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.Acetic acid bacteria (AAB) are Gram-negative, coccoid or rod-shaped, obligate aerobic bacteria that have the ability to incompletely oxidize a wide range of carbohydrates, alcohols and sugar alcohols. They are involved in the production of several fermented foods and beverages, either in a beneficial (vinegars, cocoa-based products, Kombucha and nata de coco) or detrimental (spoilage of beer, wine and cider) manner and are also used in the production of commercially important fine chemicals and bacterial cellulose. Recently, some AAB have beenAbbreviations: AAB, acetic acid bacteria; AFLP, amplified fragment length polymorphism; ERIC enterobacterial repetitive intergenic consensus sequences; FAFLP, fluorescent amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; REP, Repetitive extragenic palindromic sequences.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of LMG 23726 T is AM999342.A supplementary figure showing the (GTG...

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed in meat products during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes has been extensively reported, research on the putative involvement of the human intestinal microbiota in PhIP metabolism remains scarce. In this study, the in vitro conversion of PhIP into its microbial derivate, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3,2:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), by fecal samples from 18 human volunteers was investigated. High-performance liquid chromatography analysis showed that all human fecal samples transformed PhIP but with efficiencies ranging from 1.8 to 96% after 72 h of incubation. Two PhIP-transforming strains, PhIP-M1-a and PhIP-M1-b, were isolated from human feces and identified by fluorescent amplified fragment length polymorphism and pheS sequence analyses as Enterococcus faecium strains. Some strains from culture collections belonging to the species E. durans, E. avium, E. faecium, and Lactobacillus reuteri were also able to perform this transformation. Yeast extract, special peptone, and meat extract supported PhIP transformation by the enriched E. faecium strains, while tryptone, monomeric sugars, starch, and cellulose did not. Glycerol was identified as a fecal matrix constituent required for PhIP transformation. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Whether it is a detoxification is still a matter of debate, since PhIP-M1 has been shown to be cytotoxic toward Caco-2 cells but is not mutagenic in the Ames assay.Diet is a major risk factor in human cancer (14). Epidemiological studies indicate that the consumption of cooked meat and meat products predisposes individuals to neoplastic disease, particularly of the colon (13). Cooked muscle meats contain potent genotoxic carcinogens belonging to the heterocyclic aromatic amine (HAA) class of chemical compounds (31). Of the 19 heterocyclic amines identified so far, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most mass-abundant heterocyclic amine produced during the cooking of beef, pork, and chicken (15, 40). Experimentally, PhIP is a potent mutagen and genotoxin and has been shown to produce mammary gland, prostate, and colon tumors in rats (23, 39). For humans, less is known about the potential role of PhIP and related heterocyclic amines in tumor development. Several studies have shown that individuals who eat "well-done" meat have an increased risk of breast (52) and colorectal (18) cancers.To determine the potential health risks associated with heterocyclic amines, several dietary studies have been conducted on the metabolism and disposition of these compounds in humans. So far, most investigations have focused on the activation and ...

