Optimization of enrichment and plating procedures for the recovery of ...

Journal of Applied Microbiology 2003, 95, 949–957

doi:10.1046/j.1365-2672.2003.02065.x

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef T.M.G. Catarame1, K.A. O’Hanlon1, G. Duffy1, J.J. Sheridan1, I.S Blair2 and D.A. McDowell2 1

The National Food Centre, Teagasc, Ashtown, Dublin, Ireland, and 2NICHE, University of Ulster, Jordanstown, UK

2003/0010 received 31 March 2003, revised 30 May 2003 and accepted 13 June 2003

ABSTRACT T . M . G . C A T A R A M E , K . A . O ’ H A N L O N , G . D U F F Y , J . J . S H E R I D A N , I . S . B L A I R A N D D . A . M C D O W E L L . 2003.

Aim: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. Methods and Results: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41Æ5C in tryptone soya broth supplemented with cefixime (50 lg l)1), vancomycin (40 mg l)1) and potassium tellurite (2Æ5 mg l)1). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l)1)] and supplemented with cefixime (50 lg ml)1) and potassium tellurite (2Æ5 mg l)1). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 lg ml)1), cefsulodin (5 mg l)1) and vancomycin (8 mg l)1). Minced beef samples were inoculated with a number of strains of E. coli O26 (n ¼ 9) and O111 (n ¼ 8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. Conclusions: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. Significance and Impact of the Study: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef. Keywords: E. coli O111, E. coli O26, enrichment media, minced beef, selective agar.

INTRODUCTION Escherichia coli is most widely recognized as a common enteric commensal in animal species. However, this species also includes a significant and frequently pathogenic group of strains, the verocytoxigenic E. coli (VTEC), which, principally as food-borne pathogens, have emerged causing serious and fatal outbreaks of human infection worldwide. Of this group with more than 100 serogroups, one particular serotype, E. coli O157 is currently the most widely recognized, having been implicated in cases of human Correspondence to: T.M.G. Catarame, The National Food Centre, Teagasc, Ashtown, Dublin, Ireland (e-mail: [email protected]).

ª 2003 The Society for Applied Microbiology

infection from over 30 countries in six continents (Tozzi et al. 2001). However, other serogroups including O26, O55, O91, O103, O111, O128 and O145 (Johnson et al. 1996) are being increasingly associated with human disease syndromes including diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (Karmali 1989). The non-O157 VTEC strains are heterogeneous in the range of genotypic and phenotypic properties displayed by the wider species, and few laboratories screen for non-O157 VTEC strains in clinical or food samples (Johnson et al. 1996). Such limited surveillance in relation to non-O157 VTEC, the absence of a specific International Organisation for Standardisation (ISO) method for their detection in foods,

