Journal of Microbiological Methods 96 (2014) 6–11
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Evaluation of eight agar media for the isolation of shiga toxin—Producing Escherichia coli Alexander Gill a,⁎, George Huszczynski b, Martine Gauthier b, Burton Blais b a b
Bureau of Microbial Hazards, Health Canada, Frederick Banting Building, Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada Ontario Laboratory Network, Canadian Food Inspection Agency, 960 Carling Avenue, Bldg. 22, C.E.F., Ottawa, Ontario K1A 0C6, Canada
a r t i c l e
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Article history: Received 11 September 2013 Received in revised form 29 October 2013 Accepted 29 October 2013 Available online 5 November 2013 Keywords: Shiga toxin/verotoxin Escherichia coli Growth Agar media Selective supplements
a b s t r a c t The growth characteristics of 96 shiga toxin-producing Escherichia coli (STEC) strains representing 36 different O-types (including priority O types O26, O45, O103, O111, O121, O145 and O157) on commercial and in-house agar media were studied. The ability of the strains to grow on agar media with varying selective supplement formulations was evaluated using MacConkey Agar (MAC); Rainbow® Agar O157 (RBA); Rainbow® Agar O157 with manufacturer-recommended selective supplements (RBA-NT); Rainbow® Agar O157 with USDA-recommended selective supplements (RBA-USDA); CHROMagar STEC™ (CH STEC); Tryptone Bile agar containing cefixime and tellurite (TBA-CT); Tryptone Bile agar containing cefixime, tellurite, eosin and methylene blue (TBA-EM); and VTEC agar. All of the strains were able to grow on MAC, RBA and VTEC agar, whereas a number of strains (including some non-O157 priority O types) were unable to grow on the highly selective media CH STEC, RBA-NT, RBA-USDA, TBA-EM and TBA-CT. Only RBA-NT and CH STEC exhibited significant inhibition of background flora from ground beef enrichment. Significant inhibition of background flora from beef trim enrichment was observed with RBA-NT, RBA-USDA, CH STEC, TBA-EM and VTEC agar. With exception of E. coli O157, several different colony morphologies were observed on the differential plating media among strains of the same O type, indicating that this colony morphology is not a reliable means of identifying target STEC. These results suggest that an approach to maximize the recovery of target STEC from beef enrichment cultures is dual plating on lesser (RBA, MAC, VTEC agar) and more highly (RBA-NT, CH STEC) selective agars. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Shiga toxin-producing Escherichia coli (STEC) (also known as verotoxin producing E. coli or enterohemorrhagic E. coli) are the enteric E. coli pathotypes of the greatest public health significance in the industrialized world due to their low infectious dose, potentially severe patient outcomes and limited treatment options (Melton-Celsa et al., 2012). Development of methods for the detection and isolation of STEC has focused on E. coli O157:H7 and its nonmotile variant (E. coli O157) as this serotype has been responsible for a number of major foodborne outbreaks and is the cause of the majority of reported cases in some regions, including Canada and the USA (CDC, 2012; PHAC, 2010). Other serotypes of STEC (non-O157 STEC) are estimated to account for 50–60% of cases of STEC illness in Canada and the USA (CDC, 2012; Thompson et al., 2005; Chui et al., 2011). The serotypes of STEC that predominate as a cause of human illness vary between geographic regions (EFSA, 2007; Johnson et al., 2006).
⁎ Corresponding author. Tel.: +1 613 952 8894. E-mail address: [email protected] (A. Gill).
The diversity of STEC serotypes causing disease is high, at least 82 different O-types of STEC have been reported as clinical isolates from three or more outbreaks or unlinked sporadic cases (Bettelheim, 2007). This situation is probably a consequence of the mobility of the verotoxin genes, which are encoded by a lysogenic phage (Kaper et al., 2004). Thus, there is a high probability of novel STEC pathogens emerging, such as the verotoxin-positive enteroaggregative E. coli O104:H4 responsible for the 2011 outbreak in Germany (Beutin and Martin, 2012). Isolation of STEC from enrichment broth cultures of foods or other sample types requires an agar medium for the isolated growth of individual colonies. Such a medium should support the growth of the target organism and ideally have selective and differential characteristics to support the rapid identification of pathogen colonies in the presence of other microbial flora. There is a variety of commercially available media for E. coli O157 which meets these requirements (Heuvelink, 2012). These media are commonly based on differential characteristics typical to E. coli O157 but unusual in other E. coli, notably the absence of sorbitol fermentation and β-D-glucuronidase activity. Selectivity is achieved by the relatively high resistance of E. coli O157 to some antimicrobials, such as novobiocin and tellurite, compared to other E. coli. These differential and selective characteristics are not shared by other STEC (Gill et al., 2012) and to date the only characteristic known to
0167-7012/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.022
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
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Table 1 Characteristics of STEC strains and their percentage recovery on selective agar media compared to Brain Heart Infusion agar.a Serotypea
Strain ID
Isolation
stx1
stx2
eae
hlyA
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26:NM O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H21 O26:H21 O26:H21 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H11 O103:H21 O103:H21 O103:H25 O111:NM O111:NM O111:NM O111:NM O111:NM O111:NM O111:NM O111:H8 O111:H8 O111:H11 O121:NM O121:H1 O121:H1 O121:H10 O121:H19 O121:H19 O121:H19 O121:H19 O121:H19 O121:H19 O145:NM O145:NM O145:NM O145:NM O145:NM O145:NM O145:H2 O145:H2 O145:H25 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:NM O157:NM O157:NM O1:H20 O5:NM O6:H34 O7:H4 O8:H19 O46:H38 O55:H7 O69:H11 O76:H19
11-6009 05-7321 05-6544 03-2816 01-5870 02-6737 99-4610 11-5130 11-5593 11-5805 04-2445 05-6545 85-X-40c R3 3267-95 3285-96 89-39 99-2076 06-0434 04-2446 95-266 09-5066 11-5806 04-3973 11-4211 11-5595 03-2444 05-4161 98-8338 CFS3 00-4748 00-4440 11-5592 03-3991 3331-00 3413-07 OLC-455 03-4064 11-5594 11-5597 96-0120 11-2925 11-3925 11-4440 03-2832 00-5288 03-2642 04-1449 04-7099 03-6430 03-4699 2454-01 VT113-5 A9619.C2 75-83 2769 ATCC 35150 1011-84 HCO 59 11-1024 11-1865 EDL933 Sakai ER63-94 E32511 87-1215 91-0812 03-2682 03-5166 92-0249 09-1764 97-0757 05-0376 11-5596 09-0523
Human Human Human Human Human Human Human Human Human Human Human Human Cattle Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Unknown Unknown Unknown Human Human Human Unknown Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Unknown Human Human Unknown Unknown Human Human Human Unknown
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + + + − − − − − − − − − − + + + + + − + − − + + + − − + + + − + + + − − + + + + +
− − − − − + − − + − − − − − − − − − − − − − − − − − + − + + − − − + + − + + + + + + + + + + − − − − − + − + + + + + + + + + + + + − − + + + + − − +
+ + + + + + + + − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + + + + + + + + + − + + + + + + + + + + + + − + − − − − + + −
− + + − + + + − + + + + + + + + + + + + + + + + + + − + + − + + + + + + + + + − + + − + + + + + + + + + − + + + + + + + + + + + − + + − − + + − + +
106% 119% 123% 133% 118% 33% 133% 85% 95% 113% 135% 81% 102% 116% 72% 94% 53% 86% 79% 167% 81% 110% 89% 81% 126% 67% 126% 109% 96% 2% 34% 82% 100% 100% 103% 87% 179% 214% 75% 90% 89% 111% 113% 108% 123% 118% 116% 103% 136% 111% 98% 36% 33% 103% 117% 83% 100% 104% 143% 81% 95% 112% 87% 122% 114% 119% 97% 123% 170% 109% 83% 127% 143% 100%
124% 94% 157% 123% 95% 67% 100% 72% 158% 103% 93% 73% 131% 113% 126% 111% 41% 110% 74% 189% 104% 95% 85% 96% 92% 83% 174% 98% 82% 8% 68% 158% 71% 100% 132% 93% 53% 145% 100% 115% 88% 100% 105% 119% 74% 97% 100% 125% 130% 100% 115% 62% 58% 69% 117% 72% 103% 85% 152% 88% 18% 160% 67% 83% 129% 113% 109% 94% 128% 123% 81% 107% 129% 120%
