Abstract
GMAC, a modified version of Sorbitol MacConkey medium (SMAC), was produced with a reduced quantity of selective agents and incorporated gentiobiose. GMAC supported a higher recovery rate of heat- or acid-injured Escherichia coli O157:H7 cells than SMAC with cefixime and tellurite (CT-SMAC), while differentiating E. coli O157:H7 from sorbitol-nonfermenting Hafnia alvei.
Escherichia coli O157:H7 has emerged as one of the most significant human pathogens (9). The most common transmission route for E. coli O157:H7 is various foods, such as beef, produce, and dairy products (10). Thus, effective methods for the isolation and identification of E. coli O157:H7 in foods are important to ensure food quality and safety. Sorbitol MacConkey medium (SMAC) and Sorbitol MacConkey medium with cefixime and tellurite (CT-SMAC) have been widely used for the isolation of this pathogen. However, numerous false-positive results from sorbitol-nonfermenting bacteria, such as Hafnia alvei, Proteus spp., Providencia spp., Aeromonas spp., and Morganella morganii, have been reported (3, 4). In addition, these media perform poorly in supporting the recovery of injured cells of E. coli O157:H7 (1, 8, 11, 12).
Recognizing the limits of currently used selective and differential media, it is desirable to develop a medium that can perform well in supporting the recovery of injured cells of E. coli O157:H7. It is also desirable to improve specificity of the medium. This study describes an improved medium, GMAC, for isolation of E. coli O157:H7.
Twenty isolates of E. coli O157:H7 and two isolates of E. coli O157:NM were obtained from the School of Food Science Bacterial Culture Collection, Washington State University (Pullman, WA).
Selective agents, such as bile salts and crystal violet, may inhibit the resuscitation of injured cells of E. coli O157:H7 (11). Modified SMAC with crystal violet removed and reduced levels of bile salts from SMAC (Difco, Sparks, MD) were prepared to determine the lowest concentration of selective agent that could completely inhibit the growth of Gram-positive bacteria. Eighteen strains of Gram-positive bacteria (Table 1) were incubated in 10 ml of tryptic soy broth (TSB; Difco) at 37°C for 18 h. After incubation, one loopful of each strain was streaked onto each prepared medium and incubated at 37°C for 24 h. As shown in Table 1, bile salt concentrations from 0.15% (normal amount of bile salts in SMAC) to 0.05% completely inhibited growth of all tested Gram-positive bacteria. Below bile salt levels of 0.05%, growth of some microorganisms was observed.
TABLE 1.
Growth of Gram-positive bacteria on medium with reduced bile salt concentration
| Tested bacteria | Sourcea | Growthb on medium with a bile salt concentration (%) of: |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| 0.15 | 0.13 | 0.11 | 0.09 | 0.07 | 0.05 | 0.03 | 0.01 | ||
| Listeria monocytogenes ATCC 19115 | ATCC | − | − | − | − | − | − | − | − |
| Listeria monocytogenes ATCC 19114 | ATCC | − | − | − | − | − | − | − | − |
| Listeria monocytogenes ATCC 15313 | ATCC | − | − | − | − | − | − | − | − |
| Listeria innocua | WUCC | − | − | − | − | − | − | − | + |
| Listeria innocua | WUCC | − | − | − | − | − | − | − | + |
| Enterococcus faecalis ATCC 19433 | ATCC | − | − | − | − | − | − | + | ++ |
| Enterococcus faecalis ATCC 29212 | ATCC | − | − | − | − | − | − | + | ++ |
| Bifidobacterium longum ATCC 15707 | ATCC | − | − | − | − | − | − | + | ++ |
| Bifidobacterium bifidum ATCC 15521 | ATCC | − | − | − | − | − | − | + | + |
| Lactobacillus fermentum ATCC 9338 | ATCC | − | − | − | − | − | − | − | − |
| Staphylococcus epidermidis ATCC 12228 | ATCC | − | − | − | − | − | − | + | ++ |
| Staphylococcus epidermidis ATCC 14990 | ATCC | − | − | − | − | − | − | − | + |
| Staphylococcus aureus ATCC 12600 | ATCC | − | − | − | − | − | − | − | + |
| Staphylococcus aureus ATCC 12692 | ATCC | − | − | − | − | − | − | − | + |
| Staphylococcus aureus ATCC 49444 | ATCC | − | − | − | − | − | − | − | + |
| Bacillus cereus ATCC 10876 | ATCC | − | − | − | − | − | − | − | +++ |
| Bacillus cereus ATCC 13061 | ATCC | − | − | − | − | − | − | − | +++ |
| Bacillus cereus | WUCC | − | − | − | − | − | − | − | +++ |
ATCC, American Type Culture Collection; WUCC, Washington State University Culture Collection.
+++, excellent growth; ++, good growth; +, growth; −, no growth.