950 T . M . G . C A T A R A M E ET AL.

and slower recognition of their significance, mean that in contrast to VTEC O157, much less is known about the epidemiology of non-O157 VTEC. However, the data that are available suggest that there are geographical differences in the relative frequency of O157 and non-O157 strains of VTEC. Significant numbers of sporadic cases and outbreaks of non-O157 infection have been described in Germany (Bitzan et al. 1993), Italy (Caprioli et al. 1994), Czech Republic (Bielaszewska` et al. 1990), Australia, Centre for Disease Control (CDC 1995a), Japan (Kudoh et al. 1994) and the USA (CDC 1995b). In many countries, including Chile (Cordove´z et al. 1992), India (Kishore et al. 1992), France (Mariani-Kurkdjian et al. 1993), and Australia (Goldwater and Bettelheim 1994), HUS associated with non-O157 VTEC infections has been reported more frequently than with O157 VTEC infections. The first outbreak caused by non-O157 VTEC strain in the British Isles occurred in a cre`che in Donegal (Ireland in September 1999). The lactose fermenting organism causing four cases of diarrhoeal illness in children attending the cre`che was serotyped as E. coli O26:H11 and was shown to contain the VT1 gene but not VT2 (McMaster et al. 2001). The second reported case of non-O157 VTEC infection in Ireland occurred in September 2000, in a child admitted to a Dublin hospital with HUS. Screening of the stool samples from the child and its family failed to detect VTEC. Similarly, serology tests were negative for VTEC organisms. However, VTEC O26 which was positive for verotoxins 1 and 2, were isolated from five contact children (McNamara 2001). There were four cases of confirmed VTEC O26 reported in 2001 in Ireland (Anon. 2001). Such cases, and the wider pattern of increasingly frequent reports of serious, if not fatal, human disease caused by non-O157 VTEC, emphasize the need for progress in the detection, surveillance, epidemiology and control of these organisms. Some information is available on the occurrence and routes of transmission of non-O157 VTEC. Thus Blanco et al. (1994) has noted the frequent detection of non-O157 VTEC in cattle, and the potential role of beef and other animal products as potential routes of human infection. Progress in the detection and wider surveillance of nonO157 VTEC is increased by the development and wider availability of non-O157 (O26 and O111) antibody-labelled immunomagnetic beads, and such systems are now being used in the recovery of these pathogens from enriched food samples (Sˇafarˇikova` and Sˇafarˇik 2001). However, as yet there has been very little work in optimizing the (pre) enrichment, or plating, techniques for detection of these organisms from food. Such procedures are essential as VTEC, when present in food samples, are usually present in very low numbers, and can be difficult to recover from complex matrices in the presence of larger numbers of nonVTEC E. coli, enteric, and other bacteria.

The aim of this study was to evaluate a range of combinations of enrichment and plating media, selective agents and incubation temperatures in the recovery and detection of small numbers of E. coli O26 or O111 in minced beef.

MATERIALS AND METHODS Bacterial strains Escherichia coli serogroup O26 (n ¼ 9) and E. coli serogroup O111 (n ¼ 8) of human and clinical origin were obtained from The Department of Medical Microbiology (Foresterhill, Aberdeen, UK), The National Collection of Type Cultures (PHLS, London, UK) and Cherry Orchard Hospital (PHLS, Dublin, Ireland) (Table 1). All strains were stored on Protect Beads at )20C according to manufacturer instructions (Technical Services Consultants Ltd, Lancashire, UK).

Table 1 Source and properties of Escherichia coli O111 and O26 isolates used in study VT Strain Source E. coli 332 352 354 361 381 8620 8783 8960 M328 E. coli 359 378 8179 8008 8009 8333 8007 9703

O26 Aberdeen* Aberdeen* Aberdeen* Aberdeen* Aberdeen* NCTC NCTC NCTC Dublin O111 Aberdeen* Aberdeen* NCTC NCTC NCTC NCTC NCTC NCTC

1

2

Reaction with O-type Enterohaemolysin specific antisera production

+ ) + + + ) ) ) +

) ) ) ) ) ) ) ) +

+ ) + + + ) + ) +

+ ) + + + ) ) ) +

+ + ) ) ) ) ) )

) + ) ) ) ) ) )

+ + + + + + + +

+ + + + + + + +

*Department of Medical Microbiology, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. The National Collection of Type Cultures, PHLS, Colindale NW9, London, UK. Microbiology Unit, Cherry Orchard Hospital, Ballyfermot, Dublin 10, Ireland. All strains were positive on eosin methylene blue agar, indole positive and positive when tested by latex agglutination.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 949–957, doi:10.1046/j.1365-2672.2003.02065.x

CULTURAL DETECTION OF E. COLI O26 AND O111

Cultures and recovered isolates were confirmed as E. coli by culture on eosin methylene blue (EMB) agar and testing for the presence of indole (Oxoid, Basingstoke, UK), and as E. coli O26 or E. coli O111 by latex agglutination (Denka Seiken, Tokyo, Japan) using the relevant O-somatic antigens and an antisera test (Statens Serum Institut, Copenhagen, Denmark). Cultures and recovered isolates were confirmed as possessing VT genes by PCR (PHLS, London, UK) and for the production of enterohaemolysin on washed sheep blood agar (Oxoid). The cultures used in this study, along with up to 90% of primary VTEC isolates from patients with diarrhoea or HUS, expressed this haemolysin in concert with verotoxin (Scheutz et al. 2001).