133% 121% 20% 111% 103% 52% 108% 83% NG 105% 71% 49% 63% 60% 126% 52% NG 100% 77% NG 13% 46% 75% 56% 97% 3% 53% NG 7% NG 28% 85% 32% 30% NG 50% b0.1% 73% 49% NG 85% 32% 118% 33% 51% 21% 68% 67% 70% 29% 63% 53% NG 53% 60% 29% 18% 30% 19% 38% 108% 36% 29% 65% 114% NG 91% NG NG b0.1% 32% NG 114% NG
51% 188% 117% 52% 47% 95% 108% 71% NG 175% 55% 40% 35% 22% 27% 33% NG 96% 130% NG 23% 79% 100% 100% 100% 25% 63% NG 96% b0.1% 60% 103% 29% 39% 67% 60% 94% 111% 67% b0.1% 115% 6% 71% 77% 18% 80% 67% 49% 52% 43% 82% 32% NG 42% 39% 34% 51% 35% 78% 75% 29% 21% 78% 53% 86% 19% 92% NG b0.1% NG 47% NG 83% NG
104% 106% b0.1%% 119% 62% 52% 62% 70% NG 103% 68% 7% 19% 18% 65% 19% NG 45% 32% NG b0.1% 46% 69% 30% 92% b0.1% 42% NG 20% NG 6% 35% 26% 9% NG 27% 42% 73% 31% NG 84% 11% 125% 8% 4% 5% 48% 47% 70% 20% 13% 6% NG NG 33% 14% 9% 11% 5% 12% 8% 17% 15% 9% 57% b0.1% 51% NG NG b0.1% 23% NG 129% NG
102% 91% 87% 101% 90% 57% 67% 83% 71% 89% 132% 76% 95% 68% 81% 20% NG 103% 81% 189% 71% 115% 109% 107% 113% 37% 111% b0.1% 51% b0.1% 38% 127% 65% 52% b0.1% 53% 128% 159% 82% b0.1% 68% 61% 95% 64% 118% 33% 55% 122% 115% 95% 67% 53% NG 56% 81% 108% 36% 67% 81% 88% 60% 112% 98% 78% 171% 59% 74% 60% 87% 85% 73% 83% 57% 117%
114% 104% 97% 103% 72% 95% 100% 98% 58% 89% 103% 90% 126% 84% 102% 98% NG 83% 121% 87% 85% 115% 106% 93% 139% 45% 153% b0.1% 65% b0.1% 49% 61% 65% 74% 132% 83% 137% 168% 88% 100% 78% 70% 105% 106% 111% 92% 52% 94% 130% 78% 71% 54% NG 83% 90% 100% 45% 81% 129% 62% 80% 93% 79% 96% 86% 120% 87% 125% 113% 88% 87% 82% 86% 92%
120% 121% 150% 188% 108% 81% 100% 102% 105% 92% 132% 71% 84% 108% 63% 127% 94% 117% 145% 122% 81% 103% 115% 93% 105% 92% 137% 89% 76% 33% 36% 88% 68% 113% 156% 73% 133% 145% 88% 85% 91% 74% 20% 81% 102% 105% 106% 106% 121% 93% 81% 7% 38% 94% 88% 75% 115% 107% 86% 112% 90% 105% 71% 122% 57% 107% 98% 114% 143% 106% 108% 110% 71% 80%
(continued on next page)
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A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
Table 1 (continued) Serotypea
Strain ID
Isolation
stx1
stx2
eae
hlyA
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O91:H21 O98:H29 O100:NM O104:H4 O104:H21 O108:H11 O110:H28 O113:H21 O115:H18 O117:H7 O123:H2 O128:H2 O128:H10 O130:H11 O146:H21 O153:H25 O156:NM O165:H25 O177:NM O179:H8 O181:H49 Orough:H21
85-489 09-5073 96-0429 11-3088 3024-94 11-3580 09-409 04-1450 03-3645 02-0035 11-4968 1759 2611-99 09-1765 02-1628 09-1775 92-0389 00-4540 03-3974 09-415 09-412 11-6008
Human Human Unknown Human Unknown Human Unknown Human Human Human Human Human Human Unknown Human Unknown Unknown Human Human Unknown Unknown Human
− + − − − − − − + + + + + − + + − − − − − +
+ − + + + + + + + − − − − + − + + + + + + +
− + − − − + − − − − + − − − − − − + + − − −
+ + − − + + + + + − + + − + + + − + + + + +
113% 107% 124% 92% 110% 110% 75% 137% 93% 111% 68% 86% 115% 125% 133% 100% 113% 118% 78% 108% 119% 103%
94% 97% 119% 127% 89% 78% 100% 130% 100% 115% 97% 100% 110% 99% 110% 133% 115% 136% 93% 138% 138% 81%
b0.1% 79% 105% 42% NG 24% NG NG NG NG 40% NG NG NG NG NG NG NG 24% NG NG NG
NG 81% 117% 50% NG 24% b0.1% NG 66% NG 85% NG NG b0.1% NG b0.1% NG NG 35% b0.1% NG NG
NG 90% 80% 35% NG 29% NG NG NG NG 51% NG NG NG NG NG NG NG 34% NG NG NG
42% 134% 104% 115% 55% 60% 65% 33% 69% 63% 62% 57% b0.1% 87% 116% 67% NG 91% 68% 70% 50% 69%
83% 72% 109% 108% 93% 63% 80% 67% 66% 105% 78% 95% 101% 86% 96% 142% 75% 118% 80% 104% 117% 66%
110% 100% 114% 121% 85% 84% 55% 133% 66% 109% 81% 93% 118% 111% 133% 87% 112% 123% 93% 114% 152% 147%
MAC: MacConkey Agar. RBA: Rainbow® Agar O157. RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite. RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite. CH STEC: CHROMagar STEC™. TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite. TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose. VTEC: VTEC agar (Gill et al., 2012). a +: positive, −: negative, NG: no growth on streaked or enumeration plates, b0.1% growth on streaked plate but recovery on enumerated plates below 0.1% of BHI.
be common to all STEC and absent from other E. coli is verotoxin production. Few outbreaks of illness due to non-O157 STEC contaminated beef have been reported, compared to E. coli O157, but the development of methods for non-O157 STEC in beef is a priority due to the high rates of carriage of non-O157 STEC by cattle and their presence in raw beef (Gill and Gill, 2010). In an attempt to mitigate public health impacts of non-O157 STEC in beef, the United States Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) has implemented testing of manufacturing trim and raw ground beef for the presence of the so-called priority STEC serogroups O26, O45, O103, O111, O121, and O145, in addition to E. coli O157:H7 (USDA, 2012). While several agar media have been developed or proposed for the isolation of STEC from foods, few studies have been published on the comparative performance of these media and those that have tended to focus on a relatively small number of STEC strains of a limited range of serotypes. For this reason we have conducted a study to evaluate the ability of eight different agar media to support the growth of 96 STEC strains, representing 36 different O-types. The media were also evaluated to assess their ability to suppress the growth of background microflora from ground beef and beef trim. 2. Material and methods 2.1. Bacterial cultures The 96 strains of STEC used in this study and their virulence characteristics are listed in Table 1. Unless otherwise noted the strains used were provided from the collection of the Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba. Strains 85-X-40c R3, 3267–95, 3285–96, 89–39, 2454–01, 2769, 2454–01, EDL933, ER63-94, E32511, Sakai, 1759, 2611–99 were kindly supplied by Dr. Roger Johnson of the Public Health Agency of Canada, Laboratory of Food-Borne Zoonoses, Guelph, Ontario. Strains CFS3, 3331–00, 3413–07 were kindly supplied by Dr. P. Micheal Doyle of the University of Georgia, Griffin, Georgia. Strain 11–3088 was kindly supplied by Dr. Vanessa Gray Allen of the Public Health Ontario Laboratories,
Toronto, Ontario. The remaining strains were from the culture collection of the Health Canada, Bureau of Microbial Hazards, Ottawa, Ontario. The presence of STEC virulence genes stx1, stx2, eae, and EHEC hlyA was determined by the PCR protocol of Paton and Paton (2003). The cultures were stored as frozen glycerol stocks. For experimental use, the cultures were streaked onto brain heart infusion (BHI) agar (Difco, BD, Sparks, MD) and incubated at 37 °C for 24 h. 2.2. Recovery of STEC strains on selective agar Single colonies of the strains were individually inoculated into 10 ml of modified tryptic soy broth (mTSB: tryptic soy broth [30 g/l; Difco], bile salts no. 3 [1.5 g/l; Difco, BD], K2HPO4 [1.5 g/l; Sigma, St. Louis, MO], pH 7.4), supplemented with 10 mg/l vancomycin (Sigma) and 3 mg/l cefsulodin (Sigma) after 4 h (mTSB-VC broth) as described in Gill et al. (2012), and incubated for 18–24 h. at 42 °C. The following selective agar media were evaluated for their ability to support the growth of STEC strains. MacConkey Agar (MAC; Difco); Rainbow® Agar O157 (RBA; Biolog Inc., Hayward, California); Rainbow® Agar O157 supplemented, with 10 mg/l novobiocin (Sigma) and 0.8 mg/l potassium tellurite (Sigma) (RBA-NT) as recommended by the manufacturer for samples with high background flora; Rainbow® Agar O157 supplemented with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate (Oxoid) and 0.15 mg/l potassium tellurite (RBA-USDA) as recommended for the USDA STEC method MLG 5B.03 (USDA, 2012); CHROMagar STEC™ (CHROMagar Microbiology, Paris, France) (CH STEC); Tryptone Bile agar (Oxoid, Basingstoke, Hampshire, UK) containing 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite (TBA-CT); Tryptone Bile agar containing 0.05 mg/l cefixime, 0.5 mg/l of potassium tellurite, 0.4 g/l eosin Y (Sigma), 0.06 g/l methylene blue (Fisher Scientific, Ottawa, Ontario) and 1 g/l lactose (TBA-EM); and VTEC agar (Gill et al., 2012), 17.5 g/l vegetable special infusion powder (Sigma), 2 g/l meat extract (Sigma), 10 g/l proteose peptone (Oxoid), 10 g/l D-sorbitol (Sigma), 5 g/l sodium chloride (Sigma), 2.5 g/l sodium phosphate dibasic (Sigma), 30 mg/l neutral red (Sigma), 1 mg/l crystal violet (Sigma), 1 g/l bile salts #3 and 15 g/l agar, supplemented with 10 mg/l vancomycin and 3 mg/l cefsulodin.
