Isolation and characterization of aerobic heterotrophic bacteria from natural spring waters in the Lanjaron area (Spain)

Aerobic, heterotrophic bacteria were isolated from nine natural mineral water springs in the Lanjaron area of Spain over the period July 1980 to May 1981. The mineral waters contained few bacteria (mean counts 26-5275 cfu per 100 ml) and the bacterial flora of all nine springs was very similar. Most of the isolates were Gram-negative rods (90%), and among these Pseudomonas spp. and members of the Flavobacterium-Cytophaga-Flexibacter group were numerically dominant. Aeromonas-Vibrio and Enterobacteriaceae isolates were an important fraction of the total number, but isolates from remaining groups (Acinetobacter, Chromobacterium, Alcaligenes and Gram-positive organisms) constituted only a small proportion of the flora. The comparatively small number of species isolated, and the occurrence of no more than three or four different bacterial types in spring water of different chemical and physical composition is discussed.     

Study of the bacterial flora of a non-carbonated natural mineral water

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the prebottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Study of the bacterial flora of a non-carbonated natural mineral water.

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the pre-bottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.     

Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia.

The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperature of 55-56 degrees C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-microm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH.     

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.     

Ribotyping of Pseudomonas aeruginosa isolates from patients and water springs and genome fingerprinting of variants concerning mucoidy

Abstract Thirty-one strains of Pseudomonas aeruginosa , isolated from water springs, clinical isolates (some of which were from cystic fibrosis (CF) patients), and two type cultures, were characterized by ribotyping. After restriction of chromosomal DNA of the different isolates with Eco RI and hybridization of Southern transfer blots with 2-acetylaminofluorene labelled Escherichia coli 16S + 23S rRNA probe, eleven different ribopatterns were obtained, representing variations of a dominant profile. This largely predominant pattern included both type cultures, all six isolates from water springs, 33% of the nine CF isolates and 43% of fourteen other clinical isolates most of them from nosocomial infections. When the genomic macrorestriction fingerprints of three mucoid CF isolates, with Ase I, Dra I or Bfr I were compared with those of their spontaneous variants, concerning mucoidy, no differences were detected.     

Presence of Thermophilic Bacteria in Laundry and Domestic Hot-Water Heaters

Thermophilic bacteria resembling Thermus aquaticus were isolated from hot water taken from domestic and commercial hot-water tanks. Cold water from the same locations never yielded thermophilic bacteria, suggesting that the bacteria were growing in the tanks. In contrast to the T. aquaticus isolates from hot springs, the present isolates were rarely pigmented. In general, the hotter sources more frequently yielded bacteria.     

Assessment of inhibitory effects of fluoride-coated tubes on biofilm formation by using the in vitro dental unit waterline biofilm model

This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed. Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (P < 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.     

The Bacterial Flora of some Queensland Fish and its Ability to cause Spoilage

The bacterial flora were determined qualitatively and quantitatively on samples taken at various stages of handling several species of fish of commercial importance in Queensland. There was an overall increase in the number of bacteria during handling and processing; both the composition and quantity of the bacterial flora of individual samples taken at each stage of handling varied widely. Members of the genus Micrococcus formed the major proportion of the flora of freshly caught fish. Pseudomonas and Moraxella spp. were predominant amongst the bacterial flora able to grow at 2° and constituted the bulk of the population in samples with high bacterial counts. This psychrophilic population was markedly reduced at the filleting stage. A medium prepared by the action of trypsin on a fish muscle homogenate was used to test bacterial isolates for their ability to produce odours. Forty-three per cent of the pseudomonad isolates produced sulphydryl odours at 5°. Only small proportions of the other groups produced detectable odours. Members of the genus Pseudomonas were considered the most important fish spoilage bacteria under the conditions found in Queensland.     

