Identification Of Acetic Acid Bacteria Research Articles

Application of Molecular Methods for the Identification of Acetic Acid Bacteria Isolated from Blueberries and Citrus Fruits

Liliana Mabel Gerard , Cristina Verónica Davies , Carina Alejandra Soldá , María Belén Corrado and María Verónica Fernández . Microbiol. Biotechnol. Lett. 2020;48:193-205. https://doi.org/10.4014/mbl.1912.12006

아로니아 식초 제조를 위한 초산균 동정 및 식초 품질 특성 비교

아로니아 식초 제조를 위한 초산균 동정 및 식초 품질 특성 비교

Identification of acetic acid bacteria through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and report of Gluconobacter nephelii Kommanee et al. 2011 and Gluconobacter uchimurae Tanasupawat et al. 2012 as later heterotypic synonyms of Gluconobacter japonicus Malimas et al. 2009 and Gluconobacter oxydans (Henneberg 1897) De Ley 1961 (Approved Lists 1980) emend. Gosselé et al. 1983, respectively

The classification and identification of acetic acid bacteria (AAB) underwent much change during the last two decades. However, several closely related AAB species are difficult to differentiate and rapid and correct species level identification remains challenging. In the present study, 281 well-characterized AAB strains representing AAB species of the family Acetobacteraceae were used to construct a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification database. The database was evaluated using 270 poorly characterized AAB strains. 16S rRNA and/or multilocus gene sequence analysis and whole-genome sequencing for the calculation of average nucleotide identity values were performed on a selection of strains to verify the accuracy of the obtained results. Virtually all poorly characterized AAB strains were accurately identified to the species level. Most of the remaining strains were considered to represent novel AAB species or belonged to species represented by too few strains in the identification database. The data also revealed that Gluconobacter nephelii and Gluconobacter uchimurae are later heterotypic synonyms of Gluconobacter japonicus and Gluconobacter oxydans, respectively.

16S rRNA in situ Hybridization Followed by Flow Cytometry for Rapid Identification of Acetic Acid Bacteria Involved in Submerged Industrial Vinegar Production.

Acetic acid bacteria are involved in many biotechnological processes such as vitamin C, gluconic acid, miglitol or acetic acid production, and others. For a technologist trying to control the industrial process, the ability to follow the microbiological development of the process is thus of importance. During the past few years hybridization in a combination with flow cytometry has often been used for this purpose. Since vinegar is a liquid, it is an ideal matrix for flow cytometry analysis. In this work we have constructed a specific probe for highly acetic acid-resistant species of the acetic acid bacteria and a protocol for in situ hybridization, which in combination with flow cytometry enables direct monitoring of bacteria producing vinegar with >10% of acetic acid. The approach was successfully applied for monitoring microbiota during industrial vinegar production.

흑찰미 식초 제조를 위한 초산균주 동정 및 품질특성 비교

This study was carried out to investigate the quality characteristics and identification of acetic acid bacteria for black waxy rice vinegar. Eight (8) strains of acetic acid bacteria were isolated for the production of acetic acid and their acidities were then compared with commercial acetic acid bacteria. Among them, F1, H4, and two types of commercial bacteria (four best strains by vinegar zymogen) were selected. After analyzing the 16S rRNA sequence, both F1 and H4 strains were identified as Acetobacter genus. Therefore, the F-1 and H-4 strains were named as Acetobacter sp. F-1 and Acetobacter sp. H-4, respectively. Acidity of black waxy rice vinegar during fermentation was steadily increased up to 16 days and the acidity was then constant. Total acidity content was higher when used FV-1 strain. In the results of Hunter's color value of black waxy vinegar, L value was at 75.01 to 80.11, while (＋a) value was at 3.34 to 3.92, and (＋b) value was at 12.84 to 18.09. The major organic acid of the black waxy vinegar was acetic acid. The total organic acid content was high when used H-4, F-1, C-2 and C-1 strains. The total free amino acid content of the black waxy vinegar by strain was the highest (351.43 mg%) of F-1 vinegar strain, and the lowest (247.74 mg%) of C-2 vinegar strain. A sensory evaluation of black waxy vinegar indicated that F-1 vinegar strain was better than the other samples in aspect of flavor, color, and overall preference.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7–V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S–23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