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Broth culture studies

Diaphragm meat was obtained from a commercial abattoir in the Dublin area. The meat samples were prepared by removing the fat tissue and mincing through a sterile 5Æ0-mm steel plate mincer (Crypto Peerless Ltd, model EB 12F; London, UK). Retail meat was purchased from a range of butcher shops in the Dublin area.

The above (working) cultures of ABR E. coli O26 or O111 were serially diluted in maximum recovery diluent (MRD) to give an initial inoculum of 100 CFU ml)1 in 225 ml volume of tryptone soya broth (TSB), modified TSB (mTSB), Luria Bertani (LB) broth, modified E. coli (mEC) broth supplemented with novobiocin (Sigma, Poole, UK) at 25 mg l)1, TSB, LB or mTSB supplemented with vancomycin (Sigma) at 40 mg l)1; cefixime (Fujisawa, Osaka, Japan) at 50 lg l)1; and potassium tellurite (Sigma) at 2Æ5 mg l)1, mTSB supplemented with novobiocin (Sigma) at 20 mg l)1, TSB supplemented with vancomycin (Sigma) at 40 mg l)1; cefixime (Fujisawa) at 50 lg l)1 (O111 only). Inoculated broths were incubated at 37C or 41Æ5C for 0, 2, 4, 6 or 8 h. After incubation, 1Æ0 ml samples were removed from each culture, serially diluted and plated onto MacConkey agar containing either 50 lg ml)1 nalidixic acid (mutant O26) or 1 mg ml)1 streptomycin sulphate (mutant O111). Plates were inverted and incubated at 37C overnight. The number of ABR mutant in each enrichment broth after incubation at each time/temperature combination was calculated. These broth culture experiments were replicated on three occasions.

Preparation of antibiotic-resistant mutants

Meat culture studies

Escherichia coli O26 was rendered resistant to nalidixic acid, and E. coli O111 was rendered resistant to streptomycin sulphate. These antibiotic-resistant mutants were prepared by inoculating three randomly selected wild type E. coli O26 strains (361, 354 and 332; Table 1) and two randomly selected wild type E. coli O111 strains (359, 378; Table 1) into tubes containing 100 ml of brain–heart infusion (BHI) broth containing 100 lg ml)1 nalidixic acid or 200 lg ml)1 streptomycin sulphate, and incubated overnight at 37C. Aliquots of 100 ll were plated onto tryptone soya agar (TSA) plates containing 1 mg ml)1 streptomycin sulphate or 50 lg ml)1 nalidixic acid, incubated at 37C overnight, and examined to detect and recover an antibiotic-resistant (ABR) mutant of a human isolate strain (359) of E. coli O111 which was resistant to streptomycin sulphate (1 mg ml)1) and an ABR mutant of a human isolate strain (361) of E. coli O26 which was resistant to nalidixic acid (50 lg ml)1). These mutant strains were stored on Protect Beads (Technical Service Consultants Ltd.