Aerobic Oral Bacteria in Healthy Captive Snakes

Cotton swab samples were taken from the ventral surface of the mouth and from the proximal esophagus from 23 captive nonpoisonous snakes. The samples were cultured and investigated for aerobic bacteria. Both the mouth and the esophagus) samples of 6 snakes were negative. When the bacterial isolates of the mouth and the esophagus of the whole snake population were compared, it was found that the flora isolated from both locations were similar. However, when the samples of individual snakes were compared it was found that the same isolates were seldom found in both the mouth and the esophagus. The most common bacteria found were Pseudomonas sp., Alcaligenes-like organisms, Gram-positive rods and Gram-positive cocci belonging to the family Micrococcaceae. Important pathogens were seldom isolated. Salmonella virchow could be found from 2 snakes. The presence of bacteriologically negative samples, great variations in the composition of the flora between individual snakes, and the occurrence of typical environmental bacteria in the oral cavity all suggest that snakes lack a specific autochtonous flora: and the bacteria isolated from the oral cavity may be occasional environmental bacteria. The source of pathogens may be the environment, too.     

Assessment of Microbial Diversity in Saudi Springs by Culture-Dependent and Culture-Independent Methods

Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in two hot springs of the Aljouf region in Saudi Arabia, including Qasr Kaff and Ain Hawas. Physicochemical characteristics of the springs were examined to establish their effect on the biodiversity of thermophilic bacteria and fungi. We employed culture-dependent techniques to study microbial diversity using four different complex media for bacteria and fungi. In addition, the direct count for algal populations from two springs was investigated. We surveyed the microbial diversity in water and sediment samples from both springs by denaturing gradient gel electrophoresis (DGGE) and clone library construction. Bacillariophycaea (18 species) was the most diverse group, followed by Cyanophyceaea. Bacterial isolates closer to the genera Bacillus spp., Geobacillus, Thermoactinomyces, and unidentified actinobacteria were recovered. Fungal isolates were related to Aspergillus, Pezizaceae, Penicillium, Acremonium, Fusarium, Chrysosporium, and Stachybotrys. Using molecular-based techniques, the results were slightly different from those obtained by culture-dependent methods, and more genera were obtained. However, most genera were uncultured microbes, particularly from bacterial communities.     

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.)

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.     

Consumption of Organic Carbon Sources and Biosynthesis of Lactic Acid by the Photosynthetic BacteriumRhodobactersp. D-4

Nonsulfur photosynthetic purple bacteria isolated from the Dzhermuk mineral springs (Armenia) were grown on sugar-containing media and found to be capable of synthesizing L(+)-lactic acid. Various organic compounds were tested as possible sole sources of carbon and an electron donors required to support bacterial growth and biosynthesis of lactic acid under various growth conditions.     

Consumption of organic carbon sources and biosynthesis of lactic acid by the photosynthetic bacterium Rhodobacter sp. D-4

Nonsulfur photosynthetic purple bacteria isolated from the Dzhermuk mineral springs (Armenia) were grown on sugar-containing media and found to be capable of synthesizing L(+)-lactic acid. Various organic compounds were tested as possible sole sources of carbon and an electron donors required to support bacterial growth and biosynthesis of lactic acid under various growth conditions.     

Heterotrophic and autotrophic microbial populations in cold perennial springs of the high arctic

The saline springs of Gypsum Hill in the Canadian high Arctic are a rare example of cold springs originating from deep groundwater and rising to the surface through thick permafrost. The heterotrophic bacteria and autotrophic sulfur-oxidizing bacteria (up to 40% of the total microbial community) isolated from the spring waters and sediments were classified into four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) based on 16S rRNA gene analysis; heterotrophic isolates were primarily psychrotolerant, salt-tolerant, facultative anaerobes. Some of the isolates contained genes for thiosulfate oxidation (soxB) and anoxygenic photosynthesis (pufM), possibly enabling the strains to better compete in these sulfur-rich environments subject to long periods of illumination in the Arctic summer. Although leucine uptake by the spring water microbial community was low, CO(2) uptake was relatively high under dark incubation, reinforcing the idea that primary production by chemoautotrophs is an important process in the springs. The small amounts of hydrocarbons in gases exsolving from the springs (0.38 to 0.51% CH(4)) were compositionally and isotopically consistent with microbial methanogenesis and possible methanotrophy. Anaerobic heterotrophic sulfur oxidation and aerobic autotrophic sulfur oxidation activities were demonstrated in sediment slurries. Overall, our results describe an active microbial community capable of sustainability in an extreme environment that experiences prolonged periods of continuous light or darkness, low temperatures, and moderate salinity, where life seems to rely on chemolithoautotrophy.     

