Long-read MinION™ sequencing of 16S and 16S-ITS-23S rRNA genes provides species-level resolution of Lactobacillaceae in mixed communities.

The Lactobacillaceae are lactic acid bacteria harnessed to deliver important outcomes across numerous industries, and their unambiguous, species-level identification from mixed community environments is an important endeavor. Amplicon-based metataxonomics using short-read sequencing of partial 16S rRNA gene regions is widely used to support this, however, the high genetic similarity among Lactobacillaceae species restricts our ability to confidently describe these communities even at genus level. Long-read sequencing (LRS) of the whole 16S rRNA gene or the near complete rRNA operon (16S-ITS-23S) has the potential to improve this. We explored species ambiguity amongst Lactobacillaceae using in-silico tool RibDif2, which identified allele overlap when various partial and complete 16S rRNA gene and 16S-ITS-23S rRNA regions were amplified. We subsequently implemented LRS by MinION™ to compare the capacity of V3-V4, 16S and 16S-ITS-23S rRNA amplicons to accurately describe the diversity of a 20-species Lactobacillaceae mock community in practice. In-silico analysis identified more instances of allele/species overlap with V3-V4 amplicons (n = 43) compared to the 16S rRNA gene (n = 11) and partial (n = up to 15) or complete (n = 0) 16S-ITS-23S rRNA amplicons. With subsequent LRS of a DNA mock community, 80% of target species were identified using V3-V4 amplicons whilst the 16S rRNA gene and 16S-ITS-23S rRNA region amplicons resulted in 95 and 100% of target species being identified. A considerable reduction in false-positive identifications was also seen with 16S rRNA gene (n = 3) and 16S-ITS-23S rRNA region (n = 9) amplicons compared with V3-V4 amplicons (n = 43). Whilst the target species affected by allele overlap in V3-V4 and 16S rRNA gene sequenced mock communities were predicted by RibDif2, unpredicted species ambiguity was observed in 16S-ITS-23S rRNA sequenced communities. Considering the average nucleotide identity (ANI) between ambiguous species (~97%) and the basecall accuracy of our MinION™ sequencing protocol (96.4%), the misassignment of reads between closely related taxa is to be expected. With basecall accuracy exceeding 99% for recent MinION™ releases, the increased species-level differentiating power promised by longer amplicons like the 16S-ITS-23S rRNA region, may soon be fully realized.

Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk.

A five-isolate cocktail of Listeria monocytogenes (10(3) cfu/ml in skim or whole raw milk) was subjected to 450 MPa for 900 s or 600 MPa for 90 s. The effects of prior growth temperature, type of milk (skim vs. whole), type of recovery-enrichment media (optimized Penn State University [oPSU] broth, Listeria Enrichment Broth [LEB], Buffered LEB [BLEB], Modified BLEB [MBLEB], and milk), storage temperature and storage time on the recovery of L. monocytogenes were examined. Optimized PSU broth significantly increased the recovery of L. monocytogenes following high pressure processing (HPP), and was 63 times more likely to recover L. monocytogenes following HPP, compared to LEB, BLEB and MBLEB broths (p<0.05; Odds Ratio=63.09, C.I. 23.70-167.96). There was a significant main effect for prior growth temperature (p<0.05). However, this relationship could not be interpreted given the significant interaction effects between temperature and both pressure and milk type. HPP-injured L. monocytogenes could be recovered using both LEB and oPSU broths after storage of milk at 4, 15 and 30 degrees C, with recovery being maximal after 24 to 72 h of storage; however, recovery yield dropped to 0% after prolonged storage of milk at 4 and 30 degrees C. In contrast, storage of milk at 15 degrees C yielded the most rapid rate of recovery and the highest recovery yield (100%), which remained high throughout the 14 days of storage at 15 degrees C. The above factors need to be taken into consideration when designing challenge studies to insure complete inactivation of L. monocytogenes and possibly other foodborne pathogens during high pressure processing of foods.

Toward validation of process criteria for high-pressure processing of orange juice with predictive models.

Mathematical models were developed to predict time to inactivation (TTI) by high-pressure processing of Salmonella in Australian Valencia orange juice (pH 4.3) and navel orange juice (pH 3.7) as a function of pressure magnitude (300 to 600 MPa) and inoculum level (3 to 7 log CFU/ml). For each model, the TTI was found to increase with increasing inoculum level and decrease with increasing pressure magnitude. The U.S. Food and Drug Administration Juice Hazard Analysis and Critical Control Point Regulation requires fruit juice processors to include control measures that produce a 5-log reduction of the pertinent microorganism of public health significance in the juice. To achieve a 5-log reduction of Salmonella in navel orange juice at 20 degrees C, the models predicted hold times of 198, 19, and 5 s at 300, 450, and 600 MPa, respectively. In Valencia orange juice at 20 degrees C, a 5-log reduction of Salmonella was achieved in 369, 25, and 5 s at 300, 450, and 600 MPa, respectively. At pressures below 400 MPa, Salmonella was more sensitive to pressure in the more acidic conditions of the navel orange juice and TTIs were shorter. At higher pressures, little difference in the predicted TTI was observed. Refrigerated storage (4 degrees C) of inoculated navel orange juice treated at selected pressure/time/inoculum combinations showed that under conditions in which viable Salmonella was recovered immediately after high-pressure processing, pressure-treated Salmonella was susceptible to the acidic environment of orange juice or to chill storage temperature. These TTI models can assist fruit juice processors in selecting processing criteria to achieve an appropriate performance criterion with regard to the reduction of Salmonella in orange juice, while allowing for processing flexibility and optimization of high-pressure juice processing.