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
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Table 2 Proportion of STEC strains whose growth is inhibited on selective agar media tested.a O-type
No. strains
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26 O45 O103 O111 O121 O145 O157 Others Total
10 6 10 10 10 9 10 31 96
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
1 0 2 3 1 1 0 20 28 (29.2%)
1 0 2 1 0 1 0 15 20 (20.8%)
1 0 2 1 1 2 0 20 27 (28.1%)
0 0 1 0 0 1 0 1 3 (3.1%)
0 0 1 0 0 1 0 0 2 (2.1%)
0 0 0 0 0 0 0 0 0
a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012).
Cultures grown in mTSB-VC were streaked onto the eight selective agar media, which were then incubated for 24 h at 37 °C. The plates were examined for growth and the colony morphology recorded. To determine the relative enumerative recovery on the selective agars, the cultures were serially diluted in phosphate buffered saline (PBS) and the dilutions manually spread plated onto the eight selective agar media and BHI agar. The plates were incubated for 24 h at 37 °C and the colonies counted. The relative efficiency of recovery was calculated as the percentage of colonies on the selective agar compared to BHI. Replicate experiments were not performed systematically as little variation is expected with a descriptive study of this type. The primary potential sources of error were identified as a failure to inoculate media or pipetting errors leading to inaccurate reporting of growth inhibition. These errors could be detected by observation of turbidity in the culture test tubes and comparison of the culture dilutions plated for enumerative recovery. A small number of errors were detected and the experiments with those strains repeated and reported. 2.3. Growth of beef microflora on selective agar Experiments were conducted to evaluate the ability of the selective agar to inhibit the growth of background microflora present in enrichment cultures from ground beef and beef trim that were not inoculated with STEC. The ground beef had a 15% fat content and was purchased from an Ottawa area supermarket, Trim samples were sourced from an Ontario beef processing plant. Samples of meat 25 g were suspended in 225 ml of mTSB-VC and incubated for 18–24 h at 42 °C. The postincubation enrichment broth was then serially diluted at a 1:10 ratio in PBS (1 ml into 9 ml) from 10−1 to 10−7 and all dilutions manually spread plated on the eight selective agar and BHI agar. The agar media were incubated as described above and the total colonies on each plate enumerated. This experiment was performed with meat from three separate lots of ground beef and beef trim. 3. Results The percentage recovery of each STEC strain on the eight selective agar media is presented in Table 1. The ability of the agar media to support the growth of strains of the 7 STEC O-types designated a priority by the USDA (O26, O45, O103, O111, O121, O145, O157) is summarized in Table 2. Three of the media tested MAC, RBA and VTEC agar were able to support the growth of all 96 STEC strains tested. TBA-EM did not support the growth of two strains (O103:H2 99–2076 and O145:H2 A9619.C2). TBA-CT did not support the growth of the same O103 and O145 strains as TBA-EM, and in addition no growth was observed for O156:NM 92–0389. RBA-USDA did not support the growth of 20 (20.8%) strains tested, including strains of the serogroups O26 (1/10), O103 (2/10), O111 (1/10) and O145 (1/9). RBA-NT and CH STEC supported the growth of the least number of STEC strains, with 28 (29.2%) strains on RBA-NT and 27 (28.1%) strains on CH STEC failing to develop visible colonies after 24 h incubation. Strains inhibited on
CH STEC included O26 (1/10), O103 (2/10), O111 (1/10), O121 (1/10) and O145 (2/9). Strains inhibited on RBA-NT included O26 (1/10), O103 (2/10), O111 (3/10), O121 (1/10) and O145 (1/9). For those STEC strains which were able to grow on the selective media, the average number of colonies counted compared to BHI agar was 100% for MAC, RBA and VTEC agar (Table 3). The average number of colonies counted on TBA-EM was 91% and for TBA-CT 78% compared to BHI agar. The average number of STEC colonies counted on RBA-NT was 58% and RBA-USDA 60% compared to BHI agar, down from 100% for RBA without additional antimicrobials. CH STEC was the medium which was most inhibitory to STEC growth, with an average of 40% of colonies compared to BHI agar and O26 was the only serogroup for which the colony count was greater than 50% of the BHI agar count. The appearance of STEC colonies on the different selective media is described in Table 4. The colour of the colonies of all strains was highly uniform on MAC, TBA-CT and TBA-EM. Colonies of all strains on TBA-CT were white, and almost all were pink on MAC and TBA-EM. Colonies of strains of the same serogroup were not a consistent colour on RBA, RBANT and RBA-USDA, With the exception of the O157 serogroup, which were blue-grey. Two or more colours of colonies were observed for strains all serogroups on CH STEC and for all serogroups on STEC agar except O26, O103 and O145, which were uniformly pink. The number of colonies recovered from enrichment broths of ground beef and beef trim in mTSB-VC on the selective agar media was variable among the three samples (Table 5). This probably reflects variation in the composition of the initial flora of the meat samples prior to enrichment. For ground beef enrichment the number of colonies recovered was only significantly less (Student's t-test p b 0.05) than BHI agar on RBA-NT and CH STEC. Significantly lower numbers of colonies (p b 0.05) were recovered from beef trim enrichment compared to BHI agar on RBA-NT, RBA-USDA and CH STEC.
Table 3 Percentage recovery of STEC strains on selective agar media compared to Brain Heart Infusion agar, excluding inhibited strains.a O-type
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26 O45 O103 O111 O121 O145 O157 Others Total
106% 93% 94% 84% 122% 95% 104% 110% 104%
109% 108% 97% 98% 100% 97% 96% 110% 103%
93% 70% 58% 41% 51% 58% 49% 55% 58%
100% 35% 82% 57% 64% 51% 54% 44% 60%
75% 33% 39% 24% 43% 34% 16% 47% 40%
85% 79% 103% 50% 81% 81% 90% 72% 78%
97% 101% 97% 68% 106% 82% 85% 93% 91%
118% 98% 107% 87% 92% 82% 94% 106% 100%
a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012).
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A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
Table 4 Appearance of STEC colonies on selective agar.a Serogroup
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC agar
O26
Pink (10)
Mauve (7) Pink (2) Pink (4) Mauve White
White (9) Pin point White (6)
Pink (6)
Pink (5) White
O103
Pink (10)
Pink (4) Blue-grey (3) White Mauve
Pink (2) Blue-grey (3) White (2) Mauve
White (9)
Pink (9)
Pink (10)
O111
Pink (10)
Blue-grey (8) White Mauve
Blue-grey (6) Pin Point
Pink (3) Mauve (4)
White (7) Pin point (3)
Pink (7) Mauve Pin point (2)
Pink (9) White
O121
Pink (10)
White (9) Pin point
Pink (10)
Pink (9) Mauve
Pink (9)
Mauve (4) Blue-grey (2) Pink (2) Pin point Blue-grey (2) Mauve (6)
Pink (6) Mauve (3)
O145
Mauve (4) Blue-grey (2) Pink (3) White Blue-grey (3) Mauve (6)
Pink (3) Blue-grey (2) White Mauve Light pink Blue-grey (6) White Mauve Pin Point Mauve (2) Pink (7) Pin point
Mauve (8) Pink Mauve (3) Pink White Blue-grey Mauve Pink (7)
Pink (10)
Pink (6)
Mauve (5) Blue-grey (4) Pink (3) Mauve Blue-grey (2)
Pink (10)
O45
Mauve (5) Blue-grey (5) Pink (3) Mauve (1) Blue-grey (2)
Pink (6) Mauve (2)
White (8)
Pink (8)
Pink (9)
O157
Pink (10)
Blue-grey (10)
Blue-grey (10)
White (10)
Pink (10)
Others
Pink (30) Light pink (1)
Pink (9) Mauve (15) Blue-grey (5)
Pink (3) Mauve (3) Blue-grey (5)
Pink (6) Mauve (4) Pink (8) Mauve (3)
White (22) Pin point (8)
Pink (31)
Light pink (9) Pink Pink (29) Light pink (2)
Blue-grey (3) Mauve (4) Pink Blue-grey (10) Pink (8) Mauve Blue-grey (3) Light pink Pin point (3)
CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite. TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012). a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite.
