BRITISH SOCIETY FOR MICROBIAL TECHNOLOGY SCIENTIFIC WORKSHOP Enterics – a bog standard approach? J.D. Perry Freeman Hospital Newcastle upon Tyne An Appraisal of Enteric Culture Media 27 November 2009

In enteric microbiology, culture media may be used for isolation of: 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. 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. 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: ].

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) H N Br Cl O-octanoic acid

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. BBL™ CHROMagar™ SalmonellaSM-ID 2 for Salmonella

ABC medium (Lab M). 3,4-cyclohexenoesculetin -ß-D-galactoside: E. coli, Klebsiella, Enterobacter, Citrobacter. 5-bromo-4-chloro-3-indolyl-alpha-D- galactoside: Salmonella (green colonies). [J. Clin. Microbiol. 1999; 37: ].

Salmonella napoli isolated from a stool sample on ABC medium. Salmonella (α-GAL +) E. coli (α and ß 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 JCM 41: ] ABC medium COMPASS agar CHROMagar SalmonellaSM-ID Hektoen No. of Salmonella No. of false positives

Evaluation and Implementation of a Chromogenic Agar Medium for Salmonella Detection in Stool in Routine Laboratory Diagnostics. [ Journal of Clinical Microbiology, February 2009, p , Vol. 47, No. 2] Sensitivities and specificities of the tested media calculated from culture results after 48 h and including enrichment MediumNo. of Salmonella.Sensitivity (%) No. of false- positives Specificity (%) SS XLD Hektoen BBL SM-ID

Q: How good are chromogenic media at inhibiting the growth of commensal bacteria ?

A: Most media for Salmonella allow the growth of most Enterobacteriaceae.

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:

Composition of medium for alafosfalin MIC’s: 16 amino acids. Buffered salt solution. Purine/Pyrimidine bases Agar/Water No proteins/peptones

Average MIC results of alafosfalin for some common faecal organisms. Salmonella spp. (n=63)10.2 mg/l E.coli (n=108)0.7 mg/l Citrobacter spp. (n=28)2.8 mg/l Enterobacter spp. (n=31)6.5 mg/l Hafnia alvei (n=10)2.8 mg/l Klebsiella spp. (n=34)6 mg/l Serratia spp. (n=13)3.2 mg/l

Genera resistant to alafosfalin (MIC >32 mg/l) Morganella spp. Proteus spp. Providencia spp. Pseudomonas spp. Acinetobacter spp.

Composition of ‘Modified’ ABC medium 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 strainsHektoenABC Mod-ABC Salmonella Non-Salmonellae ß-GAL positives E.coli

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:33 Direct culture: Hektoen enteric agar:18 (54.6 %) ABC medium:17 (51.5 %) ‘Modified’ ABC:25 (75.8 %) Post enrichment: Hektoen enteric agar:32 (97 %) ABC medium:33 (100 %) ‘Modified’ ABC:33 (100 %)

Field trial 1: results False positives on direct culture and post-enrichment: Hektoen enteric agar:172 (21.6 %) ABC medium:15 (1.9 %) ‘Modified’ ABC: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 Inhibigen TM technology. [

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 TM show promise for improved detection of Salmonella but further studies with clinical samples are needed.

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 – Chromogenic Salmonella agar. XLD – 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; ). 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 ß- glucuronidase. 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 non- fermenting 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 Jul;47(7): ] 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.

Media for C. difficile: A) CLO (bioMerieux) B) Oxoid C) BBL D) CCEY (Bioconnections) E) CCEY plus lysozyme ABC DE

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 Bio- Rad.