Characteristics of some fermentative bacteria from a thermophilic methane-producing fermenter

Anaerobic bacteria from a 55C methane-producing beef waste fermenter were enumerated, isolated, and characterized. Direct microscopic bacterial counts were 5.2–6.8×10¹⁰ per g fermenter effluent. Using a nonselective roll-tube medium which contained 40% fermenter effluent, 8.5–14.1% of the microscopic count was culturable. Deletion of fermenter effluent significantly reduced the viable count. Sixty-four randomly picked strains were characterized. All were pleomorphic, gram-negative, anaerobic rods, many of which were difficult to grow in liquid media. The strains were divided into 5 major groups based on glucose fermentation, hydrogen sulfide production, starch hydrolysis, fermentation products, and morphology. Glucose was fermented by 75% of the isolates, 76% utilized starch, 25% produced hydrogen sulfide, 76% produced hydrogen, 37% produced indole, 21% hydrolyzed gelatin, and 13% were sporeformers. Ethanol, lactate, formate, acetate, and hydrogen were common fermentation products. Twenty-four representative strains had 1–12 flagella. Growth was observed between 35 and 73C. These studies indicate that species diversity among the isolated organisms was low.     

Incidence of Plasmids in Marine Vibrio spp. Isolated from an Oil Field in the Northwestern Gulf of Mexico

Presumptive marine Vibrio spp. were collected from an operational oil field and control site located in the northwestern Gulf of Mexico. Of 440 isolates analyzed for the presence of extrachromosomal deoxyribonucleic acid elements or plasmids by using the cleared lysate and agarose gel techniques, 31% showed distinct plasmid bands on agarose gels. A majority of the plasmids detected were estimated to have molecular masses of 10 × 10⁶ or less. Multiple plasmids were observed in approximately half of the plasmid-containing strains. A number of isolates contained plasmids with similar banding and mobility patterns. The oil field area had noticeably more plasmid-containing strains (35 versus 23% in the control site) and a greater number of plasmids per plasmid-containing strain (an average of 2.5 plasmids, versus 1.5 in the control site). Oil field discharges might have resulted in increased plasmid incidence and diversity.     

Characterization of some anaerobic bacteria from the liquid phase of a mesophilic anaerobic digester fed with a prefermented cheese whey substrate

Bacterial counts on the contents of an anaerobic fixed-bed digester receiving a whey substrate were conducted using the modified roll tube technique. Average anaerobic counts after 48 hours incubation on lactate containing media were 3.12 × 10⁹ and 3.7 × 10⁹ ml^–1, respectively. These counts were between 140 and 190 times higher than aerobic counts on the same media. Seventy-four strains from both media were isolated and characterized, and the relationship between the organisms was calculated according to the similarity coefficient of Sokal and Michener [20]. The organisms were clustered using the unweighted pair group method and the results were presented in the form of a simplified dendrogram. The isolates clustered in three major groups (A, B, and C) at a similarity level of 76%. A small diffuse group of five organisms was also found. The organisms in two (A and B) of the major clusters were obligate anaerobes. Cluster A represented 34% of the isolates, cluster B represented 50%, and the facultative streptococci in cluster C, 11%. The isolates in clusters A and B could only tentatively be identified as members of the generaBacteroides andPeptostreptococcus, respectively. The isolates could not be positively identified.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates ( n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant ( P > 0.05). There was a significant difference ( P > 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates (n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant (P greater than 0.05). There was a significant difference (P less than 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster (n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster ( n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

Genetic Variation of the Bloom-Forming Cyanobacterium Microcystis aeruginosa within and among Lakes: Implications for Harmful Algal Blooms

