Rep-PCR of E. coli from shellfish

Contamination of shellfish with E. coli is a problem which can result in shellfish not being able to be harvested. Identifying the source of the contamination is an important step if this contamination is to be controlled.

Repetitive extragenic palindromic-PCR (Rep-PCR), is a type of polymerase chain reaction that targets the repetitive sequences in bacterial genomes using specific primers that generate a DNA barcode like fingerprint. Mike Sadowsky at University of Minnesota has a good website (external link) which explains this method in detail.

The question we sought to address in this study was “Is genotyping of E. coli from shellfish a useful technique for determination of faecal source?”

Method

The first step is to recover E. coli from shellfish. Standard analysis of E. coli in shellfish uses enrichment techniques, which grow E. coli in broth culture and then using an MPN format, enumerate the E. coli. For genotyping, enrichment can not be used as it will alter significantly the distribution of types of E. coli types present. A direct plating method is therefore required. Note however, that without enrichment, the detection limit for recovery of E. coli will be higher.

Shellfish from two sites were collected on two different days, approximately 1 month apart, and E. coli recovered from the shellfish. The Rep-PCR profiles of these shellfish were compared with E. coli isolated from Shag faecal material (also collected on day 1), and also with isolates in our database from human sewage and cow faecal material.

Results

The Rep-PCR profiles identified at each site are shown in the table below. Each profile indicates a different Rep-PCR fingerprint or DNA banding pattern. The number in brackets indicates the number of isolates with that profile.

The shag faecal sample analysed contained two genotypes of E. coli, twenty of which were Rep-001, and two of which were Rep-018. The Rep-018 profile was also found in isolates recovered from shellfish at site A. This is shown in the figure.

Three of the Rep-PCR profiles from the day 30 sampling of site B (Rep 22, 23 and 24) clustered among municipal sewage isolates. The remaining isolates did not cluster with isolates from any known source.

Conclusion

Rep-PCR analysis requires analysis of a much larger number of isolates for more conclusive source attributions to be made. This work does however demonstrate the technical feasibility of the analysis. The degree of separation of Rep-PCR genotypes between sources remains to be quantified. However if suspected sources could be sampled concurrently with a contaminated source, it would appear that comparisons of Rep-PCR types could be made, and estimates of contributions made.

The finding of the same type (Rep18) in shag faeces and shellfish is suggestive that shag faeces was a source of contamination of the shellfish at Site A at the time of sampling.