4. Discussion The isolation of STEC from foods remains a challenge because STEC are very often present with other non-pathogenic E. coli or Enterobacteriaceae and no selective condition has been identified which is unique to the STEC pathotype of E. coli. Since no unique differential characteristic has been identified, other than verotoxin expression, this means that isolation requires that potentially large numbers of colonies must be screened individually for VT genes or expression. Diverse STEC have been successfully isolated from foods, by identifying VT positive colonies grown on a relatively permissive agar medium by colony immunoblot (Atalla et al., 2000) or colony hybridization (Barlow et al., 2006). But the lack of uptake of these methods reflects the technical challenges in adopting these methods outside of a research laboratory. Alternatively, isolation media for specific subgroups of STEC have been developed which are based on selective or differential
characteristics common in the subgroup. These include hemolysin activity (Beutin et al., 1989), carbohydrate fermentation patterns (Possé et al., 2008) or antimicrobials (Hiramatsu et al., 2002; Tzschoppe et al., 2012). This approach may be of utility when seeking the isolation of a STEC strain of known characteristics, but given the diversity of STEC strains there is serious concern that STEC pathogens may not be recovered on agar media containing inhibitory levels of antimicrobial agents or which use differential characteristics that may screen out the pathogen. It should be noted that the diversity of colony morphologies observed on the agar media in this study, even among strains of a given O type, indicates that colony appearance will not provide a reliable means of identifying target STEC in the presence of other E. coli. The problem of media being too selective was demonstrated in this study by the results obtained with CH STEC, RBA-NT and RBA-USDA. These media were unable to support the growth of a significant proportion of STEC strains (CH STEC 28.1%, RBA-NT 29.2%, RBA-USDA 20.8%),
Table 5 Recovery of flora from beef enriched in modified tryptic soy broth with 10 mg/l vancomycin and 3 mg/l cefsulodin for 24 h at 42 °C.a Log CFU/ml O-type
BHI
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
Ground beef Ground beef Ground beef Mean Beef trim Beef trim Beef trim Mean
8.5 8.9 8.8 8.8a 8.4 8.8 8.6 8.6a
8.6 8.8 8.7 8.7a 8.5 8.6 8.5 8.5ac
8.7 8.8 8.8 8.8a 8.5 8.6 8.6 8.5a
b2.0 2.3 4.7 3.0b 3.3 7.5 4.5 5.1bc
7.4 8.0 8.6 8.0a 5.6 7.6 4.8 6.0b
b2.0 3.5 4.3 3.3b 5.0 6.9 5.0 5.6b
8.5 8.9 8.7 8.7a 8.1 8.5 8.3 8.3a
8.1 8.9 8.8 8.6a 7.3 7.4 4.3 6.3b
8.1 8.9 8.8 8.6a 7.3 7.4 4.3 6.3b
BHI: Brain Heart Infusion Agar; MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012). a Averages with different superscripts are significantly different by Student's t-test (p b 0.05).
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
including strains belonging to 5 of the 6 priority serogroups (O26, O45, O103, O111, O121, O145) designated for beef testing in the US (USDA, 2012). Inhibition of a significant proportion of STEC strains was also reported in two previous publications which evaluated the performance of CH STEC and identified resistance to tellurite as the primary selective principle of the medium (Hirvonen et al., 2012; Tzschoppe et al., 2012). Hirvonen et al. (2012) reported that CH STEC inhibited the growth of 93 of 362 (25.7%) STEC strains tested and the media was not inhibitory of the single strains tested of enteropathogenic E. coli, enterotoxigenic E. coli and enteroaggregative E. coli. The inhibited STEC included O26 (2/20), O103 (19/30) and non-sorbitol fermenting O157:H7 and NM (4/183) (Hirvonen et al., 2012). Tzschoppe et al. (2012) reported that CH STEC inhibited the growth of 34 of 133 (25.5%) STEC strains, including O103 (9/18), O121 (2/4), O145 (1/10), sorbitol fermenting O157 (5/5) and diverse serotypes (17/17). When used for the purpose of isolation of target bacteria from enrichment broth, selective agents added to agar media improve the probability of recovering the target bacteria by reducing the numbers of non-target flora which can grow on the media. In this study RBANT and CH STEC were the only agar media which had significantly lower numbers of colonies from ground beef enrichment (mTSB-VC at 42 °C) compared to non-selective BHI agar. However, RBA-NT, RBAUSDA, CH-STEC, TBA-EM and VTEC agar were equally inhibitory to the growth of background microflora from beef trim enrichment broth. These results suggest that depending on the composition of the initial microflora of the sample and the enrichment conditions, selective agar may not always improve the probability of target recovery.
5. Conclusions The essential requirement of any agar media for the isolation of bacteria is the ability to support the growth of the target organism, and secondary to that is the ability to suppress the growth of non-target organisms. Consequently, we conclude that due to the significant proportion of STEC strains whose growth is inhibited on these media, including strains of priority serogroups, that the agar media RBA-USDA, RBA-NT, and CH STEC are unsuitable for application to the isolation of STEC from beef as the sole isolation media. Nonetheless, RBA-USDA, RBA-NT, and CH STEC might be applied to beef samples containing high levels of background microflora, if paired with an agar media capable of supporting the growth of a wide range of STEC, such as MAC, RBA or VTEC agar.
Acknowledgements The authors would like to thank Mahdid Meymandy of Health Canada and Mylène Deschênes of the Canadian Food Inspection Agency for their technical assistance.
11
References Atalla, H.N., Johnson, R., McEwen, S., Usborne, R.W., Gyles, C.L., 2000. Use of a shiga toxin (Stx)-enzyme-linked immunosorbent assay and immunoblot for detection and isolation of stx-producing Escherichia coli from naturally contaminated beef. J. Food Prot. 63, 1167–1172. Barlow, R.S., Gobius, K.S., Desmarchelier, P.M., 2006. Shiga toxin-producing Escherichia coli in ground beef and lamb cuts: results of a one-year study. Int. J. Food Microbiol. 111, 1–5. Bettelheim, K.A., 2007. The non-O157 Shiga-toxigenic (verocytotoxigenic) Escherichia coli; under-rated pathogens. Crit. Rev. Microbiol. 33, 67–87. Beutin, L., Martin, A., 2012. Outbreak of shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany causes a paradigm shift with regard to human pathogenicity of STEC strains. J. Food Prot. 75, 408–418. Beutin, L., Montenegro, M.A., Orskov, I., Orskov, F., Prada, J., Zimmermann, S., Stephan, R., 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27, 2559–2564. CDC, 2012. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2011 (Final Report). U.S. Department of Health and Human Services, CDC, Atlanta, Georgia. Chui, L., Lee, M.-C., Malejczyk, K., Lim, L., Fok, D., Kwong, P., 2011. Prevalence of shiga toxin-producing Escherichia coli as detected by enzyme-linked immunoassays and real-time PCR during the summer months in Northern Alberta, Canada. J. Clin. Microbiol. 49, 4307–4310. European Food Safety Authority, 2007. Scientific opinion of the panel on biological hazards on a request from EFSA on monitoring of verotoxigenic Escherichia coli. EFSA J. 579, 1–61. Gill, A.O., Gill, C.O., 2010. Non-O157 verotoxigenic Escherichia coli and beef: a Canadian perspective. Can. J. Vet. Res. 74, 161–169. Gill, A., Martinez-Perez, A., McIlwham, S., Blais, B., 2012. Development of a method for the detection of verotoxin producing Escherichia coli in food. J. Food Prot. 75, 827–837. Heuvelink, A.E., 2012. Culture media for the isolation of diarrhoeagenic Escherichia coli from foods. Ch. 16 In: Corry, J.E.L., Curtis, G.D.W., Baird, R.M. (Eds.), Handbook of Culture Media for Food and Water Microbiology, pp. 321–356. Hiramatsu, R., Matsumoto, M., Miwa, Y., Suzuki, Y., Saito, M., Miyazaki, Y., 2002. Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains. J. Clin. Microbiol. 40, 922–925. Hirvonen, J.J., Siitonen, A., Kaukoranta, S., 2012. Usability and performance of CHROMagar STEC in detection of shiga toxin-producing Escherichia coli strains. J. Clin. Microbiol. 50, 3586–3590. Johnson, K.E., Thorpe, C.M., Sears, C.L., 2006. The emerging clinical importance of nonO157 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis. 43, 1587–1595. Kaper, J.B., Nataro, J.P., Mobley, H.L., 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2, 123–140. Melton-Celsa, A., Mohawk, K., Teel, L., O'Brien, A., 2012. Pathogenesis of shiga-toxin producing Escherichia coli. Curr. Top. Microbiol. Immunol. 357, 67–103. Paton, A.W., Paton, J.C., 2003. Detection and characterization of STEC in stool samples using PCR. An overview. Methods Mol. Med. 73, 45–54. Possé, B., De Zutter, L., Heyndrickx, M., Herman, L., 2008. Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol positive and negative O157. FEMS Microbiol. Lett. 282, 124–131. Public Health Agency of Canada, 2010. National enteric surveillance program (NESP). Annual Summary 2010. (http://www.nml-lnm.gc.ca/NESP-PNSME/surveillance-2010eng.html. Accessed 11 March 2013). Thompson, L.H., Giercke, S., Beaudoin, C., Woodward, D., Wylie, J.L., 2005. Enhanced surveillance of non-O157 verotoxin-producing Escherichia coli in human stool samples from Manitoba. Can. J. Infect. Dis. Med. Microbiol. 16, 329–334. Tzschoppe, M., Martin, A., Beutin, L., 2012. A rapid procedure for the detection and isolation of enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables. Int. J. Food Microbiol. 152, 19–30. USDA, 2012. MLG 5B.03 detection and isolation of non-O157 shiga toxin-producing Escherichia coli (STEC) from meat products. http://www.fsis.usda.gov/PDF/MLG-5B. pdf (Accessed 17 March 2013).
