rambach SMID
100
100
96.8
100
87.9
77.4
79.8
96.5
95.7
93.6
98.7
99.6
96.3
95.4
98.1
100
Stool sample containing Salmonella on
Rambach agar
Stool sample containing Salmonella on SM-ID agar.
Stool sample containing Salmonella on ABC medium.
Stool sample containing Salmonella on XLD medium.
A new approach to the selective
isolation of Salmonella using
suicide substrates.
Principle of ‘suicide substrate’
• A non-inhibitory substrate which is cleaved
enzymatically to release an inhibitory product.
• Resistance may be due to:
• Resistance of the target site to the inhibitory
agent.
• Lack of an appropriate enzyme for cleavage
of the substrate.
• Reduced uptake of the substrate by the
bacterial cell.
Identification of a novel selective agent:
L-alanyl-1-aminoethylphosphonic acid
or alafosfalin.
Fig 1: Structure of alafosfalin:
H 2N
CH 3
O
C
C
H
CH 3
O
N
C
P
H
H
OH
OH
Composition of medium for
alafosfalin MIC’s:
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16 amino acids.
Buffered salt solution.
Purine/Pyrimidine bases
Agar/Water
No proteins/peptones
Average MIC results of
alafosfalin for some common
faecal organisms.
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Salmonella spp. (n=63)
E.coli (n=108)
Citrobacter spp. (n=28)
Enterobacter spp. (n=31)
Hafnia alvei (n=10)
Klebsiella spp. (n=34)
Serratia spp. (n=13)
10.2 mg/l
0.7 mg/l
2.8 mg/l
6.5 mg/l
2.8 mg/l
6 mg/l
3.2 mg/l
Genera resistant to alafosfalin
(MIC >32 mg/l)
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Morganella spp.
Proteus spp.
Providencia spp.
Pseudomonas spp.
Acinetobacter spp.
Composition of ‘Modified’
ABC medium
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16 amino acids.
Buffered salt solution.
Purine/Pyrimidine bases
Agar/Water
Alafosfalin (1 mg/l)
Chromogenic mix
0.5 % Sodium deoxycholate
Selectivity of various
Salmonella agars
% strains recovered
No. of strains
Hektoen
ABC
Mod-ABC
Salmonella
63
95.2
100
98.4
Non-Salmonellae
372
70.4
87.9
54.6
ß-GAL positives
235
73.6
95.7
46.0
E.coli
108
45.4
96.3
10.2
Field trial study 1: Newcastle, UK.
• 797 consecutive liquid stool samples from
Newcastle PHL were cultured onto:
• Hektoen enteric, ABC medium, ‘Modified’
ABC medium.
• Selenite broth enrichment followed by:
Hektoen enteric, ABC medium, ‘Modified’
ABC medium.
Field trial 1 results:
Total Salmonella isolated:
Direct culture:
Hektoen enteric agar:
ABC medium:
‘Modified’ ABC:
Post enrichment:
Hektoen enteric agar:
ABC medium:
‘Modified’ ABC:
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18 (54.6 %)
17 (51.5 %)
25 (75.8 %)
32 (97 %)
33 (100 %)
33 (100 %)
Field trial 1: results
False positives on direct culture and
post-enrichment:
Hektoen enteric agar:
ABC medium:
‘Modified’ ABC:
172 (21.6 %)
15 (1.9 %)
4 (0.5 %)
Stool sample containing Salmonella on
Rambach agar
Stool sample containing Salmonella on SM-ID agar.
Stool sample containing Salmonella on ABC medium.
Stool sample containing Salmonella on
Hektoen Enteric agar
Stool sample containing Salmonella on XLD medium.
Stool sample containing Salmonella on
Modified ABC medium (containing alafosfalin).
Stool sample containing Salmonella on Rambach agar.
Stool sample containing Salmonella on SM-ID medium.
Stool sample containing Salmonella on ABC medium.
Stool sample containing Salmonella on
Hektoen Enteric agar.
Stool sample containing Salmonella on XLD medium.
Stool sample containing Salmonella on
Modified ABC medium (containing alafosfalin).