Identification of acetic acid bacteria isolated in Thailand and assigned to the genus Acetobacter by groEL gene sequence analysis

Twenty-three strains of acetic acid bacteria were isolated by an enrichment culture approach from fruits, flowers, mushrooms, and fermented rice products collected in Thailand. All were assigned to the genus Acetobacter and examined taxonomically for 16S rRNA gene and groEL gene sequences. Based on analysis of 16S rRNA gene and groEL gene sequences, the strains isolated were divided into ten groups: Group 1 comprised four isolates identified as A. tropicalis, Group 2 consisted of three isolates identified as A. indonesiensis, Group 3 had two isolates identified as A. persici, Group 4 was two isolates (A. orientalis), Group 5 was one isolate (A. cibinongensis), Group 6 contained three isolates (A. pasteurianus), Group 7 was one isolate of A. papayae, Group 8 was two isolates identified as A. fabarum, Group 9 was one isolate of A. okinawensis, and Group 10 was four isolates identified as A. ghanensis.

Optimization of denaturing high performance liquid chromatography technique for rapid detection and identification of acetic acid bacteria of interest in vinegar production

This paper evaluates the use of denaturing high performance liquid chromatography (DHPLC) technology for the discrimination of genetic differences in the 16S rRNA and alcohol dehydrogenase (AdhA) genes among bacterial species based on its efficiency and sensitivity to enable the detection and discrimination of different genetic sequences. In order to optimize DHPLC protocols for the analysis of 16S rRNA gene fragments amplified from bacteria, DNA isolated from 22 different strains representing main bacterial groups of interest in food microbiology was analyzed. While the use of 16S rRNA gene did not allow to difference two wild strains of <em>Acetobacter malorum</em>, this region revealed as useful to differentiate them from some pathogenic bacteria as <em>Escherichia coli</em>, <em>Salmonella typhimurium</em>, <em>Listeria monocytogenes</em>, <em>Listeria innocua</em>, <em>Clostridium perfringens </em>or <em>Sthapylococcus aureus</em>, from spoilage microorganisms as <em>Xantomonas vesicatoria</em> and <em>Alicyclobacillus</em> spp., and also from lactic acid bacteria as <em>Lactobacillus plantarum</em>, <em>Lactobacillus casei</em>, <em>Lactobacillus sakei</em>, <em>Lactobacillus acidophilus</em>, <em>Streptococcus thermophilus </em>and <em>Lactococcus lactis</em> that may suppose technological risk during vinegar production. The results demonstrate that 16S rRNA gene region is not adequate for the discrimination of the acetic acid bacteria (AAB) strains, so AdhA gene was selected to identify the two wild strains of <em>Acetobacter malorum</em>. Also 6 different reference strains of AAB were separated based on differences in AdhA gene region. DHPLC technology is able to discriminate between these two wild strains of <em>A. malorum</em> based on differences existing in the AdhA gene region. The data obtained indicate that the technique is capable of identifying most bacteria at species level and even at strain level with optimization of the protocols. This is of particular relevance in the case of AAB due to their poor recovery on culture media and difficulties in detection of viable but non cultivable cells.

