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Metataxonomic Profiling of Native and Starter Microbiota During Ripening of Gouda Cheese Made With Listeria monocytogenes-Contaminated Unpasteurized Milk.

ABSTRACT: Unpasteurized milk is used to produce aged artisanal cheeses, which presents a safety concern due to possible contamination with foodborne pathogens, especially Listeria monocytogenes. The objective of this study was to examine the composition of the bacterial community in unpasteurized milk used to prepare Gouda cheese artificially contaminated with L. monocytogenes (~1 log CFU/ml) and assess the community dynamics and their potential interaction with L. monocytogenes during a 90-day ripening period using targeted 16S rRNA sequencing. The diversity of bacterial taxa in three batches of unpasteurized milk was not significantly different, and the microbiomes were dominated by species of Lactococcus, Streptomyces, Staphylococcus, and Pseudomonas. The highest relative abundances were observed for Pseudomonas fluorescens (31.84-78.80%) and unidentified operational taxonomic units (OTUs) of Pseudomonas (7.56-45.27%). After manufacture, both with and without L. monocytogenes-contaminated unpasteurized milk, Gouda cheese was dominated by starter culture bacteria (including Lactococcus lactis subsp. cremoris, lactis, lactis bv. diacetylactis, and Streptococcus thermophilus), in addition to unassigned members in the taxa L. lactis and Streptococcus. During ripening there was an overall decrease in L. lactis abundance and an increase in the number of taxa with relative abundances >0.1%. After 90-day ripening, a total of 82 and 81 taxa were identified in the Gouda cheese with and without L. monocytogenes, respectively. Of the identified taxa after ripening, 31 (Gouda cheese with L. monocytogenes) and 56 (Gouda cheese without L. monocytogenes) taxa had relative abundances >0.1%; 31 were shared between the two types of Gouda cheese, and 25 were unique to the Gouda cheese without added L. monocytogenes. No unique taxa were identified in the Gouda cheese with the added L. monocytogenes. This study provides information on the dynamics of the bacterial community in Gouda cheese during ripening, both with and without the addition of L. monocytogenes.

SUBMITTER: Salazar JK

PROVIDER: S-EPMC7994605 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The eradication of Listeria monocytogenes from food chains is still a great challenge for the food industry and control authorities since some clonal complexes (CCs) are either better adapted to food processing environments (FPEs) or are globally widespread. In this work, we focus on the in-house evolution of L. monocytogenes genotypes collected from a heavily contaminated FPE whose contamination pattern underwent a massive and yet unexplained change. At the beginning of the sampling in 2010, a high variety of most likely transient L. monocytogenes genotypes was detected belonging to sequence type (ST) 1, ST7, ST21, ST37. After several efforts to intensify the hygiene measures, the variability was reduced to L. monocytogenes ST5 that was dominant in the following years 2011 and 2012. We aimed to elucidate possible genetic mechanisms responsible for the high abundance and persistence of ST5 strains in this FPE. Therefore, we compared the genomes of six L. monocytogenes ST5 strains to the less frequently occurring transient L. monocytogenes ST37 and ST204 from the same FPE as well as the highly abundant ST1 and ST21 isolated in 2010. Whole genome analysis indicated a high degree of conservation among ST5 strains [average nucleotide identity (ANI) 99.93-99.99%; tetranucleotide correlation 0.99998-0.99999]. Slight differences in pulsed field gel electrophoresis (PFGE) patterns of two ST5 isolates could be explained by genetic changes in the tRNA-Arg-TCT prophages. ST5 and ST204 strains harbored virtually identical 91 kbp plasmids related to plasmid group 2 (pLM80 and pLMUCDL175). Interestingly, highly abundant genotypes present in the FPE in 2010 did not harbor any plasmids. The ST5 plasmids harbored an efflux pump system (bcrABC cassette) and heavy metal resistance genes possibly providing a higher tolerance to disinfectants. The pLM80 prototype plasmids most likely provide important genetic determinants for a better survival of L. monocytogenes in the FPE. We reveal short-term evolution of L. monocytogenes strains within the same FPE over a 3 year period and our results suggest that plasmids are important for the persistence of ST5 strains in this FPE.