Genomic and Functional Diversity of Bacteria Isolated from Hot Springs in Odisha,India

This study focused on culture-dependent survey of important bacterial community diversity of hot springs of Odisha. India. Molecular and cultural techniques were employed for assessing and exploiting the genetic and functional variability among the isolates obtained from three alkaline and mesophilic hot springs. A total of 48 isolates belonging to family Bacillaceae, Paenibacillaceae, Planococcaceae, Pseudomonadaceae and Enterobacteriaceae were identified. Majority of the bacterial isolates were affiliated with the genus Bacillus. Morphologically all the isolated bacteria were either Gram-positive spore-forming rods, or Gram-negative rods. The optimum temperature for growth of the isolates varied between 37°C to 50°C. The functional diversity revealed that many of the predominant and scarce isolates produced a variety of extracellular enzymes such as amylase, cellulase, lipase, phosphatase and protease, and genus Bacillus dominated for extracellular enzymatic activity. We employed two molecular markers to characterize the isolates. The hsp60 universal target sequence was found to be more discriminatory than 16s rRNA gene sequences. The cultivable bacterial community structure that colonized in the investigated thermal springs did not reveal much overlapping. Our results indicate that bacteria in the geothermal environment are metabolically active and cultivable populations may have great potential in biotechnology.     

Some aspects of the gastrointestinal microflora of germfree mice associated with cultured microfloras

Attempts are described to 'normalize' germfree mice by association with 3, 21 and 71 different intestinal bacterial cultures isolated from mice with an SPF flora. Germfree mice associated naturally with an SPF flora served as controls. Vital bacterial counts were determined by aerobic and anaerobic culture. Stomach and small intestine contained fewer bacteria per gram than caecum and large intestine. Aerobic vital counts from caecum and large intestine were higher in the experimental groups than in control mice. The aerobic and anaerobic flora in stomach and small intestine comprised mainly Gram-positive non-fusiform shaped rods. In the caecum and colon Gram-positive cocci predominated in the aerobic culture while in the anaerobic culture fusiform-shaped rods were prominent. Scanning electron microscopy of oesophagus, ileum, caecum and faeces demonstrated colonization of the oesophageal epithelium only after association with 71 bacterial strains; the filamentous bacteria present in the ileum of SPF mice were not found in the experimental groups and caecum and faeces contained mainly fusiform-shaped bacteria. Non-bacterial matter decreased in the caecum and faeces with increase in the complexity of the flora.     

Opportunistic Pathogens and Elements of the Resistome that Are Common in Bottled Mineral Water Support the Need for Continuous Surveillance

Several differences concerning bacterial species, opportunistic pathogens, elements of the resistome as well as variations concerning the CFU/mL counts were identified in some of the five most marketed bottled mineral water from Araraquara city, São Paulo, Brazil. Two out of five brands tested were confirmed as potential source of opportunistic pathogens, including Mycobacterium gordonae, Ralstonia picketti and Burkholderia cepacia complex (Bcc). A total of one hundred and six isolates were recovered from four of these bottled mineral water brands. Betaproteobacteria was predominant followed by Alphaproteobacteria, Gammaproteobacteria and Firmicutes. Ninety percent of the bacteria isolated demonstrated resistance to seventeen of the nineteen antimicrobials tested. These antimicrobials included eight different classes, including 3rd and 4th generation cephalosporins, carbapenems and fluoroquinolones. Multidrug resistant bacteria were detected for fifty-nine percent of isolates in three water brands at counts up to 10³ CFU/ml. Of major concern, the two bottled mineral water harboring opportunistic pathogens were also source of elements of the resistome that could be directly transferred to humans. All these differences found among brands highlight the need for continuous bacteriological surveillance of bottled mineral water.