Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan-MGB probes.

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10(2)-10(3) cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S-23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA. The RT-PCR assay used in this study is a reliable, specific and fast method for quantifying these five AAB species in wine and vinegar.

Quantification of the expression of reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth conditions.

AIMS: The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)-alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB). METHODS AND RESULTS: Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adhA was measured in Acetobacter aceti, Acetobacter pasteurianus, Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adhA expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adhA expression. CONCLUSIONS: The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter. SIGNIFICANCE AND IMPACT OF THE STUDY: Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.

Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation.

AIMS: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. METHODS AND RESULTS: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. CONCLUSIONS: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.

Identification of acetic acid bacteria by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA.

Acetic acid bacteria (AAB) irreversibly spoil wines and represent a serious problem. Limited studies on the ecology of AAB during winemaking have been done due to the lack of rapid and precise techniques for their identification. RFLP analysis of PCR-amplified fragment of 16S rDNA was performed on AAB reference strains. The amplified rDNAs were approximately 870-bp long for all AAB species while no amplicons were detected for lactic acid bacteria and yeasts. Out of the four restriction enzymes tested, TaqI was the most efficient one and divided the studied AAB into six groups. However, complete differentiation among collection strains of Acetobacter pasteurianus and Gluconoacetobacter hansenii was not possible.