Working cultures of ABR E. coli O26 or ABR E. coli O111 were serially diluted and inoculated into 25 g of minced diaphragm meat to initial concentrations of 100 CFU g)1. The inoculated meat was stomached aseptically (Stomacher 400 Circulator; Seward Medical UAC House, London, UK) at 230 rev min-2 for 2 min, with 225 ml of TSB, mTSB, LB, TSB supplemented with vancomycin (Sigma) at 40 mg l)1 and potassium tellurite (Sigma) at 2Æ5 mg l)1 (O111 only), TSB supplemented with vancomycin (Sigma) at 40 mg l)1 cefixime (Fujisawa) at 50 lg l)1 and potassium tellurite (Sigma) at 2Æ5 mg l)1 (O26 only) in a sterile filter bag (Seward Lab Systems), and incubated at 37 or 41Æ5C, for 0, 2, 4, 6 or 8 h. After incubation, 1 ml samples were removed from each culture, serially diluted and plated onto MacConkey agar containing either 50 lg ml)1 nalidixic acid (mutant O26) or 1 mg ml)1 streptomycin sulphate (mutant O111). Plates were inverted, incubated at 37C overnight and examined to provide estimates of ABR mutant numbers in each enrichment broth after incubation at each time/ temperature combination. These meat culture experiments were replicated on three occasions.

Meat samples

Preparation of inocula A Protect bead of one or other of the above ABR mutants was incubated overnight in 10 ml of BHI at 37C overnight, and a 1 ml aliquot of the resultant culture was transferred into a fresh 10 ml of BHI and further incubated at 37C overnight. This process yielded cultures containing VTEC concentrations between 109 and 1010 CFU ml)1.

Statistical analysis The bacteria counts were normalized by transforming the data using log10. The mean value (from the three replicates) was plotted against time by linear regression analysis using



Genstat 5 (Rothamsted Experimental Station, Harpenden, UK). The slope of the line was calculated to obtain a growth rate for the organism under each experimental condition (the units for growth rates are log10 CFU ml)1 h)1). The t-test (Genstat 5) was used to compare the growth rates for the pathogens under varying conditions of enrichment media/time/temperature combination. Selective agars Escherichia coli O26 (n ¼ 9; Table 1); E. coli O111 (n ¼ 8; Table 1), E. coli O157 and a number of common meat contaminants i.e. Proteus spp., Shigella sonnei, Enterobacter clocae, Citrobacter spp., Staphylococcus aureus, Serratia marcescens, Enterobacter faecalis and Salmonella Typhimurium were streaked onto a range of commercially available agars used in the detection of bacterial contamination of foods, i.e. chromocult agar (Merck, Darmstadt, Germany), chromogenic agar (Oxoid), MacConkey agar (Oxoid), blood agar (Oxoid), haemorrhagic colitis agar (Oxoid), Sorbitol MacConkey (Oxoid) and Rainbow agar-O157 (Biolog, Hayward, CA, USA) and MacConkey agar (lactose replaced by rhamnose at levels of 10, 20, 30, 40 and 50 g l)1). A range of selective antibiotics [cefixime (50 lg ml)1), potassium tellurite (2Æ5 mg l)1), cefsulodin (5 mg l)1) and vancomycin (8 mg l)1)] were added to the selective agars. Plates were incubated at 37C overnight and the morphology of the VTEC colonies were compared with the morphology of other recovered bacterial species. Application of developed techniques for recovery of E. coli O26 and E. coli O111 from minced beef Enrichment media/time/temperature combinations supporting the recovery/growth of ABR mutants of VTEC, and selective/differential agars identified in pure culture studies using E. coli O26 and E. coli O111 were applied (Fig. 1) in the examination of minced beef, prepared as above, and inoculated to ca 10 CFU g)1 with wild type strains of E. coli O26 (n ¼ 9; Table 1) or E. coli O111 (n ¼ 8; Table 1). Samples of inoculated mince beef (25 g) and an uninoculated (control) minced beef were stomached in 225 ml volumes of enrichment broths, as described above, and incubated at 41Æ5C for 6 or 24 h. In order to aid the selective isolation of the innoculated E. coli O26 and O111 from retail minced beef, immunomagnetic separation (IMS) using immunomagnetic beads (Seiken particles; Denka Seiken) selective for either E. coli O26 or E. coli O111 were used. This involved removing 1Æ0 ml aliquots of the enriched culture to 1Æ5 ml eppendorf tubes containing one drop (about 25 ll) of the IMS bead suspension and incubating for 30 min at room temperature.