Diversity of acetic acid bacteria present in healthy grapes from the Canary Islands

The identification of acetic acid bacteria (AAB) from sound grapes from the Canary Islands is reported in the present study. No direct recovery of bacteria was possible in the most commonly used medium, so microvinifications were performed on grapes from Tenerife, La Palma and Lanzarote islands. Up to 396 AAB were isolated from those microvinifications and identified by 16S rRNA gene sequencing and phylogenetic analysis. With this method, Acetobacter pasteurianus, Acetobacter tropicalis, Gluconobacter japonicus and Gluconacetobacter saccharivorans were identified. However, no discrimination between the closely related species Acetobacter malorum and Acetobacter cerevisiae was possible. As previously described, 16S-23S rRNA gene internal transcribed spacer (ITS) region phylogenetic analysis was required to classify isolates as one of those species. These two species were the most frequently occurring, accounting for more than 60% of the isolates. For typing the AAB isolates, both the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and (GTG)5-PCR techniques gave similar resolution. A total of 60 profiles were identified. Thirteen of these profiles were found in more than one vineyard, and only one profile was found on two different islands (Tenerife and La Palma).

Differentiation of species of the family Acetobacteraceae by AFLP DNA fingerprinting: Gluconacetobacter kombuchae is a later heterotypic synonym of Gluconacetobacter hansenii

Amplified fragment length polymorphism (AFLP) DNA fingerprinting was investigated as a tool for fast and accurate identification of acetic acid bacteria (AAB) to the species level. One hundred and thirty five reference strains and 15 additional strains, representing 50 recognized species of the family Acetobacteraceae, were subjected to AFLP analysis using the restriction enzyme combination ApaI/TaqI and the primer combination A03/T03. The reference strains had been previously subjected to either DNA-DNA hybridization or 16S-23S rRNA spacer region gene sequence analysis and were regarded as being accurately classified at the species level. The present study revealed that six of these strains should be reclassified, namely Gluconacetobacter europaeus LMG 1518 and Gluconacetobacter xylinus LMG 1510 as Gluconacetobacter xylinus and Gluconacetobacter europaeus, respectively; Gluconacetobacter kombuchae LMG 23726(T) as Gluconacetobacter hansenii; and Acetobacter orleanensis strains LMG 1545, LMG 1592 and LMG 1608 as Acetobacter cerevisiae. Cluster analysis of the AFLP DNA fingerprints of the reference strains revealed one cluster for each species, showing a linkage level below 50 % with other clusters, except for Acetobacter pasteurianus, Acetobacter indonesiensis and Acetobacter cerevisiae. These three species were separated into two, two, and three clusters, respectively. At present, confusion exists regarding the taxonomic status of Gluconacetobacter oboediens and Gluconacetobacter intermedius; the AFLP data from this study supported their classification as separate taxa. The 15 additional strains could all be identified at the species level. AFLP analysis further revealed that some species harboured genetically diverse strains, whereas other species consisted of strains showing similar banding patterns, indicating a more limited genetic diversity. It can be concluded that AFLP DNA fingerprinting is suitable for accurate identification and classification of a broad range of AAB, as well as for the determination of intraspecific genetic diversity.

Validation of the (GTG) 5-rep-PCR fingerprinting technique for rapid classification and identification of acetic acid bacteria, with a focus on isolates from Ghanaian fermented cocoa beans

Amplification of repetitive bacterial DNA elements through the polymerase chain reaction (rep-PCR fingerprinting) using the (GTG) 5 primer, referred to as (GTG) 5-PCR fingerprinting, was found a promising genotypic tool for rapid and reliable speciation of acetic acid bacteria (AAB). The method was evaluated with 64 AAB reference strains, including 31 type strains, and 132 isolates from Ghanaian, fermented cocoa beans, and was validated with DNA:DNA hybridization data. Most reference strains, except for example all Acetobacter indonesiensis strains and Gluconacetobacter liquefaciens LMG 1509, grouped according to their species designation, indicating the usefulness of this technique for identification to the species level. Moreover, exclusive patterns were obtained for most strains, suggesting that the technique can also be used for characterization below species level or typing of AAB strains. The (GTG) 5-PCR fingerprinting allowed us to differentiate four major clusters among the fermented cocoa bean isolates, namely A. pasteurianus (cluster I, 100 isolates), A. syzygii- or A. lovaniensis-like (cluster II, 23 isolates), and A. tropicalis-like (clusters III and IV containing 4 and 5 isolates, respectively). A. syzygii-like and A. tropicalis-like strains from cocoa bean fermentations were reported for the first time. Validation of the method and indications for reclassifications of AAB species and existence of new Acetobacter species were obtained through 16S rRNA sequencing analyses and DNA:DNA hybridizations. Reclassifications refer to A. aceti LMG 1531, Ga. xylinus LMG 1518, and Ga. xylinus subsp. sucrofermentans LMG 18788 T.

