Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0 % for 16S rRNA gene clones and 64.3 % for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54 % of the total volatile fatty acids and methane was 57.20 % of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.     

Phylogenetic Comparison of the Methanogenic Communities from an Acidic, Oligotrophic Fen and an Anaerobic Digester Treating Municipal Wastewater Sludge

Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

Anaerobic cellulolytic microbial communities decomposing the biomass of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

Four previously isolated methanogenic anaerobic consortia, which were originally cultivated on a cellulose-containing substrate (filter paper), were used as inocula for the anaerobic conversion of the biomass of Anabaena variabilis into biogas at 55°C. The cumulative methane yield in the biogas produced by the most active consortia reached 64%. However, the biotransformation was only efficient in the course of the single inoculation and pretreatment of the cyanobacterial biomass by its concentration and freeze-thawing. The DGGE analysis of the structure of the selected microbial consortia, cultivated on the filter paper, revealed qualitative variations in the biodiversity of predominant Bacteria, showing differences in band number and intensity. The composition of methanogenic Archaea in these consortia was similar, with the presence of the genera Methanoculleus and Methanosarcina. The efficiency of the microbial consortia selection, and the role of the various microbial trophic groups in bioconversion of the substrates, such as cellulose and the biomass of phototrophic microorganisms are discussed.     

Enhanced Anaerobic Biodegradation of Benzene-Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

Syntrophic-Methanogenic Associations along a Nutrient Gradient in the Florida Everglades

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68:6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.     

Diversity of Benzylsuccinate Synthase-Like (bssA) Genes in Hydrocarbon-Polluted Marine Sediments Suggests Substrate-Dependent Clustering

The potential of hydrocarbon biodegradation in marine sediments was determined through the detection of a functional biomarker, the bssA gene, coding for benzylsuccinate synthase, the key enzyme of anaerobic toluene degradation. Eight bssA clone libraries (409 sequences) were constructed from polluted sediments affected by the Prestige oil spill in the Atlantic Islands National Park and from hydrocarbon-amended sediment microcosms in Mallorca. The amplified products and database-derived bssA-like sequences grouped into four major clusters, as determined by phylogenetic reconstruction, principal coordinate analysis (PCoA), and a subfamily prediction tool. In addition to the classical bssA sequences that were targeted, we were able to detect sequences homologous to the naphthylmethylsuccinate synthase gene (nmsA) and the alkylsuccinate synthase gene (assA), the bssA homologues for anaerobic 2-methylnaphthalene and alkane degradation, respectively. The detection of bssA-like variants was determined by the persistence and level of pollution in the marine samples. The observed level of gene diversity was lower in the Mallorca sediments, which were dominated by assA-like sequences. In contrast, the Atlantic Islands samples, which were highly contaminated with methylnaphthalene-rich crude oil, showed a high proportion of nmsA-like sequences. Some of the detected genes were phylogenetically related to Deltaproteobacteria communities, previously described as the predominant hydrocarbon degraders at these sites. Differences between all detected bssA-like genes described to date indicate separation between marine and terrestrial sequences and further subgrouping according to taxonomic affiliation. Global analysis suggested that bssA homologues appeared to cluster according to substrate specificity. We observed undetected divergent gene lineages of bssA homologues, which evidence the existence of new degrader groups in these environments.     

Molecular characterization of the nitrifying bacterial consortia employed for the activation of bioreactors used in brackish and marine aquaculture systems

The addition of commercial nitrifying bacterial products has resulted in significant improvement of nitrification efficiency in recirculating aquaculture systems (RAS). We developed two nitrifying bacterial consortia (NBC) from marine and brackish water as start up cultures for immobilizing commercialized nitrifying bioreactors for RAS. In the present study, the community compositions of the NBC were analyzed by universal 16S rRNA gene and bacterial amoA gene sequencing and fluorescence in situ hybridization (FISH). This study demonstrated that both the consortia involved autotrophic nitrifiers, denitrifiers as well as heterotrophs. Abundant taxa of the brackish water heterotrophic bacterial isolates were Paenibacillus and Beijerinckia spp. whereas in the marine consortia they were Flavobacterium, Cytophaga and Gramella species. The bacterial amoA clones were clustered together with high similarity to Nitrosomonas sp. and uncultured beta Proteobacteria. FISH analysis detected ammonia oxidizers belonging to β subclass of proteobacteria and Nitrosospira sp. in both the consortia, and Nitrosococcus mobilis lineage only in the brackish water consortium and the halophilic Nitrosomonas sp. only in the marine consortium. However, nitrite oxidizers, Nitrobacter sp. and phylum Nitrospira were detected in both the consortia. The metabolites from nitrifiers might have been used by heterotrophs as carbon and energy sources making the consortia a stable biofilm.     