Following incubation, the magnetic beads were washed twice with 1Æ0 ml sterile saline solution using a magnetic separator MPC-M (Dynal, Oslo, Norway). Finally, the beads were suspended in 100 ll saline buffer and 50 ll was plated onto the selected agars and spread with a sterile cotton swab. Plates were inverted and incubated overnight at 37C. Three colonies from each plate exhibiting typical presumptive positive characteristics of E. coli O26 or O111 were picked and restreaked onto EMB for indole analysis, and enterohaemolysin agar and nutrient agar for latex agglutination and antiserum agglutination tests.

RESULTS ABR mutant growth in enrichment media Escherichia coli O26. The rates of growth of an ABR mutant of E. coli O26 under a range of enrichment conditions are shown in Table 2. No growth occurred in mTSB + novobiocin, mEC broth + novobiocin, LB broth + cefixime, vancomycin and potassium tellurite (CVPt) or mTSB + CVPt and these broths are not included in the table. There were no significant differences among the growth rates of E. coli O26 in broth cultures in TSB (1Æ12 ± 0Æ22), LB (1Æ08 ± 0Æ21) or mTSB (0Æ62 ± 0Æ07). There was a significantly higher growth rate (P < 0Æ5) in TSB (1Æ12 ± 0Æ22) than TSB+CVPt (0Æ52 ± 0Æ08). There were no significant differences among the growth rates of this organism in meat-derived cultures in any of the enrichment media examined at either incubation temperatures examined. There were no significant differences among the rates of growth at 37 or 41Æ5C in broth and meat for E. coli O26. The lag phases for E. coli O26 in meat culture were similar in TSB (0Æ54 h) and TSB + CVPt (0Æ44 h) at 41Æ5C. Escherichia coli O111. The rates of growth of an ABR mutant of E. coli O111 under a range of enrichment conditions are presented in Table 3. No growth occurred in mTSB + novobiocin, mEC broth + novobiocin, mTSB + CVPt, TSB + CVPt, LB + CVPt or TSB + VPt and they are not included in the table. The growth rate of E. coli O111 in (unsupplemented) TSB (0Æ91 ± 0Æ13) was significantly higher (P < 0Æ05) than in mTSB (0Æ17 ± 0Æ04), or LB (0Æ28 ± 0Æ02). There was no significant difference between the growth rates in TSB and TSB + CV. In meat-derived cultures, a similar pattern was observed, with strains exhibiting significantly higher growth rates (P < 0Æ001) in TSB than in mTSB or LB at both incubation temperatures examined and no significant difference between the growth rates in TSB and TSB + CV. There were no significant differences among the rates of growth at 37 or 41Æ5C for



953

Minced beef (25 g)

O26

O111

Tryptone soya broth (225 ml) with

Tryptone soya broth (225 ml) with

cefixime (50 mg l–1), vancomycin (40 mg l–1)

cefixime (50 mg l–1), vancomycin (40 mg l–1)

and potassium tellurite (2·5 mg l–1 ) at

41·5°C for 6 h and 24 h

41·5°C for 6 and 24 h ↓

↓

IMS extraction O26

IMS extraction O111

↓

↓

O26 Selective Agar

O111 Selective Agar

Rhamnose 20 g l–1 MacConkey agar +

Chromocult agar + Cefixime 50 mg l–1,

Cefixime 50 mg–1 ml, potassium tellurite

–1 –1 Cefsulodine 50 mg l , vancomycin 8 mg l

2·5 mg l–1 (CT-RMAC)

(CCV-Chromocult)

↓

↓

Brown or red suspect colonies

Purple suspect colonies

↓

↓ O111 Serological/Biochemical Tests

O26 Serological/Biochemical Tests EMB agar

EMB agar

Indole test

Indole test

Latex and antiserum agglutination

Latex and antiserum agglutination

(for O26 antigen)

