Pr oject Summar y. Escherichia coli O157 indicator organism. Principal Investigator: Terrance M. Arthur and Mohammad Koohmaraie

Transcription

1 Pr oject Summar y Escherichia coli O157 indicator organism Principal Investigator: Terrance M. Arthur and Mohammad Koohmaraie U.S. Department of Agriculture, Agricultural Research Service Study Completed May 2003 Funded by The Beef Checkoff 1

2 Escherichia coli O157 indicator organism Background Predictive microbiology has long sought to determine the quality of a food product based on the presence or absence of various spoilage organisms or their byproducts. Similar attempts have been used for food safety. However, detection of certain foodborne pathogens can be complicated due to the low numbers at which they are usually found. In order to circumvent this complication, researchers have searched for suitable marker organisms for various pathogens. Early studies determined that there were two classes of organisms one could look for: indicator and index organisms. Indicator organisms refer to single or multiple bacterial species that can be used to gauge the effectiveness of antimicrobial interventions. The assumption is made that the target organism and the indicator organism will react in a similar fashion to the intervention. Therefore, any reductions in the indicator organism population in response to application of an antimicrobial intervention are thought to be mirrored by the population of the target organism. Index organisms must be present only when the target organism is present and be absent when the target organism is absent. Moreover, the index organism must exist in sufficient numbers for detection, be detectable by rapid methods, and be easily differentiable from other microflora. While true index organisms may never be found for E. coli O157:H7, it may be possible to identify indicator organisms that also meet many, but not all, or the requirements of index organisms. In order to identify either an indicator or index organism for E. coli O157:H7, it will be necessary to thoroughly examine the microbial populations that cohabitate with the pathogen. Traditionally, this was done by culture methods using enrichments and plating on semisolid agar in an attempt to isolate pure cultures of each inhabitant. The biochemical characteristics of the isolates were then used to identify the organism and to establish phylogenetic relationships. However, due to the fastidious nature of most bacterial species, these methods underestimated the true bacterial diversity present in most samples. It has been reported that the number of colonies recovered from a sample was less than one percent of those identified by direct counting. The advent of molecular microbiology has removed the bias imposed by needing to culture a bacterial species in order for identification. It has been determined that the nucleotide sequence of the RNA component of the ribosomal small subunit (16S rdna) has regions that are highly conserved among species as well as those that diverge. It is believed that such a sequence could be used as an evolutionary clock to determine the phylogeny of species. Sequences of 16S rdna have since been used extensively for identification of members of bacterial populations that had previously been recalcitrant to culture by traditional methods. The stated objectives for this work were: To identify members of bacterial populations in cattle feces and present on carcasses in order to select a bacterial strain that can serve as an indicator organism for Escherichia coli O157. Methodology Sample collection. Samples were collected during visits to large Midwestern beef processing plants (6 total visits spread among 3 different processing plants). The samples were collected during the summer (June to August, 2002), the peak season for E. coli O157:H7 prevalence. Samples were obtained as previously described, with some modifications. Fecal samples were obtained by removing 11g of fecal material from the distal portion of the colon after evisceration. Carcasses were sampled prior to evisceration by swabbing with a Speci-sponge (NASCO, Ft. 2