Colorless colonies of bacteria other than E. coli O157:H7 observed on SMAC were isolated from food samples (ground beef, pork, and chicken) purchased from a local market (Seoul, South Korea). Isolated bacteria were identified using the API 20E system (bioMérieux SA, Marcy l'Etoile, France). Among isolated background bacteria, H. alvei bacteria were the most numerous. Characteristics of carbohydrate fermenting of E. coli O157:H7, O157:NM, and H. alvei were compared using the API CH50 system (bioMérieux SA). All 22 strains of E. coli O157:H7 and O157:NM do not ferment gentiobiose, whereas 50 strains of 54 H. alvei strains fermented gentiobiose (Tables 2 and 3).
TABLE 2.
Characteristics and growth of E. coli O157:H7
| Bacterium | No. of strains tested | No. of strains that fermenta: |
No. of strains on SMAC that showed colorless colonies | No. of strains on GMAC that showed: |
||
|---|---|---|---|---|---|---|
| Sorbitol | Gentiobiose | Pink colonies | Colorless colonies | |||
| E. coli O157:H7 | 20 | 0 | 0 | 20 | 0 | 20 |
| E. coli O157:NM | 2 | 0 | 0 | 2 | 0 | 2 |
Tested by API 50CH.
TABLE 3.
Characteristics and growth of H. alvei
| Source of H. alvei bacteria | No. of strains tested | No. of strains that fermenta: |
No. of strains on SMAC that showed colorless colonies | No. of strains on GMAC that showed: |
||
|---|---|---|---|---|---|---|
| Sorbitol | Gentiobiose | Pink colonies | Colorless colonies | |||
| Ground beef | 20 | 0 | 20 | 20 | 20 | 0 |
| Ground pork | 29 | 0 | 26 | 29 | 26 | 3 |
| Chicken | 5 | 0 | 4 | 5 | 4 | 1 |
Tested by API 50CH.
The ingredients of GMAC are as follows: 15.5 g peptone (Difco), 10 g d-sorbitol (Difco), 0.5 g bile salts (Fluka, Paris, France), 5 g sodium chloride (Samchun Chemical Co. Ltd., Pyeongtaek-si, South Korea), 15 g agar (Difco), 0.03 g neutral red (Samchun Chemical), and 5 g β-d-gentiobiose (Carbosynth, Berkshire, United Kingdom) per liter.
Twenty-two strains of E. coli O157:H7 and O157:NM and 54 strains of H. alvei were incubated in 10 ml of TSB at 37°C for 18 h. After incubation, one loopful of each strain was streaked onto SMAC and GMAC and incubated at 37°C for 24 h. All 22 E. coli O157:H7 and O157:NM strains streaked onto GMAC were colorless (Table 2). Of 54 H. alvei strains, 50 strains produced pink colonies on GMAC (Table 3). Figures 1 and 2 show colonies of E. coli O157:H7 and H. alvei on SMAC and GMAC.
FIG. 1.
Both E. coli O157:H7 (A) and H. alvei (B) produced colorless colonies on SMAC.
FIG. 2.
Colonies of E. coli O157:H7 and H. alvei on GMAC. Colonies of E. coli O157:H7 are colorless (A) compared to colonies of H. alvei, which are pink (B).
To compare the numbers of background bacteria, food samples (25 g), including ground beef, pork, and chicken, were incubated in stomacher bags containing 225 ml of buffered peptone water (BPW; Difco) at 37°C for 18 h. After incubation, samples were homogenized with a stomacher for 2 min. Samples were serially 10-fold diluted in BPW, and 0.1 ml of diluents was spread plated onto SMAC and GMAC. The plates were incubated at 37°C for 24 h. After incubation, the numbers of colorless and pink colonies on SMAC and GMAC were estimated. The number of colorless colonies on GMAC was lower than that on SMAC in all samples, and overall, there were no significant differences in bacterial counts between SMAC and GMAC (Table 4).
TABLE 4.