(for O111 antigen)

Enterohaemolysin agar

Enterohaemolysin agar

Fig. 1 Enrichment and detection procedures for Escherichia coli O26 and E. coli O111 in minced beef

Table 2 Growth rates for Escherichia coli O26 (log10 CFU ml)1 h)1) over an 8-h period under various enrichment conditions

Sample Broth Meat Meat

Temp. (C) 37 37 41Æ5

TSB

mTSB a

1Æ12 ± 0Æ22 1Æ34 ± 0Æ23d 1Æ39 ± 0Æ14e

LB ab

0Æ62 ± 0Æ07 1Æ13 ± 0Æ12d 1Æ12 ± 0Æ06e

TSB + CVPt ab

1Æ08 ± 0Æ21 1Æ19 ± 0Æ15d 1Æ22 ± 0Æ12e

0Æ52 ± 0Æ08bc 1Æ08 ± 0Æ19d 1Æ16 ± 0Æ09e

Values on the same row sharing a letter are not significantly different. Values on the same row not sharing a letter are significantly different (P < 0Æ05). TSB, tryptone soya broth; MTSB, modified TSB; LB, Luria Bertani broth; TSB + CVPt, TSB with cefixime 50 lg l)1, vancomycin 40 mg l)1 and potassium tellurite 2Æ5 mg l)1. ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 949–957, doi:10.1046/j.1365-2672.2003.02065.x


Sample Temp. (C) TSB Broth Meat Meat

37 37 41Æ5

MTSB

LB

TSB + CV

TSB + VT

0Æ91 ± 0Æ13a 0Æ17 ± 0Æ04b 0Æ28 ± 0Æ02b 0Æ48 ± 0Æ19ab 0Æ49 ± 0Æ11ab 1Æ1 ± 0Æ16d 0Æ35 ± 0Æ05e 0Æ28 ± 0Æ06e 0Æ74 ± 0Æ11de nm 1Æ12 ± 0Æ10f 0Æ34 ± 0Æ06g 0Æ32 ± 0Æ07g 0Æ97 ± 0Æ07fg nm

Table 3 Growth rates for Escherichia coli O111 (log10 CFU ml)1 h)1) over an 8-h period under various enrichment conditions

Values on the same row sharing a letter are not significantly different. Values not sharing a letter on the same row are significantly different (P < 0Æ05). nm, not measured. TSB, tryptone soya broth; mTSB, modified TSB; LB ¼ Luria Bertani broth; TSB + CV, TSB with cefixime 50 lg l)1 and vancomycin 40 mg l)1; TSB + VT, TSB with vancomycin 40 mg l)1 and potassium tellurite 2Æ5 mg l)1.

E. coli O111. The lag phases for E. coli O111 in meat culture were similar in TSB (0Æ56 h) and TSB + CV (0Æ50 h) at 41Æ5C. Wild type non-O157 E. coli growth in selective agars Escherichia coli O26 wild type isolates grew on chromocult agar, chromogenic agar, MacConkey agar, blood agar, haemorrhagic colitis agar, sorbitol MacConkey and Rainbow agar forming colonies of different sizes and morphologies. Such cultures could not be reliably and consistently distinguished from one or more members of the range of common meat contaminant species examined in the study. The most consistent differentiation of E. coli O26 was achieved on MacConkey agar containing rhamnose (20 g l)1) instead of lactose and supplemented with cefixime 50 lg ml)1 and potassium tellurite 2Æ5 mg l)1 (CT-RMAC). On this medium, all examined E. coli O26 strains grew as brown colonies with a yellow halo. The common meat contaminant species, either did not grow on this medium, or produced easily differentiated red colonies with no halos. Other VTEC strains examined gave red colonies (O157 strains, n ¼ 3; O111 strains, n ¼ 4) or failed to grow (O111 strains, n ¼ 4) on this medium (CT-RMAC). Escherichia coli O111 wild type cultures grew on chromogenic agar, MacConkey agar, blood agar, haemorrhagic colitis agar, sorbitol MacConkey, Rainbow agar-O157 and rhamnose MacConkey (10–50 g rhamnose per litre) forming colonies of different sizes and morphologies. Such cultures could not be reliably and consistently distinguished from one or more members of the range of common meat contaminant species examined in the study. The most consistent differentiation of E. coli O111 was achieved on chromocult agar. All E. coli O111 strains examined, E. coli, E. coli O26 and Proteus spp. grew as purple colonies on chromocult agar containing cefixime 50 lg ml)1, cefsulodin 5 mg l)1 and vancomycin 8 mg l)1 (CCV-chromocult). E. coli O157 could be differentiated from E. coli O111 as it grew as pink colonies on chromocult agar.