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Evaluation of eight agar media for the isolation of shiga toxin—Producing Escherichia coli Alexander Gill a,⁎, George Huszczynski b, Martine Gauthier b, Burton Blais b a b
Bureau of Microbial Hazards, Health Canada, Frederick Banting Building, Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada Ontario Laboratory Network, Canadian Food Inspection Agency, 960 Carling Avenue, Bldg. 22, C.E.F., Ottawa, Ontario K1A 0C6, Canada
a r t i c l e
i n f o
Article history: Received 11 September 2013 Received in revised form 29 October 2013 Accepted 29 October 2013 Available online 5 November 2013 Keywords: Shiga toxin/verotoxin Escherichia coli Growth Agar media Selective supplements
a b s t r a c t The growth characteristics of 96 shiga toxin-producing Escherichia coli (STEC) strains representing 36 different O-types (including priority O types O26, O45, O103, O111, O121, O145 and O157) on commercial and in-house agar media were studied. The ability of the strains to grow on agar media with varying selective supplement formulations was evaluated using MacConkey Agar (MAC); Rainbow® Agar O157 (RBA); Rainbow® Agar O157 with manufacturer-recommended selective supplements (RBA-NT); Rainbow® Agar O157 with USDA-recommended selective supplements (RBA-USDA); CHROMagar STEC™ (CH STEC); Tryptone Bile agar containing cefixime and tellurite (TBA-CT); Tryptone Bile agar containing cefixime, tellurite, eosin and methylene blue (TBA-EM); and VTEC agar. All of the strains were able to grow on MAC, RBA and VTEC agar, whereas a number of strains (including some non-O157 priority O types) were unable to grow on the highly selective media CH STEC, RBA-NT, RBA-USDA, TBA-EM and TBA-CT. Only RBA-NT and CH STEC exhibited significant inhibition of background flora from ground beef enrichment. Significant inhibition of background flora from beef trim enrichment was observed with RBA-NT, RBA-USDA, CH STEC, TBA-EM and VTEC agar. With exception of E. coli O157, several different colony morphologies were observed on the differential plating media among strains of the same O type, indicating that this colony morphology is not a reliable means of identifying target STEC. These results suggest that an approach to maximize the recovery of target STEC from beef enrichment cultures is dual plating on lesser (RBA, MAC, VTEC agar) and more highly (RBA-NT, CH STEC) selective agars. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Shiga toxin-producing Escherichia coli (STEC) (also known as verotoxin producing E. coli or enterohemorrhagic E. coli) are the enteric E. coli pathotypes of the greatest public health significance in the industrialized world due to their low infectious dose, potentially severe patient outcomes and limited treatment options (Melton-Celsa et al., 2012). Development of methods for the detection and isolation of STEC has focused on E. coli O157:H7 and its nonmotile variant (E. coli O157) as this serotype has been responsible for a number of major foodborne outbreaks and is the cause of the majority of reported cases in some regions, including Canada and the USA (CDC, 2012; PHAC, 2010). Other serotypes of STEC (non-O157 STEC) are estimated to account for 50–60% of cases of STEC illness in Canada and the USA (CDC, 2012; Thompson et al., 2005; Chui et al., 2011). The serotypes of STEC that predominate as a cause of human illness vary between geographic regions (EFSA, 2007; Johnson et al., 2006).
⁎ Corresponding author. Tel.: +1 613 952 8894. E-mail address: [email protected] (A. Gill).
The diversity of STEC serotypes causing disease is high, at least 82 different O-types of STEC have been reported as clinical isolates from three or more outbreaks or unlinked sporadic cases (Bettelheim, 2007). This situation is probably a consequence of the mobility of the verotoxin genes, which are encoded by a lysogenic phage (Kaper et al., 2004). Thus, there is a high probability of novel STEC pathogens emerging, such as the verotoxin-positive enteroaggregative E. coli O104:H4 responsible for the 2011 outbreak in Germany (Beutin and Martin, 2012). Isolation of STEC from enrichment broth cultures of foods or other sample types requires an agar medium for the isolated growth of individual colonies. Such a medium should support the growth of the target organism and ideally have selective and differential characteristics to support the rapid identification of pathogen colonies in the presence of other microbial flora. There is a variety of commercially available media for E. coli O157 which meets these requirements (Heuvelink, 2012). These media are commonly based on differential characteristics typical to E. coli O157 but unusual in other E. coli, notably the absence of sorbitol fermentation and β-D-glucuronidase activity. Selectivity is achieved by the relatively high resistance of E. coli O157 to some antimicrobials, such as novobiocin and tellurite, compared to other E. coli. These differential and selective characteristics are not shared by other STEC (Gill et al., 2012) and to date the only characteristic known to
0167-7012/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.022
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
7
Table 1 Characteristics of STEC strains and their percentage recovery on selective agar media compared to Brain Heart Infusion agar.a Serotypea
Strain ID
Isolation
stx1
stx2
eae
hlyA
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26:NM O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H21 O26:H21 O26:H21 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H11 O103:H21 O103:H21 O103:H25 O111:NM O111:NM O111:NM O111:NM O111:NM O111:NM O111:NM O111:H8 O111:H8 O111:H11 O121:NM O121:H1 O121:H1 O121:H10 O121:H19 O121:H19 O121:H19 O121:H19 O121:H19 O121:H19 O145:NM O145:NM O145:NM O145:NM O145:NM O145:NM O145:H2 O145:H2 O145:H25 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:NM O157:NM O157:NM O1:H20 O5:NM O6:H34 O7:H4 O8:H19 O46:H38 O55:H7 O69:H11 O76:H19