Enumeration of background bacteria on SMAC and GMAC
| Food samples | No. of microorganisms (CFU/ml) on: |
|||||
|---|---|---|---|---|---|---|
| SMAC |
GMAC |
|||||
| Total colonies | Pink colonies | Colorless colonies | Total colonies | Pink colonies | Colorless colonies | |
| Ground beef | 1.3 × 105 | 8.1 × 104 | 5.1 × 104 | 1.3 × 105 | 1.3 × 105 | 4.0 × 103 |
| Ground beef | 2.1 × 105 | 4.5 × 104 | 1.7 × 105 | 1.9 × 105 | 1.9 × 105 | 4.0 × 103 |
| Ground beef | 1.8 × 105 | 5.4 × 104 | 1.3 × 105 | 1.2 × 105 | 1.2 × 105 | 1.0 × 103 |
| Ground beef | 3.4 × 105 | 2.3 × 105 | 1.1 × 105 | 3.7 × 105 | 2.1 × 105 | 1.6 × 104 |
| Ground beef | 2.8 × 105 | 2.7 × 105 | 9.0 × 103 | 4.3 × 105 | 4.3 × 105 | 2.0 × 103 |
| Ground beef | 2.4 × 105 | 7.8 × 104 | 1.6 × 105 | 5.8 × 105 | 5.8 × 105 | 3.0 × 103 |
| Ground beef | 5.6 × 105 | 1.3 × 105 | 4.3 × 105 | 7.5 × 105 | 7.4 × 105 | 1.2 × 104 |
| Ground pork | 1.3 × 106 | 1.2 × 106 | 1.3 × 105 | 1.0 × 106 | 1.0 × 106 | 3.5 × 104 |
| Ground pork | 1.5 × 106 | 1.2 × 105 | 1.4 × 106 | 2.8 × 106 | 2.8 × 106 | 8.0 × 104 |
| Ground pork | 2.1 × 105 | 5.2 × 104 | 1.6 × 105 | 2.3 × 105 | 2.1 × 105 | 2.1 × 104 |
| Chicken | 1.7 × 105 | 6.7 × 104 | 1.0 × 105 | 3.7 × 105 | 3.7 × 105 | 8.0 × 103 |
| Chicken | 1.9 × 106 | 1.3 × 105 | 1.8 × 105 | 6.7 × 105 | 6.5 × 105 | 1.7 × 104 |
In the recovery test of heat- or acid-injured E. coli O157:H7 cells, three strains of E. coli O157:H7 (ATCC 35150, ATCC 43889, ATCC 43890) were transferred to 5 ml of TSB and incubated at 37°C for 24 h. Cells were harvested by centrifugation at 4,000 × g for 20 min at 4°C. To investigate the recovery of heat-injured E. coli O157:H7 cells, the pellets were resuspended in 5 ml of BPW. One milliliter of suspension (diluted to ca. 108 to 109 CFU/ml) was added to test tubes containing 5 ml of BPW that had been preheated at 60°C and held for 1.5 min. To estimate the recovery of acid-injured E. coli O157:H7 cells, the pellets were resuspended in 5 ml of BPW adjusted to a pH of 2.26 with 1% lactic acid and held for 10 min. After each treatment, the broth was serially 10-fold diluted in BPW, and 0.1 ml of diluents was spread plated onto tryptic soy agar (TSA; Difco), CT-SMAC, and GMAC. Plates were incubated at 37°C for 24 h, and colonies were counted. This experiment was repeated three times. Data were analyzed by analysis of variance (ANOVA) using the Statistical Analysis System software (SAS Institute, Cary, NC) and separation of means by Duncan's multiple-range test at P values of <0.05. As shown in Table 5, there were no significant (P > 0.05) differences in the recovery of injured E. coli O157:H7 cells between TSA (7.41 log10 CFU/ml) and GMAC (7.08 log10 CFU/ml) when heat treated at 60°C for 1.5 min. Significantly (P < 0.05) fewer numbers of heat-injured E. coli O157:H7 CFU (5.13 log10 CFU/ml) were recovered on CT-SMAC. There was a significant (P < 0.05) difference in the numbers of acid-injured E. coli O157:H7 CFU for GMAC (5.82 log10 CFU/ml) and TSA (7.80 log CFU/ml). However, GMAC supported a significantly (P < 0.05) greater recovery of acid-injured E. coli O157:H7 CFU than did CT-SMAC (more than 2.40 log10 CFU/ml).
TABLE 5.
The recovery of heat- and acid-injured E. coli O157:H7 cells on TSA, CT-SMAC, and GMAC
| Medium | No. of CFU/ml recovereda |
|
|---|---|---|
| Heat treatment (60°C for 1.5 min) | Acid treatment (1% lactic acid for 10 min) | |
| TSA | 7.41 ± 0.07a | 7.80 ± 0.04a |
| GMAC | 7.08 ± 0.03a | 5.82 ± 0.30b |
| CT-SMAC | 5.13 ± 0.30b | 3.40 ± 0.41c |
Means ± standard deviations with different letters within a column are significantly different (P < 0.05).
Methods to detect injured E. coli O157:H7 cells are important due to the low infectious dose of the organism (8). GMAC does not contain antibiotics, such as cefixime and tellurite, and the concentration of bile salts was reduced from 0.15% to 0.05%. Also, GMAC contains gentiobiose as a differentiation marker. As a result, this GMAC better supported the recovery of heat- or acid-injured E. coli O157:H7 cells than did CT-SMAC and increased specificity.
Recently, numerous rapid methods have been developed to detect and identify specific pathogens present in foods (7). These methods include convenience-based, antibody-based, and nucleic acid-based assays (5). However, the rapid methods described above are used primarily as a screening tool; thus, further confirmation by traditional culture methods is required for presumptive positive microorganisms (2). Also, an enrichment step is needed for the antibody-based and nucleic acid-based assays to increase sensitivity. Therefore, conventional plating methods that offer advantages, including cost-effectiveness, ease of use, and familiarity, among users, are important (6). GMAC can supplement existing selective and differential media for isolation of E. coli O157:H7.
Acknowledgments
This work was supported by grant no. R32-2008-000-10183-0 from the World Class University (WCU) project of the Ministry of Education, Science & Technology (MEST) and the KOSEF through Seoul National University.
Footnotes
Published ahead of print on 27 October 2010.
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