Application of developed techniques for recovery of E. coli O26 and O111 from minced beef After 6 h enrichment, IMS and plating, all colonies picked were confirmed as E. coli O26 by latex agglutination. After 24 h enrichment, all colonies picked were also confirmed as E. coli O26. After 6 h enrichment, IMS and plating, all colonies picked were confirmed as E. coli O111 by latex agglutination. After 24 h incubation, only half of the colonies picked were confirmed as E. coli O111. DISCUSSION Pathogens are generally present in foods at low levels in the midst of large numbers of other microflora organisms. The pathogen is generally below the detection limit of both cultural and rapid detection methods and so is placed in a liquid medium to allow growth of the pathogen to a level at which it is detectable. Enrichment is a balance between providing conditions which are optimal for growth of the pathogen of choice and the inhibition of the competitive microflora. This is particularly the case with raw meat samples which have a large competitive microflora which can inhibit the recovery and detection of the pathogen of concern (Baylis et al. 2001). In this study, the enrichment broth analysis showed that the growth rate of E. coli O26 was similar in TSB, mTSB and mEC. This is in agreement with previous studies carried out by Hara-Kudo et al. (2000). However, contrary to the work of Hara-Kudo et al. (1999) on E. coli O157, this study recorded a significantly higher growth rate (P < 0Æ001) for E. coli O111 at 41Æ5C in TSB (1Æ12 ± 0Æ10) than mTSB (0Æ34 ± 0Æ06). There were no significant differences between the rates of growth of E. coli O111 strain in TSB or TSB supplemented with cefixime and vancomycin indicating that these selective agents were not suppressing the growth of the pathogen. All E. coli O26 strains (i.e. nine of nine) were resistant to potassium tellurite (MIC > 100 lg ml)1), while the majority (i.e. six of eight) E. coli O111 strains tested were sensitive