11-6009 05-7321 05-6544 03-2816 01-5870 02-6737 99-4610 11-5130 11-5593 11-5805 04-2445 05-6545 85-X-40c R3 3267-95 3285-96 89-39 99-2076 06-0434 04-2446 95-266 09-5066 11-5806 04-3973 11-4211 11-5595 03-2444 05-4161 98-8338 CFS3 00-4748 00-4440 11-5592 03-3991 3331-00 3413-07 OLC-455 03-4064 11-5594 11-5597 96-0120 11-2925 11-3925 11-4440 03-2832 00-5288 03-2642 04-1449 04-7099 03-6430 03-4699 2454-01 VT113-5 A9619.C2 75-83 2769 ATCC 35150 1011-84 HCO 59 11-1024 11-1865 EDL933 Sakai ER63-94 E32511 87-1215 91-0812 03-2682 03-5166 92-0249 09-1764 97-0757 05-0376 11-5596 09-0523
Human Human Human Human Human Human Human Human Human Human Human Human Cattle Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Unknown Unknown Unknown Human Human Human Unknown Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Unknown Human Human Unknown Unknown Human Human Human Unknown
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + + + − − − − − − − − − − + + + + + − + − − + + + − − + + + − + + + − − + + + + +
− − − − − + − − + − − − − − − − − − − − − − − − − − + − + + − − − + + − + + + + + + + + + + − − − − − + − + + + + + + + + + + + + − − + + + + − − +
+ + + + + + + + − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + + + + + + + + + − + + + + + + + + + + + + − + − − − − + + −
− + + − + + + − + + + + + + + + + + + + + + + + + + − + + − + + + + + + + + + − + + − + + + + + + + + + − + + + + + + + + + + + − + + − − + + − + +
106% 119% 123% 133% 118% 33% 133% 85% 95% 113% 135% 81% 102% 116% 72% 94% 53% 86% 79% 167% 81% 110% 89% 81% 126% 67% 126% 109% 96% 2% 34% 82% 100% 100% 103% 87% 179% 214% 75% 90% 89% 111% 113% 108% 123% 118% 116% 103% 136% 111% 98% 36% 33% 103% 117% 83% 100% 104% 143% 81% 95% 112% 87% 122% 114% 119% 97% 123% 170% 109% 83% 127% 143% 100%
124% 94% 157% 123% 95% 67% 100% 72% 158% 103% 93% 73% 131% 113% 126% 111% 41% 110% 74% 189% 104% 95% 85% 96% 92% 83% 174% 98% 82% 8% 68% 158% 71% 100% 132% 93% 53% 145% 100% 115% 88% 100% 105% 119% 74% 97% 100% 125% 130% 100% 115% 62% 58% 69% 117% 72% 103% 85% 152% 88% 18% 160% 67% 83% 129% 113% 109% 94% 128% 123% 81% 107% 129% 120%
133% 121% 20% 111% 103% 52% 108% 83% NG 105% 71% 49% 63% 60% 126% 52% NG 100% 77% NG 13% 46% 75% 56% 97% 3% 53% NG 7% NG 28% 85% 32% 30% NG 50% b0.1% 73% 49% NG 85% 32% 118% 33% 51% 21% 68% 67% 70% 29% 63% 53% NG 53% 60% 29% 18% 30% 19% 38% 108% 36% 29% 65% 114% NG 91% NG NG b0.1% 32% NG 114% NG
51% 188% 117% 52% 47% 95% 108% 71% NG 175% 55% 40% 35% 22% 27% 33% NG 96% 130% NG 23% 79% 100% 100% 100% 25% 63% NG 96% b0.1% 60% 103% 29% 39% 67% 60% 94% 111% 67% b0.1% 115% 6% 71% 77% 18% 80% 67% 49% 52% 43% 82% 32% NG 42% 39% 34% 51% 35% 78% 75% 29% 21% 78% 53% 86% 19% 92% NG b0.1% NG 47% NG 83% NG
104% 106% b0.1%% 119% 62% 52% 62% 70% NG 103% 68% 7% 19% 18% 65% 19% NG 45% 32% NG b0.1% 46% 69% 30% 92% b0.1% 42% NG 20% NG 6% 35% 26% 9% NG 27% 42% 73% 31% NG 84% 11% 125% 8% 4% 5% 48% 47% 70% 20% 13% 6% NG NG 33% 14% 9% 11% 5% 12% 8% 17% 15% 9% 57% b0.1% 51% NG NG b0.1% 23% NG 129% NG
102% 91% 87% 101% 90% 57% 67% 83% 71% 89% 132% 76% 95% 68% 81% 20% NG 103% 81% 189% 71% 115% 109% 107% 113% 37% 111% b0.1% 51% b0.1% 38% 127% 65% 52% b0.1% 53% 128% 159% 82% b0.1% 68% 61% 95% 64% 118% 33% 55% 122% 115% 95% 67% 53% NG 56% 81% 108% 36% 67% 81% 88% 60% 112% 98% 78% 171% 59% 74% 60% 87% 85% 73% 83% 57% 117%
114% 104% 97% 103% 72% 95% 100% 98% 58% 89% 103% 90% 126% 84% 102% 98% NG 83% 121% 87% 85% 115% 106% 93% 139% 45% 153% b0.1% 65% b0.1% 49% 61% 65% 74% 132% 83% 137% 168% 88% 100% 78% 70% 105% 106% 111% 92% 52% 94% 130% 78% 71% 54% NG 83% 90% 100% 45% 81% 129% 62% 80% 93% 79% 96% 86% 120% 87% 125% 113% 88% 87% 82% 86% 92%
120% 121% 150% 188% 108% 81% 100% 102% 105% 92% 132% 71% 84% 108% 63% 127% 94% 117% 145% 122% 81% 103% 115% 93% 105% 92% 137% 89% 76% 33% 36% 88% 68% 113% 156% 73% 133% 145% 88% 85% 91% 74% 20% 81% 102% 105% 106% 106% 121% 93% 81% 7% 38% 94% 88% 75% 115% 107% 86% 112% 90% 105% 71% 122% 57% 107% 98% 114% 143% 106% 108% 110% 71% 80%
(continued on next page)
8
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
Table 1 (continued) Serotypea
Strain ID
Isolation
stx1
stx2
eae
hlyA
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O91:H21 O98:H29 O100:NM O104:H4 O104:H21 O108:H11 O110:H28 O113:H21 O115:H18 O117:H7 O123:H2 O128:H2 O128:H10 O130:H11 O146:H21 O153:H25 O156:NM O165:H25 O177:NM O179:H8 O181:H49 Orough:H21
85-489 09-5073 96-0429 11-3088 3024-94 11-3580 09-409 04-1450 03-3645 02-0035 11-4968 1759 2611-99 09-1765 02-1628 09-1775 92-0389 00-4540 03-3974 09-415 09-412 11-6008
Human Human Unknown Human Unknown Human Unknown Human Human Human Human Human Human Unknown Human Unknown Unknown Human Human Unknown Unknown Human
− + − − − − − − + + + + + − + + − − − − − +
+ − + + + + + + + − − − − + − + + + + + + +
− + − − − + − − − − + − − − − − − + + − − −
+ + − − + + + + + − + + − + + + − + + + + +
113% 107% 124% 92% 110% 110% 75% 137% 93% 111% 68% 86% 115% 125% 133% 100% 113% 118% 78% 108% 119% 103%
94% 97% 119% 127% 89% 78% 100% 130% 100% 115% 97% 100% 110% 99% 110% 133% 115% 136% 93% 138% 138% 81%
b0.1% 79% 105% 42% NG 24% NG NG NG NG 40% NG NG NG NG NG NG NG 24% NG NG NG
NG 81% 117% 50% NG 24% b0.1% NG 66% NG 85% NG NG b0.1% NG b0.1% NG NG 35% b0.1% NG NG
NG 90% 80% 35% NG 29% NG NG NG NG 51% NG NG NG NG NG NG NG 34% NG NG NG
42% 134% 104% 115% 55% 60% 65% 33% 69% 63% 62% 57% b0.1% 87% 116% 67% NG 91% 68% 70% 50% 69%
83% 72% 109% 108% 93% 63% 80% 67% 66% 105% 78% 95% 101% 86% 96% 142% 75% 118% 80% 104% 117% 66%
110% 100% 114% 121% 85% 84% 55% 133% 66% 109% 81% 93% 118% 111% 133% 87% 112% 123% 93% 114% 152% 147%
MAC: MacConkey Agar. RBA: Rainbow® Agar O157. RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite. RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite. CH STEC: CHROMagar STEC™. TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite. TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose. VTEC: VTEC agar (Gill et al., 2012). a +: positive, −: negative, NG: no growth on streaked or enumeration plates, b0.1% growth on streaked plate but recovery on enumerated plates below 0.1% of BHI.