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Reclassification ofA polyphasic study revealed taxonomic heterogeneity among reference strains of the species Lactobacillus brevis. Representative strains of L. brevis and related taxa were investigated by partial sequence analysis of the housekeeping gene encoding the alpha-subunit of phenylalanyl-tRNA synthase (pheS). Species-specific clusters were delineated for all taxa studied except for two L. brevis strains, LMG 11494 and LMG 11984, respectively isolated from cheese and wheat, which occupied a distinct position. Their phylogenetic affiliation was determined using 16S rRNA gene sequence analysis and it was found that both strains (with 99?9 % gene sequence similarity between them) belonged to the Lactobacillus buchneri group, with nearest neighbours Lactobacillus hammesii and L. brevis (gene sequence similarities of 99?2 and 98?1 %, respectively). Further genotypic and phenotypic studies, including fluorescent amplified fragment length polymorphism, DNA-DNA hybridization and DNA G+C content, clearly demonstrated that the two strains represent a single novel taxon for which the name Lactobacillus parabrevis sp. nov. is proposed (type strain LMG 11984 T =ATCC 53295 T ).Lactobacillus brevis strains are frequently isolated from the spoilage microbiota in wine and beer fermentations, but also occur in fermented foods and feed such as sourdoughs, sour starch, cheeses, olives and silage (Stiles & Holzapfel, 1997). Identification of strains of L. brevis using conventional phenotypic methods often leads to ambiguous results (Kandler & Weiss, 1986;Pot et al., 1994). Molecular approaches have proved to be much more reliable (Vogel et al., 1994;Sohier et al., 1999;Guarneri et al., 2001) and have enabled 'L. brevis-like' isolates to be assigned to novel taxa such as Lactobacillus acidifarinae, Lactobacillus hammesii, Lactobacillus spicheri and Lactobacillus zymae (Meroth et al., 2004;Valcheva et al., 2005;Vancanneyt et al., 2005). In the present study, reference strains of L. brevis available from the BCCM/LMG Bacteria Collection (http://www.belspo. be/bccm/lmg.htm) were screened genotypically and the results demonstrated that two strains, LMG 11494 and LMG 11984, occupied a distinct position. Further genomic and phenotypic research revealed that these strains represent a single novel species.Strain LMG 11494 (=NCFB 1058) was isolated from farmhouse red Cheshire cheese and was originally deposited in the NCFB culture collection as L. brevis by A. Hayward in 1957. Strain LMG 11984 (=ATCC 53295) was isolated from wheat and deposited in the ATCC as L. brevis (originally named Sporolactobacillus sp.) by M. Spiller in 1992. It is a patent strain used for the production of leavening barm (Spiller, 1987). Both strains and related reference strains were cultivated and maintained on de Man, Rogosa and Sharpe (MRS) agar medium (pH 6?5;de Man et al., 1960) and incubated at 30 uC for 24-48 h, unless otherwise indicated.Sequence analysis of the phenylalanyl-tRNA synthase alphasubunit (pheS) housekeeping gene has been proved to be a...

Sourdough is a mixture of flour and water that is fermented with lactic acid bacteria (LAB) (Gänzle et al., 1998;Vogel et al., 1999). Sourdough fermentations improve dough properties, bread texture and flavour, retard the staling process and protect bread from mould and bacterial spoilage (Corsetti et al., 1998; Hammes & Gänzle, 1998;Rosenquist & Hansen, 1998). Because of their artisanal and regiondependent handling, sourdoughs are a huge source of diverse LAB species and strains.Lactobacillus brevis is one of the common taxa found in several sourdoughs such as artisanal Greek wheat sourdoughs (De Vuyst et al., 2002) and Belgian sourdoughs (our ongoing study). In the latter two surveys, most strains identified as L. brevis showed the characteristic taxonomic properties of the species. Three isolates were aberrant in their phenotypic and genotypic properties and were assigned as L. brevis-like. In the present study, their taxonomic position was investigated.The three strains studied were isolated on different occasions from three different artisanal wheat sourdoughs. One strain, ACA-DC 3411 t1 (=LMG 22199), was isolated in 1997 from Greek sourdough (De Vuyst et al., 2002). The strain was purified after suspension (1 : 10, w/v) and serial dilutions in saline (0?9 % NaCl, w/v), plating on MRS agar supplemented with 2 % (w/v) maltose and incubation at 30 uC for 48 h. Two further strains, LMG 22198 T and LMG 22200 T , were isolated in 2003 from Belgian sourdoughs. They were purified after suspension (1 : 10, w/v) and serial dilution in peptone/water (0?1 %, w/v), plating on MRS agar (pH 5?4) supplemented with 1 % (w/v) maltose and fructose and 0?1 % (w/v) cycloheximide and incubation at 37 u C for 48 h. Bacteriological purity of all isolates was checked by plating and examining living and Gram-stained cells. Cultivation conditions for further experiments and Abbreviations: AFLP, amplified fragment length polymorphism; LAB, lactic acid bacteria.