to potassium tellurite (MIC < 1 lg ml)1) (results not shown). Among the latter serogroup E. coli O111, the two potassium tellurite resistant strains (359 and 378) were also the only O111 VT producers (Table 1). Such interserotype variations in sensitivity to potassium tellurite have been previously reported (De Boer and Heuvelink 2000). In other cases, intra-serotype variations have been noted, e.g. Zadik et al. (1993) found that E. coli O157 was resistant to potassium tellurite in solid Cefixime Tellurite Sorbitol MacConkey (CT-SMAC) medium, but was sensitive to this selective agent in liquid culture. Such differences between and/or within serogroups may well be related to presence/ absence and extent of expression of the genes coding for resistance to potassium tellurite. In E. coli O157, resistance to potassium tellurite is probably phage encoded (Ohnishi et al. 2001), while in other members of the Enterobacteriaceae, resistance is often plasmid (Walter and Taylor 1992) or chromosomally mediated (Burian et al. 1998). The results of this study suggest that tellurite resistance is not consistently present, nor (when present) consistently expressed, among the Enterobacteriaceae, including VTEC. This selective agent should therefore not be included in media designed to recover a wide range of VTEC including non-O157 E. coli such as E. coli O111. Similarly, this study has shown that tellurite containing media currently in use for the enrichment and selection of E. coli O157, are likely to specifically suppress a number of non-O157 serotypes of VTEC. The observed linkage between tellurite resistance and VT production (Table 1) should be further investigated, as a means of allowing the increasingly important differentiation of those strains/serotypes which are of most specific pathogenic significance i.e. the verotoxin producers, within an expanding range of infection associated serotypes. All the strains of E. coli O26 (n ¼ 9) and O111 (n ¼ 8) tested in this study were able to grow in TSB broth in the presence of cefixime (50 lg l)1) and vancomycin (40 mg l)1), suggesting that these antibiotics, which suppress other Gram-negative aerobes such as Proteus spp. (Zadik et al. 1993), and elements of the Gram-positive microflora of meat (Yao and Moellering 1991), can assist in the selective recovery of some VTEC. This study observed that E. coli O26, unlike the other VTEC examined, was unable to ferment rhamnose. Such differences in carbohydrate metabolizing abilities among VTEC strains/serotypes have been previously reported, suggesting that it should be possible to exploit this serospecific characteristic in differentiating O26 from other confirmed VTEC isolates (Hiramatsu et al. 2002). Observations made in this study using CT-RMAC formulations containing a range of concentrations of rhamnose indicated that E. coli O26 grew as distinctive brown colonies and could be more easily differentiated from other VTEC on CT-RMAC containing a concentration of 20 g l)1.

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This study was unable to find as effective a carbohydrate source for the selection of E. coli O111 on agar. All examined members of this serogroup grew on, and could be most reliably distinguished from most competing bacteria on chromocult agar. The typical morphology of E. coli O111 on chromocult agar is purple colonies. The enrichment conditions noted above in relation to growth in TSB, the presence of cefixime and vancomycin was valuable in fsuppressing a range of Gram-positive and Gram-negative contaminants, during recovery of E. coli O111 on chromocult agar. This study has identified procedures for enriching E. coli O26 and O111 which when used in combination with IMS are very effective in concentrating the target E. coli O26 and O111 cells for plating onto selective agar. The fact that this study was not able to define one or more temperature/time/media/supplement combinations which was completely effective in the selective recovery and/or differentiation of VTEC, probably reflects the overall and increasing diversity within this group. As discussed by McDowell and Sheridan (2001), the set of genes which make this group of organisms significant pathogens, are continuing to spread into a widening population within strains of E. coli, species within the genus Escherichia, and beyond, to more distant species such as Citrobacter freundii (Schmidt et al. 1993). Such gene diffusion, leading to the acquisition and expression of pathogenic genes, e.g. VT genes, within a range of otherwise diverse strains and species, poses increasing problems in culture (phenotypic) based methods in microbiology. In the future, it may become increasingly necessary to develop culture/phenotypic independent methods, which focus on the detection of undesirable and otherwise pathogenic genes, within otherwise heterogeneous groups of bacteria. However, in the short-term, it may be more important to recognize the extent of such gene diffusion, and its impact on current selective/differential culture based strategies. Thus it may be necessary to continue to review culture based approaches, to ensure that they continue to reflect the relationships which initially made them valid. In relation to VTEC, for example, sorbitol fermenting E. coli O157 organisms are now being identified, and there is a danger that clones exhibiting this atypical phenotype could be overlooked or misidentified in commonly used culture schemes using SMAC or SMACderived media (Bielaszewska` et al. 1998). ACKNOWLEDGEMENTS The Food Institutional Research Measure (FIRM) administered by the Irish Department of Agriculture and Rural Development is acknowledged for funding this research. We thank Fujisawa (Japan) for kindly supplying the cefixime, and Oxoid (Germany) for supplying the enterohaemolysin



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