16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

Validation of a PCR-based method for the detection of various rendered materials in feedstuffs using a forensic DNA extraction kit.

A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the positive control DNA samples could be detected with the ovine primers. The multispecies primer pair was validated in three laboratories for use with bovine meat and bone meal and lamb meal but not with PMBM. The three laboratories had an average accuracy of 98.9% for bovine meat and bone meal, 97.8% for lamb meal, and 63.3% for PMBM. When examined on an individual laboratory basis, one of these four laboratories could not identify a single feed sample containing PMBM by using the multispecies primer, whereas the other laboratory identified only one PMBM-fortified sample, suggesting that the limit of detection for PMBM with this primer pair is around 0.1% (wt/wt). The results of this study demonstrated that the DNA forensic kit can be used to extract DNA from animal feed, which can then be used for PCR analysis to detect animal-derived protein present in the feed sample.

Development of a custom 16S rRNA gene library for the identification and molecular subtyping of Salmonella enterica.

The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500 bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500 bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility.

Produce isolates of the Escherichia coli Ont:H52 serotype that carry both Shiga toxin 1 and stable toxin genes.

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.

Isolation and characterization of Listeria monocytogenes isolates from ready-to-eat foods in Florida.

Of 3,063 ready-to-eat food samples tested, 91 (2.97%) were positive for Listeria monocytogenes, and lineage 1 strains outnumbered lineage 2 strains 57 to 34. Seventy-one isolates (78%) exhibited multiple antibiotic resistance, and an L. monocytogenes-specific bacteriophage cocktail lysed 65 of 91 (71%) isolates. Determining phage, acid, and antibiotic susceptibility phenotypes enabled us to identify differences among strains which were otherwise indistinguishable by conventional methods.