1. Evaluation of Culture Conditions and Enumeration Methods of
Enteric Bacteria Isolated from Ground Beef
Taylor A. Winslow, Paige L. Stanley, Indiren Pillay
Department of Biological & Environmental Sciences, Georgia College & State University, Milledgeville, GA
Abstract
Materials and Methods
Results & Discussion
An established protocol for the enrichment of Escherichia coli using E.coli Media with Novobiocin (ECM) was compared to a more convenient universal enrichment media, Universal Pre-enrichment Broth (UPB). Additionally, microbial growth after various incubation times of both media at 24, 36, and 48 hours was compared. There is no significant difference between ECM and UPB for the enrichment of E. coli isolated from raw meat sources. However, ECM appears to be better at enriching and isolating presumptive pathogens, such as E.coli O157:H7. Furthermore, we evaluated a commercially available Colilert-18 enumeration system and a novel, ready to use, Compact Dry™ system. Because Colilert-18 is routinely used for water analysis and Compact Dry™ is designed for the enumeration of coliforms from raw materials and environmental surfaces, we were therefore interested to see which of these systems would be most effective using samples collected from raw meat sources. Preliminary results indicate that for highly concentrated meat samples, Compact Dryª media may be more efficient at obtaining coliform and E.coli counts.
ECM-enriched samples yielded more presumptive O157:H7 colonies than UPB. Additionally, no significant difference in growth of E.coli O157:H7 among the 24, 36 and 48 hour intervals was observed.
Compact Dry™ plates enabled enumeration of O157:H7 colonies.
The Colilert-18 assay of samples spiked with E. coli O157:H7, rendered only total coliform counts, and was unable to distinguish O157:H7 colonies.
From the enriched meat samples, no accurate counts could be obtained from the Colilert-18 trays because of excessive cells despite dilutions.
However, accurate counts could be obtained from the Compact Dry™ at similar dilutions.
Evaluation of Culture Conditions
Ground beef samples were homogenized in a stomacher with modified ECM or UPB.
Homogenates were incubated at intervals of 24, 36, and 48 hours at 35°C.
At each time interval, samples from the enriched homogenate were inoculated onto Sorbitol MacConkey agar with Cefeximine and Tellurite (CT-SMAC), and incubated for 24 hours at 35°C.
CT-SMAC is routinely used for the selective and differential isolation of enterohemorrhagic Escherichia coli O157.
Each of the enrichment methods were analyzed temporally for relative amounts of colorless, presumptive E.coli O157:H7 colonies.
*No growth of non-O157:H7 E.coli was expected because the samples were specifically spiked with E.coli O157:H7
**The basis of the colorimetric indicator that Colilert-18 uses for coliform bacteria is the metabolism of ONPG (ortho-Nitrophenyl-beta-galactoside). However, the identification and enumeration of E.coli is based on the metabolism of MUG (4-methylumbelliferyl-beta-D-glucoronide). Most strains of E.coli produce the enzyme beta-D-glucoronidase, which hydrolyzes this molecule and fluoresces blue under UV light. For this experiment, we spiked the sample with E.coli O157:H7, which does not produce this enzyme and therefore cannot be enumerated by this Colilert-18.
Evaluation of Colilert-18 and Compact Dry™ Systems
Macerated raw ground beef samples in modified ECM media were spiked with a log culture of E.coli O157:H7.
Three replicates of serial dilutions at 10-3, 10-6 and 10-9 were made in 100 ml bottles of sterile saline.
1ml of each dilution was extracted and pipetted onto Compact Dry™ Plates according to the manufacturer’s protocol.
These same bottles were combined with the proprietary Colilert reagent and sealed in the Colliert-18 trays.
The Colilert-18 trays and the Compact Dry™ plates were incubated for 24 hours at 35°C.
After 24 hours, were analyzed for total coliform, E.coli, and E.coli O157:H7 colonies. For the Colilert-18 trays, total coliforms counts were obtained by visually counting the number of trays that turned yellow.
Counts of E.coli were obtained by counting the fluorescent blue wells under a UV light.
Most probable numbers were extrapolated from the Colilert-18 chart and the dilution factor.
From the Compact Dry™ plates, coliform bacteria was counted by the blue colonies and E.coli O157:H7 was counted by the magenta colonies. Total counts were obtained by counting all discernible colonies.
Background
Universal Pre-Enrichment Broth (UPB) is commonly used for the enrichment of food-borne bacteria such as Salmonella and E.coli. Studies have shown that there is no significant difference in the enumeration of E.coli when comparing UPB to other, more specific, enrichment media [1]. The United States Department of Agriculture’s standardized protocols for the identification and isolation of foodborne E.coli O157:H7, use modified E.coli media with novobiocin for the enrichment of E.coli O157:H7 [2]. We conducted a series of comparative experiments to determine if the more cost-effective and more universal UPB was as effective for the enrichment of E.coli O157:H7 as ECM.
Compact Dry™ EC and Colilert-18 are both commercially available systems for the enumeration of coliform bacteria and E.coli. Compact Dry™ EC is a relatively new method for the direct enumeration of E.coli from raw food materials. Coliform bacteria produce blue-green colonies on the basis of the enzymeβ- glucuronidase reacting with X-Gluc. E.coli O157:H7 produces pink colonies in the presence of Magenta-Gal. Colilert-18 is an indirect system based upon the principle of Most Probable Number (MPN) and is commonly used for the enumeration of bacterial coliforms from water samples. Colilert-18 also utilizes a colorimetric assay to distinguish coliform counts from E.coli counts. Metabolism of ONPG (ortho-Nitrophenyl-β-galactoside) by coliform bacteria will turn each of the wells in the tray yellow, and metabolism of MUG (4-methyumbelliferyl-β-D-glucoronide) by most strains of E.coli will fluoresce the wells blue under UV light. However, E.coli O157:H7 does not produce the enzyme β-D-glucoronidase, and therefore will not hydrolyze MUG and will not fluoresce blue. Both of these commercial enumeration systems, were evaluated to determine each of their enumeration efficacies for E.coli from raw, ground beef samples.
°Growth within trays was too extensive to obtain an accurate count. Numbers represent that growth was more than the maximum amount Colilert-18 was able to count at that dilution.
Conclusion
The optimum conditions for E. coli enrichment from meat samples appear to be modified ECM for 24hrs at 35°C.
Compact Dry™ plates enabled enumeration of both E. coli and E. coli O157:H7.
Colilert-18 does not allow for the enumeration of E.coli O157:H7 specifically.
Using the Compact Dry™ enumeration also requires only 1ml aliquots versus the 100ml needed for Colilert-18.
Therefore, Colilert-18 does not appear to be adaptable for the enumeration of E.coli from meat samples. Its application is effective for the enumeration of organisms from water samples.
However, Compact Dry™ appears suitable for enumeration of E. coli, E.coli O157:H7 and total coliforms from raw meat samples.
The experiment was repeated using three replicates of unspiked ground meat samples enriched in ECM.
After 24 hr enrichment at 35°C, serial dilutions were made.
The dilutions were then incubated using the Compact Dry™ and Colilert-18 systems, and analyzed as described above.
Citations
Acknowledgements
Nam, H. M., et al. (2004). "Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples." Foodborne Pathog Dis 1(1):
Food and Drug Administration. (2012) Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. Second Edition. [Enterohemorrhagic Escherichia coli (EHEC), pp. 75 ].
Sergio Patitucci
Brennan Poon-Kwong
Dr. Dave Bachoon’s Lab
Dr. Pillay’s Lab