Acetobacter cibinongensisBacteremia in Human

<i>Acetobacter cibinongensis</i>Bacteremia in Human

Application of molecular methods for routine identification of acetic acid bacteria

Recently many new species of Acetic acid Bacteria have been described. The description and identification as new species was based on molecular techniques (sequencing of the 16S rRNA gene, DNA base ratio (% GC) determinations and DNA–DNA hybridisation) and phenotypic characterization. In the present paper, we propose a fast and reliable method for the identification most of the species currently described based on the RFLP–PCR of the 16S rRNA. According to the proposed protocol, 1 species can be identified with the use of a single enzyme, 13 with a combination of 2 enzymes, 2 species with a combination of 3 enzymes, 2 with a combination of 4 enzymes. To differentiate 5 more species RFLP–PCR of the ITS was also needed, after using 3 enzymes. Finally, a pair of species ( Acetobacter pasteurianus and Acetobacter pomorum) could not be distinguished with the proposed method. However, doubts can be raised about their differentiation as separate species. Keeping these limitations in mind, the method is fast and reliable, allowing the processing of large number of samples in relatively short periods of time (less than 24 h after the isolation).

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S–23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S–23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNA Ile and tRNA Ala, the sequences of 16S–23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. aceti-specific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.

Identification of acetic acid bacteria isolated from fruits collected in Thailand.

Identification of acetic acid bacteria isolated from fruits collected in Thailand.

The enumeration and identification of acetic acid bacteria from South African red wine fermentations

Acetic acid bacteria are microorganisms that can profoundly influence the quality of wine. Surprisingly, little research has been done on these microorganisms in the winemaking field. The object of this study was to investigate the occurrence of acetic acid bacteria in South African red wine fermentations and to identify the dominant species occurring. Acetic acid bacteria were isolated and enumerated from small-scale and commercial red must fermentations in 1998 and 1999, respectively. The initial occurrence of acetic acid bacteria in the must was shown to vary with cell numbers ranging from 10 6–10 7 to 10 4–10 5 cfu/ml for the 1998 and 1999 musts, respectively. The acetic acid bacteria decreased to 10 2–10 3 cfu/ml in musts having a low pH (≤3.6), whereas in some musts having a high pH (≥3.7), the cell numbers increased during fermentation. During the process of cold soaking, the cell numbers of acetic acid bacteria also increased until inoculation with commercial wine yeast. Gluconobacter oxydans dominated in the fresh must and Acetobacter pasteurianus and A. liquefaciens during fermentation . This study showed that A. liquefaciens and A. hansenii were present in significant numbers, which has not been reported before.

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.

Identification of acetic acid bacteria isolated from Indonesian sources, especially of isolates classified in the genus Gluconobacter.

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.

Isolation and identification of acetic acid bacteria for submerged acetic acid fermentation at high temperature.

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.

Isolation and identification of acetic acid bacteria from different ecosystems

Forty-five cultures of acetic acid bacteria were isolated from different ecosystems including fruits, vegetables, flowers, vinegars, etc. On the basis of morphological, physiological and biochemical characteristics they were identified asA. aceti, A. xylinum, A. suboxydans, A. melanogenum andBacterium xylinoides. It was obvious thatA. aceti andA. xylinum were prevalent in fruits, vinegars and cane juice whileA. rancens was to be found in flowers and vegetables. Grapes seem to be a fine source for isolation of common species. Alcohol tolerance and ability to oxidize alcohol to acetic acid showed thatA. aceti andA. xylinum possess higher alcohol tolerance and produce more acetic acid than the other species.