Unusual bacterial populations observed in a full-scale municipal sludge digester affected by intermittent seawater inputs

This study investigated the bacterial community of a full-scale anaerobic digester, which suffers from intermittent seawater contaminations, using 16S rRNA gene clone analysis over different seasons. Bacteroidetes, Proteobacteria, and unclassifiable bacteria were the three major bacterial groups within the clone library (a total of 290 clones). A significant portion of the total clones (29.3%) was not affiliated to any previously reported phylum, and 55.3% of the unclassifiable clones (16.9% of the total clones) showed potential relations to the species of Thermotogae, rarely present under normal mesophilic anaerobic conditions. These results suggested that the novel populations may have the potential to play an important role in anaerobic processes, particularly under abnormal environmental conditions. Additionally, statistical analysis supported that seasonal variations in influent characteristics, and potential competitions among different populations, may be related to the unusual bacterial diversity and community dynamics observed over the study period.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Microbial Community Analysis of Anaerobic Reactors Treating Soft Drink Wastewater

The anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors containing methanogenic microbial consortia were applied to treat synthetic soft drink wastewater, which contains polyethylene glycol (PEG) and fructose as the primary constituents. The AP and HP reactors achieved high COD removal efficiency (>95%) after 80 and 33 days of the operation, respectively, and operated stably over 2 years. 16S rRNA gene pyrotag analyses on a total of 25 biofilm samples generated 98,057 reads, which were clustered into 2,882 operational taxonomic units (OTUs). Both AP and HP communities were predominated by Bacteroidetes, Chloroflexi, Firmicutes, and candidate phylum KSB3 that may degrade organic compound in wastewater treatment processes. Other OTUs related to uncharacterized Geobacter and Spirochaetes clades and candidate phylum GN04 were also detected at high abundance; however, their relationship to wastewater treatment has remained unclear. In particular, KSB3, GN04, Bacteroidetes, and Chloroflexi are consistently associated with the organic loading rate (OLR) increase to 1.5 g COD/L-d. Interestingly, KSB3 and GN04 dramatically decrease in both reactors after further OLR increase to 2.0 g COD/L-d. These results indicate that OLR strongly influences microbial community composition. This suggests that specific uncultivated taxa may take central roles in COD removal from soft drink wastewater depending on OLR.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Bacterial Diversity within Feedlot Manure

In this study, we identified the predominant culturable anaerobic bacteria and enumerated the total culturable anaerobic bacterial population present in samples of feedlot manure from Southern Queensland. Sixteen bacterial isolates were cultured from feedlot pad material with species of Lactobacillus, Clostridium and Bacillus predominating. From a library of 123 clones, produced by the amplification, cloning and partial DNA sequencing of the 16S rRNA gene, only 3% were closely related to previously described species and 21% to known genera. Of the total clone library, 96% were apparently Gram-positive and fell within families whose members were generally anaerobes. The majority (71%) of the clone library was related to either members of the family Clostridiaceae or lactic acid-producing bacteria (Lactobacillus or Lactosphaera). It was concluded that Gram-positive clostridial and lactic acid-producing bacteria predominate in feedlot pad manure. The overwhelming majority of species are novel and have not been obtained in culture. It would appear that the most likely source of the sickly-sweet nuisance odours (particularly from butyric acid) that emanate from feedlots is the by-product of anaerobic fermentation by clostridia. Gut-inhabiting and Gram-negative bacteria do not appear to survive for lengthy periods of time under the environmental conditions present in feedlot manure.     

Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda

The type III secretion system (T3SS) of Edwardsiella tarda plays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation of E. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface of E. tarda and is required for biofilm formation by E. tarda in Dulbecco''s modified Eagle''s medium (DMEM). Biofilm formation by E. tarda in DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody to E. tarda cultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody to E. tarda cultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation.     

Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME‐1 group

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate‐reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME‐1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82–90% of a composite ANME‐1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME‐1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron‐accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]‐hydrogenases are lacking in the dataset, genes for an [FeFe]‐hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c‐type cytochromes were identified, which have not yet been correlated with methanogenesis‐related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME‐1 to the bacterial sulfate‐reducing partner.     