be common to all STEC and absent from other E. coli is verotoxin production. Few outbreaks of illness due to non-O157 STEC contaminated beef have been reported, compared to E. coli O157, but the development of methods for non-O157 STEC in beef is a priority due to the high rates of carriage of non-O157 STEC by cattle and their presence in raw beef (Gill and Gill, 2010). In an attempt to mitigate public health impacts of non-O157 STEC in beef, the United States Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) has implemented testing of manufacturing trim and raw ground beef for the presence of the so-called priority STEC serogroups O26, O45, O103, O111, O121, and O145, in addition to E. coli O157:H7 (USDA, 2012). While several agar media have been developed or proposed for the isolation of STEC from foods, few studies have been published on the comparative performance of these media and those that have tended to focus on a relatively small number of STEC strains of a limited range of serotypes. For this reason we have conducted a study to evaluate the ability of eight different agar media to support the growth of 96 STEC strains, representing 36 different O-types. The media were also evaluated to assess their ability to suppress the growth of background microflora from ground beef and beef trim. 2. Material and methods 2.1. Bacterial cultures The 96 strains of STEC used in this study and their virulence characteristics are listed in Table 1. Unless otherwise noted the strains used were provided from the collection of the Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba. Strains 85-X-40c R3, 3267–95, 3285–96, 89–39, 2454–01, 2769, 2454–01, EDL933, ER63-94, E32511, Sakai, 1759, 2611–99 were kindly supplied by Dr. Roger Johnson of the Public Health Agency of Canada, Laboratory of Food-Borne Zoonoses, Guelph, Ontario. Strains CFS3, 3331–00, 3413–07 were kindly supplied by Dr. P. Micheal Doyle of the University of Georgia, Griffin, Georgia. Strain 11–3088 was kindly supplied by Dr. Vanessa Gray Allen of the Public Health Ontario Laboratories,
Toronto, Ontario. The remaining strains were from the culture collection of the Health Canada, Bureau of Microbial Hazards, Ottawa, Ontario. The presence of STEC virulence genes stx1, stx2, eae, and EHEC hlyA was determined by the PCR protocol of Paton and Paton (2003). The cultures were stored as frozen glycerol stocks. For experimental use, the cultures were streaked onto brain heart infusion (BHI) agar (Difco, BD, Sparks, MD) and incubated at 37 °C for 24 h. 2.2. Recovery of STEC strains on selective agar Single colonies of the strains were individually inoculated into 10 ml of modified tryptic soy broth (mTSB: tryptic soy broth [30 g/l; Difco], bile salts no. 3 [1.5 g/l; Difco, BD], K2HPO4 [1.5 g/l; Sigma, St. Louis, MO], pH 7.4), supplemented with 10 mg/l vancomycin (Sigma) and 3 mg/l cefsulodin (Sigma) after 4 h (mTSB-VC broth) as described in Gill et al. (2012), and incubated for 18–24 h. at 42 °C. The following selective agar media were evaluated for their ability to support the growth of STEC strains. MacConkey Agar (MAC; Difco); Rainbow® Agar O157 (RBA; Biolog Inc., Hayward, California); Rainbow® Agar O157 supplemented, with 10 mg/l novobiocin (Sigma) and 0.8 mg/l potassium tellurite (Sigma) (RBA-NT) as recommended by the manufacturer for samples with high background flora; Rainbow® Agar O157 supplemented with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate (Oxoid) and 0.15 mg/l potassium tellurite (RBA-USDA) as recommended for the USDA STEC method MLG 5B.03 (USDA, 2012); CHROMagar STEC™ (CHROMagar Microbiology, Paris, France) (CH STEC); Tryptone Bile agar (Oxoid, Basingstoke, Hampshire, UK) containing 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite (TBA-CT); Tryptone Bile agar containing 0.05 mg/l cefixime, 0.5 mg/l of potassium tellurite, 0.4 g/l eosin Y (Sigma), 0.06 g/l methylene blue (Fisher Scientific, Ottawa, Ontario) and 1 g/l lactose (TBA-EM); and VTEC agar (Gill et al., 2012), 17.5 g/l vegetable special infusion powder (Sigma), 2 g/l meat extract (Sigma), 10 g/l proteose peptone (Oxoid), 10 g/l D-sorbitol (Sigma), 5 g/l sodium chloride (Sigma), 2.5 g/l sodium phosphate dibasic (Sigma), 30 mg/l neutral red (Sigma), 1 mg/l crystal violet (Sigma), 1 g/l bile salts #3 and 15 g/l agar, supplemented with 10 mg/l vancomycin and 3 mg/l cefsulodin.
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
9
Table 2 Proportion of STEC strains whose growth is inhibited on selective agar media tested.a O-type
No. strains
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26 O45 O103 O111 O121 O145 O157 Others Total
10 6 10 10 10 9 10 31 96
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
1 0 2 3 1 1 0 20 28 (29.2%)
1 0 2 1 0 1 0 15 20 (20.8%)
1 0 2 1 1 2 0 20 27 (28.1%)
0 0 1 0 0 1 0 1 3 (3.1%)
0 0 1 0 0 1 0 0 2 (2.1%)
0 0 0 0 0 0 0 0 0
a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012).
Cultures grown in mTSB-VC were streaked onto the eight selective agar media, which were then incubated for 24 h at 37 °C. The plates were examined for growth and the colony morphology recorded. To determine the relative enumerative recovery on the selective agars, the cultures were serially diluted in phosphate buffered saline (PBS) and the dilutions manually spread plated onto the eight selective agar media and BHI agar. The plates were incubated for 24 h at 37 °C and the colonies counted. The relative efficiency of recovery was calculated as the percentage of colonies on the selective agar compared to BHI. Replicate experiments were not performed systematically as little variation is expected with a descriptive study of this type. The primary potential sources of error were identified as a failure to inoculate media or pipetting errors leading to inaccurate reporting of growth inhibition. These errors could be detected by observation of turbidity in the culture test tubes and comparison of the culture dilutions plated for enumerative recovery. A small number of errors were detected and the experiments with those strains repeated and reported. 2.3. Growth of beef microflora on selective agar Experiments were conducted to evaluate the ability of the selective agar to inhibit the growth of background microflora present in enrichment cultures from ground beef and beef trim that were not inoculated with STEC. The ground beef had a 15% fat content and was purchased from an Ottawa area supermarket, Trim samples were sourced from an Ontario beef processing plant. Samples of meat 25 g were suspended in 225 ml of mTSB-VC and incubated for 18–24 h at 42 °C. The postincubation enrichment broth was then serially diluted at a 1:10 ratio in PBS (1 ml into 9 ml) from 10−1 to 10−7 and all dilutions manually spread plated on the eight selective agar and BHI agar. The agar media were incubated as described above and the total colonies on each plate enumerated. This experiment was performed with meat from three separate lots of ground beef and beef trim. 3. Results The percentage recovery of each STEC strain on the eight selective agar media is presented in Table 1. The ability of the agar media to support the growth of strains of the 7 STEC O-types designated a priority by the USDA (O26, O45, O103, O111, O121, O145, O157) is summarized in Table 2. Three of the media tested MAC, RBA and VTEC agar were able to support the growth of all 96 STEC strains tested. TBA-EM did not support the growth of two strains (O103:H2 99–2076 and O145:H2 A9619.C2). TBA-CT did not support the growth of the same O103 and O145 strains as TBA-EM, and in addition no growth was observed for O156:NM 92–0389. RBA-USDA did not support the growth of 20 (20.8%) strains tested, including strains of the serogroups O26 (1/10), O103 (2/10), O111 (1/10) and O145 (1/9). RBA-NT and CH STEC supported the growth of the least number of STEC strains, with 28 (29.2%) strains on RBA-NT and 27 (28.1%) strains on CH STEC failing to develop visible colonies after 24 h incubation. Strains inhibited on
CH STEC included O26 (1/10), O103 (2/10), O111 (1/10), O121 (1/10) and O145 (2/9). Strains inhibited on RBA-NT included O26 (1/10), O103 (2/10), O111 (3/10), O121 (1/10) and O145 (1/9). For those STEC strains which were able to grow on the selective media, the average number of colonies counted compared to BHI agar was 100% for MAC, RBA and VTEC agar (Table 3). The average number of colonies counted on TBA-EM was 91% and for TBA-CT 78% compared to BHI agar. The average number of STEC colonies counted on RBA-NT was 58% and RBA-USDA 60% compared to BHI agar, down from 100% for RBA without additional antimicrobials. CH STEC was the medium which was most inhibitory to STEC growth, with an average of 40% of colonies compared to BHI agar and O26 was the only serogroup for which the colony count was greater than 50% of the BHI agar count. The appearance of STEC colonies on the different selective media is described in Table 4. The colour of the colonies of all strains was highly uniform on MAC, TBA-CT and TBA-EM. Colonies of all strains on TBA-CT were white, and almost all were pink on MAC and TBA-EM. Colonies of strains of the same serogroup were not a consistent colour on RBA, RBANT and RBA-USDA, With the exception of the O157 serogroup, which were blue-grey. Two or more colours of colonies were observed for strains all serogroups on CH STEC and for all serogroups on STEC agar except O26, O103 and O145, which were uniformly pink. The number of colonies recovered from enrichment broths of ground beef and beef trim in mTSB-VC on the selective agar media was variable among the three samples (Table 5). This probably reflects variation in the composition of the initial flora of the meat samples prior to enrichment. For ground beef enrichment the number of colonies recovered was only significantly less (Student's t-test p b 0.05) than BHI agar on RBA-NT and CH STEC. Significantly lower numbers of colonies (p b 0.05) were recovered from beef trim enrichment compared to BHI agar on RBA-NT, RBA-USDA and CH STEC.
Table 3 Percentage recovery of STEC strains on selective agar media compared to Brain Heart Infusion agar, excluding inhibited strains.a O-type
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
O26 O45 O103 O111 O121 O145 O157 Others Total
106% 93% 94% 84% 122% 95% 104% 110% 104%
109% 108% 97% 98% 100% 97% 96% 110% 103%
93% 70% 58% 41% 51% 58% 49% 55% 58%
100% 35% 82% 57% 64% 51% 54% 44% 60%
75% 33% 39% 24% 43% 34% 16% 47% 40%
85% 79% 103% 50% 81% 81% 90% 72% 78%
97% 101% 97% 68% 106% 82% 85% 93% 91%
118% 98% 107% 87% 92% 82% 94% 106% 100%
a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012).