Project description:Industrial production of Gouda cheeses mostly relies on a rotated use of different mixed-strain lactic acid bacteria starter cultures to avoid phage infections. However, it is unknown how the application of these different starter culture mixtures affect the organoleptic properties of the final cheeses. Therefore, the present study assessed the impact of three different starter culture mixtures on the batch-to-batch variations among Gouda cheeses from 23 different batch productions in the same dairy company. Both the cores and rinds of all these cheeses were investigated after 36, 45, 75, and 100 weeks of ripening by metagenetics based on high-throughput full-length 16S rRNA gene sequencing accompanied with an amplicon sequence variant (ASV) approach as well as metabolite target analysis of non-volatile and volatile organic compounds. Up to 75 weeks of ripening, the acidifying Lactococcus cremoris and Lactococcus lactis were the most abundant bacterial species in the cheese cores. The relative abundance of Leuconostoc pseudomesenteroides was significantly different for each starter culture mixture. This impacted the concentrations of some key metabolites, such as acetoin produced from citrate, and the relative abundance of non-starter lactic acid bacteria (NSLAB). Cheeses with the least Leuc. pseudomesenteroides contained more NSLAB, such as Lacticaseibacillus paracasei that was taken over by Tetragenococcus halophilus and Loigolactobacillus rennini upon ripening time. Taken together, the results indicated a minor role of leuconostocs in aroma formation but a major impact on the growth of NSLAB. The relative abundance of T. halophilus (high) and Loil. rennini (low) increased with ripening time from rind to core. Two main ASV clusters of T. halophilus could be distinguished, which were differently correlated with some metabolites, both beneficial (regarding aroma formation) and undesirable ones (biogenic amines). A well-chosen T. halophilus strain could be a candidate adjunct culture for Gouda cheese production.

Project description:BACKGROUND:The more quickly bacterial pathogens responsible for foodborne illness outbreaks can be linked to a vehicle of transmission or a source, the more illnesses can be prevented. Whole genome sequencing (WGS) based approaches to source tracking have greatly increased the speed and resolution with which public health response can pinpoint the vehicle and source of outbreaks. Traditionally, WGS approaches have focused on the culture of an individual isolate before proceeding to DNA extraction and sequencing. For Listeria monocytogenes (Lm), generation of an individual isolate for sequencing typically takes about 6?days. Here we demonstrate that a hybrid, "quasimetagenomic" approach ie; direct sequencing of microbiological enrichments (first step in pathogen detection and recovery) can provide high resolution source tracking sequence data, 5 days earlier than response that focuses on culture and sequencing of an individual isolate. This expedited approach could save lives, prevent illnesses and potentially minimize unnecessary destruction of food. METHODS:Naturally contaminated ice cream (from a 2015 outbreak) was enriched to recover Listeria monocytogenes following protocols outlined in the Bacteriological Analytic Manual (BAM). DNA from enriching microbiota was extracted and sequenced at incremental time-points during the first 48?h of pre-enrichment using the Illumina MiSeq platform (2 by 250), to evaluate genomic coverage of target pathogen, Listeria monocytogenes. RESULTS:Quasimetagenomic sequence data acquired from hour 20 were sufficient to discern whether or not Lm strain/s were part of the ongoing outbreak or not. Genomic data from hours 24, 28, 32, 36, 40, 44, and 48 of pre-enrichments all provided identical phylogenetic source tracking utility to the WGS of individual isolates (which require an additional 5?days to culture). CONCLUSIONS:The speed of this approach (more than twice as fast as current methods) has the potential to reduce the number of illnesses associated with any given outbreak by as many as 75% percent of total cases and potentially with continued optimization of the entire chain of response, contribute to minimized food waste.

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Metataxonomic Profiling of Native and Starter Microbiota During Ripening of Gouda Cheese Made With Listeria monocytogenes-Contaminated Unpasteurized Milk.

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