3 Atkinson, Wis.) wetted in 25 ml of buffered peptone water (BPW; Difco, Becton, Dickinson, Sparks, Md.). Pre-evisceration carcass samples were obtained by swabbing approximately 8,000 cm 2 of the inside and outside round area. All samples were stored on ice for transport to laboratory. DNA isolation. Upon arrival at the lab, the samples were massaged by hand to homogenize. For the pre-evisceration samples, 1 ml was removed prior to addition of enrichment media. Microbial DNA was extracted from the 1-mL sample using a DNeasy Tissue Kit (Qiagen). For fecal samples, 1 g of feces was removed and diluted with 5 ml of sterile water. 1.5 ml of the fecal slurry was centrifuged for 3 min at 500 x g to remove debris. One milliliter of supernatant was processed for DNA extraction suing a Wizard Genomic DNA extraction kit (Promega). PCR. To minimize bias, two separate 16S universal primer sets were used for each sample (Table 1). Therefore, two PCR reactions were set up for each sample. Between 100 and 300 ng of extracted genomic DNA was used in 50 µl 16S rdna amplification reactions. Each PCR reaction also consisted of 200 µm deoxyribonucleotides, 1.5 mm MgCl 2, 500 nm each primer, and 2.5 U of HotStar Taq (Quiagen). The PCR program consisted of the following steps: 94 C for 15 min to denature the DNA and activate the polymerase; 25 cycles of 94 C for 45 s, 55 C for 30 s, 72 C for 2 min; then 72 C for 10 min. Cloning and sequencing. The two PCR reactions for each sample were combined and mixed. The PCR products were cloned into the pcr4 TOPO vector (Invitrogen) in a reaction consisting of 2.5 µl of PCR DNA, 1 µl salt solution, 2 µl sterile water, and 0.5 µl vector. The reactions were incubated at room temperature for 15 min, then 2 µl was transformed via heat shock into 50 µl of competent cells (One Shot TOP10, Invitrogen). In order to minimize chances of picking sibling colonies, three separate ligation/transformations were performed for each sample and outgrowth for all transformations was limited to 30 min. One hundred microliters of each transformation were plated onto LB agar plates containing 100 µg/ml carbencillin. The plates were incubated overnight at 37 C. From each plated transformation, 32 colonies were picked for sequencing for a total of 96 colonies per sample. Enrichment and recovery of E. coli O157:H7. Samples were analyzed for the presence of E. coli O157:H7 using the previously described MRU method. Tryptic soy broth (TSB; Difco) was added to all samples and the enrichments were incubated at 25 C for 2 h, then at 42 C for 5 h prior to being held at 4 C overnight. IMS was performed to recover E. coli O157:H7 according to the manufacturer s directions (Dynal, Lacke Success, N.Y.), except that protamine (100 µl of a 50- µg/ml solution; Sigma) was added to pre-evisceration enrichments prior to adding the selective beads. After IMS, E. coli O157:H7 beads were spread directly onto (a) ntrainbow: Rainbow agar (Biolog, Inc., Hayward, Calif.) supplemented with novobiocin (20mg/liter; Sigma) and potassium tellurite (0.8mg/liter; Sigma) and (b) ctsmac: sorbitol MacConkey agar (Difco) supplemented with cefixime (0.05 mg/liter) and potassium tellurite (2.5 mg/liter; Dynal). Confirmation of E. coli O157:H7. Up to three suspect colonies (based on colony phenotype on either or both agars) were tested per animal using DrySpot latex agglutination tests (Oxoid). Growth from colonies identified as potentially positive was streaked for isolation on ctsmac for further testing. Confirmation as E. coli O157:H7 was accomplished using multiplex PCR to detect stx1, stx2, eae, flic H7 and rfbe O157 genes. Samples were considered positive if at least one recovered isolate: (a) carried at least one stx gene, (b) carried the rfbe O157 gene, (c) and carried the flic H7 gene. Findings Genomic DNA extraction procedures were optimized for both pre-evisceration (carcass) sponge samples and fecal samples. Four methods were evaluated based on quantity of DNA recovered and suitability of the recovered DNA for use in PCR. The methods used were the 3

4 DNeasy Tissue kit from Qiagen, The Wizard Genomic DNA Isolation kit from Promega, the Fecal UltraClean DNA Extraction kit from MoBio, and traditional Phenol extraction followed by alcohol precipitation. Bacterial DNA was best isolated from carcass samples using the DNeasy Tissue kit, while the Wizard Genomic DNA kit was used for fecal samples. Samples were collected from two large commercial processing plants. A total of 120 carcass samples and 90 fecal samples were collected and assayed for the presence of E. coli O157:H7. Isolates of E. coli O157:H7 were obtained from 63 (53%) of the carcass samples and 16 (18%) of the fecal samples (Table 2). Total genomic DNA was extracted from all 210 samples and stored at -20 C. Following completion of the O157:H7 culture procedures, 15 O157:H7-positive and 15 O157:H7-negative samples were selected from both the fecal and pre-evisceration sample sets. The attempt was made during this selection to choose samples from as many different processing plants as possible. The final group of 60 samples was used for 16S rdna PCR, cloning, and sequencing. Bi-directional sequencing was performed for 96 clones from each sample. Each clone potentially represented a unique genus or species of bacteria. In all 11,520 sequences were generated from 5,760 clones. Two sets of universal primers were chosen to amplify the 16S rdna sequences from the sample DNA. By using the two primer sets, bias toward specific genera should be minimized. The TOPO TA cloning kit from Invitrogen was used for shotgun cloning of the 16S rdna PCR products. Sequencing was done in a 384 well plate format using vector-based primers. For data retrieval and analysis, a database was designed to accumulate the sequence information. This database was used for bulk BLAST searching of the 16S rdna sequences against the entire NCBI database and archiving the results based on percent similarity and length of overlap between sequences. Besides BLAST analysis of the sequences, the DNA sequences were also aligned using the Clustal W program. From this alignment, various types of phylogenetic trees are being evaluated (neighbor-joining, maximum parsimony, and maximum likelihood). An initial phylogram (Figure 1) was generated using an alignment of 16S rdna sequences from samples 281 and 282. The sequences from sample 281 are indicated with the filled squares. A distinct clustering can be seen in the preliminary analysis. All samples will now be aligned and a master phylogram will be generated. If the clustering of sequences continues to be seen, this will indicate that there is a high likelihood of identifying indicator organisms for E. coli O157:H7. Implications This project was initiated with the goal of producing a more rapid and simple method to detect the bacterial pathogen E. coli O157:H7 associated with beef cattle. For this purpose, the identities of several members of the bacterial community found in cattle feces and on carcasses were determined. Once the community members were identified, the community census was evaluated to identify those bacteria which were commonly present when E. coli O157 was also present. From the analysis, it seems as if there are some bacterial species that are present only when E. coli O157 is present and some that are present only when E. coli O157 is absent. Further work will be conducted to determine if rapid detection methods can be developed for these bacteria and if they will be useful in monitoring E. coli O157 presence. 4

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7 For more information contact: National Cattlemen's Beef Association A Contractor to the Beef Checkoff 9110 East Nichols Avenue Centennial, Colorado (303)