Conclusions on the use of the ‘suicide
substrate’ alafosfalin as a selective agent.
Advantages
• Cost of inhibitor (1 mg/l): 10 p per litre of agar.
• Highly stable agent.
• Improves selectivity and specificity.
Disadvantages
• Inhibits Salmonella typhi. (or use di-alanyl
fosfalin).
• Of limited use post-enrichment.
• Specialised medium is required.
OSCM II using InhibigenTM technology.
[www.oxoid.com]
Conclusions on use of Salmonella
media:
• Chromogenic media offer a means of detecting
Salmonella with high specificity and reduced labour time
when compared with conventional agars.
• There is a lack of evidence that they have any superiority
for detection of Salmonella when compared with
conventional agars e.g. XLD / Hektoen and some studies
suggest a lack of sensitivity.
• Large evaluation studies are lacking – particularly with
some newer media.
• Suicide substrates or Inhibigens show promise for
improved detection of Salmonella but further studies with
clinical samples are needed.
TM
Media for Shigella:
• Examples include Hektoen enteric agar, DCA,
SS agar, XLD etc and all are based on similar
principles e.g. non fermentation of lactose /
sucrose.
• Comparative studies are lacking.
• No specific chromogenic media are
commercially available.
SALSA (AES CHEMUNEX)
ASAP –
XLD –
Chromogenic
Salmonella
agar.
For Salmonella
and Shigella
detection.
A range of media is available for
isolation of Campylobacter.
• Traditional media were blood-based and employed
a cocktail of antimicrobials including polymyxin,
vancomycin, trmethoprim, cephalothin &
amphotericin.
• Charcoal was shown to be a useful substitute for
blood (Bolton et al. JCM; 19; 169-71).
• Superior selective agents have been identified e.g.
cefoperazone.
• Modified charcoal cefoperazone deoxycholate
agar (CCDA) is widely used.
• Preferred temperature of incubation is 42°C
under microaerophilic conditions (typically
5% oxygen / 10% carbon dioxide and 85%
nitrogen).
• No single medium and no single set of
incubation conditions will isolate all
Campylobacter.
Chromogenic media for
Campylobacter
• These have been developed for enumeration
of Campylobacter in food and there are no
journal reports describing evaluation with
clinical samples.
Stool sample on Campylosel
Same stool sample on CCDA
Same stool sample on Campy Food ID
CASA (AES CHEMUNEX)
Brilliance Campycount Agar
(Oxoid)
Media for E. coli O157:H7
• Media are designed on the principles that this
serotype differs from most E. coli in that:
• Most strains of E. coli O157 fail to generate
acid from sorbitol.
• Most strains of E. coli O157 fail to produce ßglucuronidase.
• Other serotypes generating verotoxin will
generally not be detected on media designed
for E. coli O157.
Media for E. coli O157:H7
• Sorbitol MacConkey is the most commonly
used selective medium.
• Tellurite and cefixime are commonly used
for increased selectivity.
• Specificity remains limited as some species
such as E. hermannii and Proteus species
occasionally grow and produce nonfermenting colonies.
CHROMagar E. coli O157
BBL™ CHROMagar™ O157
Rapid and sensitive detection of Shiga toxin-producing Escherichia
coli from non-enriched stool specimens by real-time PCR in
comparison to enzyme immunoassay and culture.
[J Clin Microbiol. 2009 Jul;47(7):2008-12.]
• Used BBL™ CHROMagar™ O157 and
EIA to validate a PCR method but the study
was not designed for evaluation of the
medium.
• Chromogenic media for E. coli O157 show
good specificity when pure strains are tested
but evaluations with clinical samples are
lacking.
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B
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Media for C. difficile:
A) CLO (bioMerieux)
B) Oxoid
C) BBL
D) CCEY (Bioconnections
E) CCEY plus lysozyme
D
E
Transparency Declaration:
In the last ten years, the presenter
has received financial support for
research or consultancy
from suppliers of chromogenic
culture media including bioMérieux,
Becton Dickinson, Lab M and BioRad.