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A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
Table 4 Appearance of STEC colonies on selective agar.a Serogroup
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC agar
O26
Pink (10)
Mauve (7) Pink (2) Pink (4) Mauve White
White (9) Pin point White (6)
Pink (6)
Pink (5) White
O103
Pink (10)
Pink (4) Blue-grey (3) White Mauve
Pink (2) Blue-grey (3) White (2) Mauve
White (9)
Pink (9)
Pink (10)
O111
Pink (10)
Blue-grey (8) White Mauve
Blue-grey (6) Pin Point
Pink (3) Mauve (4)
White (7) Pin point (3)
Pink (7) Mauve Pin point (2)
Pink (9) White
O121
Pink (10)
White (9) Pin point
Pink (10)
Pink (9) Mauve
Pink (9)
Mauve (4) Blue-grey (2) Pink (2) Pin point Blue-grey (2) Mauve (6)
Pink (6) Mauve (3)
O145
Mauve (4) Blue-grey (2) Pink (3) White Blue-grey (3) Mauve (6)
Pink (3) Blue-grey (2) White Mauve Light pink Blue-grey (6) White Mauve Pin Point Mauve (2) Pink (7) Pin point
Mauve (8) Pink Mauve (3) Pink White Blue-grey Mauve Pink (7)
Pink (10)
Pink (6)
Mauve (5) Blue-grey (4) Pink (3) Mauve Blue-grey (2)
Pink (10)
O45
Mauve (5) Blue-grey (5) Pink (3) Mauve (1) Blue-grey (2)
Pink (6) Mauve (2)
White (8)
Pink (8)
Pink (9)
O157
Pink (10)
Blue-grey (10)
Blue-grey (10)
White (10)
Pink (10)
Others
Pink (30) Light pink (1)
Pink (9) Mauve (15) Blue-grey (5)
Pink (3) Mauve (3) Blue-grey (5)
Pink (6) Mauve (4) Pink (8) Mauve (3)
White (22) Pin point (8)
Pink (31)
Light pink (9) Pink Pink (29) Light pink (2)
Blue-grey (3) Mauve (4) Pink Blue-grey (10) Pink (8) Mauve Blue-grey (3) Light pink Pin point (3)
CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite. TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012). a MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite.
4. Discussion The isolation of STEC from foods remains a challenge because STEC are very often present with other non-pathogenic E. coli or Enterobacteriaceae and no selective condition has been identified which is unique to the STEC pathotype of E. coli. Since no unique differential characteristic has been identified, other than verotoxin expression, this means that isolation requires that potentially large numbers of colonies must be screened individually for VT genes or expression. Diverse STEC have been successfully isolated from foods, by identifying VT positive colonies grown on a relatively permissive agar medium by colony immunoblot (Atalla et al., 2000) or colony hybridization (Barlow et al., 2006). But the lack of uptake of these methods reflects the technical challenges in adopting these methods outside of a research laboratory. Alternatively, isolation media for specific subgroups of STEC have been developed which are based on selective or differential
characteristics common in the subgroup. These include hemolysin activity (Beutin et al., 1989), carbohydrate fermentation patterns (Possé et al., 2008) or antimicrobials (Hiramatsu et al., 2002; Tzschoppe et al., 2012). This approach may be of utility when seeking the isolation of a STEC strain of known characteristics, but given the diversity of STEC strains there is serious concern that STEC pathogens may not be recovered on agar media containing inhibitory levels of antimicrobial agents or which use differential characteristics that may screen out the pathogen. It should be noted that the diversity of colony morphologies observed on the agar media in this study, even among strains of a given O type, indicates that colony appearance will not provide a reliable means of identifying target STEC in the presence of other E. coli. The problem of media being too selective was demonstrated in this study by the results obtained with CH STEC, RBA-NT and RBA-USDA. These media were unable to support the growth of a significant proportion of STEC strains (CH STEC 28.1%, RBA-NT 29.2%, RBA-USDA 20.8%),
Table 5 Recovery of flora from beef enriched in modified tryptic soy broth with 10 mg/l vancomycin and 3 mg/l cefsulodin for 24 h at 42 °C.a Log CFU/ml O-type
BHI
MAC
RBA
RBA-NT
RBA-USDA
CH STEC
TBA-CT
TBA-EM
VTEC
Ground beef Ground beef Ground beef Mean Beef trim Beef trim Beef trim Mean
8.5 8.9 8.8 8.8a 8.4 8.8 8.6 8.6a
8.6 8.8 8.7 8.7a 8.5 8.6 8.5 8.5ac
8.7 8.8 8.8 8.8a 8.5 8.6 8.6 8.5a
b2.0 2.3 4.7 3.0b 3.3 7.5 4.5 5.1bc
7.4 8.0 8.6 8.0a 5.6 7.6 4.8 6.0b
b2.0 3.5 4.3 3.3b 5.0 6.9 5.0 5.6b
8.5 8.9 8.7 8.7a 8.1 8.5 8.3 8.3a
8.1 8.9 8.8 8.6a 7.3 7.4 4.3 6.3b
8.1 8.9 8.8 8.6a 7.3 7.4 4.3 6.3b
BHI: Brain Heart Infusion Agar; MAC: MacConkey Agar; RBA: Rainbow® Agar O157; RBA-NT: Rainbow® Agar O157 with 10 mg/l novobiocin and 0.8 mg/l potassium tellurite; RBA-USDA: Rainbow® Agar O157 with 5 mg/l novobiocin, 0.05 mg/l cefixime trihydrate and 0.15 mg/l potassium tellurite; CH STEC: CHROMagar STEC™; TBA-CT: Tryptone Bile agar with 0.05 mg/l cefixime and 0.5 mg/l of potassium tellurite; TBA-EM: TBA-CT with 0.4 g/l eosin Y, 0.06 g/l methylene blue and 1 g/l lactose; VTEC: VTEC agar (Gill et al., 2012). a Averages with different superscripts are significantly different by Student's t-test (p b 0.05).
A. Gill et al. / Journal of Microbiological Methods 96 (2014) 6–11
including strains belonging to 5 of the 6 priority serogroups (O26, O45, O103, O111, O121, O145) designated for beef testing in the US (USDA, 2012). Inhibition of a significant proportion of STEC strains was also reported in two previous publications which evaluated the performance of CH STEC and identified resistance to tellurite as the primary selective principle of the medium (Hirvonen et al., 2012; Tzschoppe et al., 2012). Hirvonen et al. (2012) reported that CH STEC inhibited the growth of 93 of 362 (25.7%) STEC strains tested and the media was not inhibitory of the single strains tested of enteropathogenic E. coli, enterotoxigenic E. coli and enteroaggregative E. coli. The inhibited STEC included O26 (2/20), O103 (19/30) and non-sorbitol fermenting O157:H7 and NM (4/183) (Hirvonen et al., 2012). Tzschoppe et al. (2012) reported that CH STEC inhibited the growth of 34 of 133 (25.5%) STEC strains, including O103 (9/18), O121 (2/4), O145 (1/10), sorbitol fermenting O157 (5/5) and diverse serotypes (17/17). When used for the purpose of isolation of target bacteria from enrichment broth, selective agents added to agar media improve the probability of recovering the target bacteria by reducing the numbers of non-target flora which can grow on the media. In this study RBANT and CH STEC were the only agar media which had significantly lower numbers of colonies from ground beef enrichment (mTSB-VC at 42 °C) compared to non-selective BHI agar. However, RBA-NT, RBAUSDA, CH-STEC, TBA-EM and VTEC agar were equally inhibitory to the growth of background microflora from beef trim enrichment broth. These results suggest that depending on the composition of the initial microflora of the sample and the enrichment conditions, selective agar may not always improve the probability of target recovery.
5. Conclusions The essential requirement of any agar media for the isolation of bacteria is the ability to support the growth of the target organism, and secondary to that is the ability to suppress the growth of non-target organisms. Consequently, we conclude that due to the significant proportion of STEC strains whose growth is inhibited on these media, including strains of priority serogroups, that the agar media RBA-USDA, RBA-NT, and CH STEC are unsuitable for application to the isolation of STEC from beef as the sole isolation media. Nonetheless, RBA-USDA, RBA-NT, and CH STEC might be applied to beef samples containing high levels of background microflora, if paired with an agar media capable of supporting the growth of a wide range of STEC, such as MAC, RBA or VTEC agar.
Acknowledgements The authors would like to thank Mahdid Meymandy of Health Canada and Mylène Deschênes of the Canadian Food Inspection Agency for their technical assistance.
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