Archaeal community compositions at different depths (up to 30 m) of a municipal solid waste landfill in Taiwan as revealed by 16S rDNA cloning analyses

Archaeal 16S rDNA clone libraries were constructed for samples taken at 10, 20 and 30 m depth in a landfill, which corresponded approximately 3, 6 and 9 years operation, respectively. Sequencing and phylogenetic analyses of representative clones showed that all of the rDNAs were closely related to typical methanogens. The distributions of phylotypes in clone libraries were similar to each other. Dominant clones in all the clone libraries were closely related to thermophilic species, such as Methanothermobacter thermautotrophicus, suggesting that the temperatures at these sites were high. This was supported by the results of H₂-dependent methanogenic activity tests, showing that the activities of all samples at 55 °C were much higher than those at 25 °C.     

Diversity of the Microeukaryotic Community in Sulfide-Rich Zodletone Spring (Oklahoma)

The microeukaryotic community in Zodletone Spring, a predominantly anaerobic sulfide and sulfur-rich spring, was examined using an 18S rRNA gene cloning and sequencing approach. The majority of the 288 clones sequenced from three different locations at Zodletone Spring belonged to the Stramenopiles, Alveolata, and Fungi, with members of the phylum Cercozoa, order Diplomonadida, and family Jakobidae representing a minor fraction of the clone library. No sequences suggesting the presence of novel kingdom level diversity were detected in any of the three libraries. A large fraction of stramenopile clones encountered were monophyletic with either members of the genus Cafeteria (order Bicosoecida) or members of the order Labyrinthulida (slime nets), both of which have so far been encountered mainly in marine habitats. The majority of the observed fungal clone sequences belonged to the ascomycetous yeasts (order Saccharomycetales), were closely related to yeast genera within the Hymenobasidiomycetes (phylum Basidiomycetes), or formed a novel fungal lineage with several previously published or database-deposited clones. To determine whether the unexpected abundance of fungal sequences in Zodletone Spring clone libraries represents a general pattern in anaerobic habitats, we generated three clone libraries from three different anaerobic settings (anaerobic sewage digester, pond sediment, and hydrocarbon-exposed aquifer sediments) and partially sequenced 210 of these clones. Phylogenetic analysis indicated that clone sequences belonging to the kingdom Fungi represent a significant fraction of all three clone libraries, an observation confirmed by phospholipid fatty acid and ergosterol analysis. Overall, this work reveals an unexpected abundance of Fungi in anaerobic habitats, describes a novel, yet-uncultured group of Fungi that appears to be widespread in anaerobic habitats, and indicates that several of the previously considered marine protists could also occur in nonmarine habitats.     

Microbial Diversity in the Deep Marine Sediments from the Qiongdongnan Basin in South China Sea

The continental shelf and slope in the northern South China Sea is well known for its prospect of oil/gas/gas-hydrate resources. To study microbial communities and their roles in carbon cycling, a 4.9-m sediment core was collected from the Qiongdongnan Basin on the continental slope of the South China Sea during our cruise HY4-2005-5 in 2005. Geochemical, mineralogical, and molecular phylogenetic analyses were carried out. Sulfate concentration in pore water decreased with depth. Abundant authigenic carbonates and pyrite were observed in the sediments. The bacterial community was dominated by aerobic and facultative organisms. Bacterial clone sequences belonged to the Gamma-, Alpha-, Deltaproteobacteria and Firmicutes group, and they were related to Fe(III) and/or Mn(IV) reducers, sulfate reducers, aromatic hydrocarbon degraders, thiosulfate/sulfite oxidizers, and denitrifiers. Archaeal clone sequences exhibited greater overall diversity than the bacterial clones with most sequences related to Deep-Sea Archaeal Group (DSAG), Miscellaneous Crenarchaeotic Group (MCG), and Uncultured Euryarchaeotic Clusters (UECs). Archaeal sequences related to Methanosarcinales, South African Gold Mine Euryarchaeotic Group (SAGMEG), Marine Benthic Group-D (MBG-D) were also present. Most of these groups are commonly present in deep-sea sediments, particularly in methane/organic-rich or putative methane hydrate-bearing sediments.     

Molecular Typing of Aeromonas Isolates in Natural Mineral Waters

A total of 103 isolates of Aeromonas spp. were obtained over a 3-year period from a natural mineral water and from surface streams located within the boundaries of the watershed of the natural mineral water wells and were typed by macrorestriction analysis of genomic DNA with XbaI and by pulsed-field gel electrophoresis. All Aeromonas caviae isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among Aeromonas hydrophila isolates. These two clones expressed no hemolytic or cytotoxic activities. Aeromonas isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water. The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer. Molecular fingerprinting of drinking water isolates is a useful tool in explaining possible reasons for bacterial occurrences.