Typification of Oenococcus oeni strains by multiplex RAPD-PCR and study of population dynamics during malolactic fermentation

Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred.IMPORTANCE This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni, which is crucial for understanding their real contribution to the unique properties of wines.

Section: Discussionmentioning

confidence: 72%

Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions

Khoury

Campbell-Sills

Salin

et al. 2017

“…LAB species were identified by species-specific PCR tests using primers matching the rpoB gene (26). O. oeni strain typing was carried out by using multiplex randomly amplified polymorphic DNA (RAPD)-PCR (25). Eight HDC ϩ O. oeni strains isolated during this work, designated strains A to H, were stored in the IOEB collection under reference numbers IOEB 0605 to IOEB 0612, respectively.…”

Section: Methodsmentioning

confidence: 99%

High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype

Lucas

Claisse

Lonvaud‐Funel

2008

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 10 7 CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 10 3 CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 10 3 per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.

“…This was supported by the levels of DNA-DNA homology and by similarities of the genetic maps of diverse strains (7,50). In addition, differentiation of O. oeni strains can only be achieved by using methods targeting minor genotypic differences: random amplification of polymorphic DNA (RAPD) (36,48,49), DNA fingerprinting (46), amplified fragment length polymorphism (3), or pulsed-field gel electrophoresis (PFGE) patterns of low-frequency restricted genomes (14, 20-22, 38, 45). However, the concept of genetic homogeneity was repeatedly challenged by the detection of groups of strains-often two major groups-distinguished on the basis of molecular and/or phenotypic/metabolic characteristics, leading authors to suggest that O. oeni might be divided into two subspecies (35,45).…”

mentioning

confidence: 99%

Multilocus Sequence Typing of Oenococcus oeni : Detection of Two Subpopulations Shaped by Intergenic Recombination

Bilhere

Lucas

Claisse

et al. 2009

Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.