To measure genetic variation within and among populations of the bloom-forming cyanobacterium Microcystis aeruginosa, we surveyed a suite of lakes in the southern peninsula of Michigan that vary in productivity (total phosphorus concentrations of ~10 to 100 μg liter⁻¹). Survival of M. aeruginosa isolates from lakes was relatively low (i.e., mean of 7% and maximum of 30%) and positively related to lake total phosphorus concentration (P = 0.014, r² = 0.407, n = 14). In another study (D. F. Raikow, O. Sarnelle, A. E. Wilson, and S. K. Hamilton, Limnol. Oceanogr. 49:482-487, 2004), survival rates of M. aeruginosa isolates collected from an oligotrophic lake (total phosphorus of ~10 μg liter⁻¹ and dissolved inorganic nitrogen:total phosphorus ratio of 12.75) differed among five different medium types (G test, P of <0.001), with higher survival (P = 0.003) in low-nutrient media (28 to 37% survival) than in high-nutrient media. Even with the relatively low isolate survivorship that could select against detecting the full range of genetic variation, populations of M. aeruginosa were genetically diverse within and among lakes (by analysis of molecular variance, Φ_sc = 0.412 [Φ_sc is an F-statistic derivative which evaluates the correlation of haplotypic diversity within populations relative to the haplotypic diversity among all sampled populations], P = 0.001), with most clones being distantly related to clones collected from lakes directly attached to Lake Michigan (a Laurentian Great Lake) and culture collection strains collected from Canada, Scotland, and South Africa. Ninety-one percent of the 53 genetically unique M. aeruginosa clones contained the microcystin toxin gene (mcyA). Genotypes with the toxin gene were found in all lakes, while four lakes harbored both genotypes possessing and genotypes lacking the toxin gene.     

Characterization of aerobic and facultative anaerobic bacteria from the liquid phase of an anaerobic fixed-bed digester treating a cheese whey substrate

Bacterial counts on the liquid phase of an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted on two different media under aerobic and facultative conditions. Average counts of 16.6×10⁶ and 26.5×10⁶ ml^–1 were obtained on the two media, with the nutritionally poorer of the two media giving the highest average count. Seventy-five isolates from both media, incubated aerobically as well as in anaerobic jars, were obtained. These isolates as well as 13 reference strains were phenotypically characterized. The similarities between cultures were calculated using the similarity coefficient of Sokal and Michener [16]. The organisms were clustered using the unweighted pair group method, and the results presented as a simplified dendrogram. The isolates could be divided into 3 main groups: gram-negative fermentative rods, mainlyEnterobacter, Klebsiella, andCitrobacter, withKlebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods of the generaPseudomonas, Alcaligenes, andAcinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.     

Characterization of Subsurface Bacteria Associated with Two Shallow Aquifers in Oklahoma

The bacterial microflora of two shallow aquifers (saturated subsurface zones) in Oklahoma was characterized by direct observation with light and electron microscopy, by plating, and by examination of colony morphology and distribution. Isolated bacterial strains were also examined. Total cell counts varied only slightly (2.9 × 10⁶ to 9.8 × 10⁶ g [dry wt]⁻¹) from sample to sample, whereas colony counts varied widely (6.3 × 10² to 6.5 × 10⁶ CFU g [dry wt]⁻¹). Colony counts on nutritionally rich media were lower than on low-nutrient media, especially in samples from the saturated zone. The variety of colony types growing on nutritionally rich media decreased with increasing depth and saturation. Colony counts of anaerobic bacteria also decreased with depth but were at least 100-fold lower than aerobic counts on most media. Cell morphologies of bacteria grown aerobically on plates included short rods, cocci, and actinomycete-like forms. Direct light microscopic observation of sediments revealed short, rod-shaped, and coccoid bacterial cells; endospores, actinomycete spores, and eucaryotic forms were not observed by light microscopy. Electron microscopic observation of bacteria released from the samples revealed that 85 to 90% of them were coccoid, gram-positive, Arthrobacter-like organisms, some of which were dividing or contained completed division septa; other types of gram-positive and gram-negative bacteria were present in lower numbers. Isolated bacterial strains were able to grow on both nutritionally rich and low-nutrient media. A higher proportion of gram-negative organisms was isolated than gram-positive organisms. Most of the isolates were capable of storing polyphosphate, poly-β-hydroxybutyrate, or polysaccharide. The results of this study suggest that the microbial population of these two shallow aquifers is dominated by aerobic, nutritionally versatile bacteria that can subsist on low concentrations of organic compounds without forming specialized resting cells. Other types of microorganisms, such as facultatively anaerobic bacteria and microeucaryotes, may also be present, but they represent only a small fraction of the microflora.     

Cultivation and Growth Characteristics of a Diverse Group of Oligotrophic Marine Gammaproteobacteria

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day⁻¹), and none of them grew in high-nutrient media (>351 mg of C liter⁻¹). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 μm³). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Enterotoxigenicity and Genetic Relatedness of Clostridium perfringens Isolates from Retail Foods in the United States

Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45°C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa. None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe⁺ strains in retail foods and the genetic diversity among nonoutbreak strains.     

Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria.

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.     

Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.     

Physiological Diversity and Distributions of Heterotrophic Bacteria in Deep Cretaceous Sediments of the Atlantic Coastal Plain

A series of 23 intact core segments was obtained from two distinct deep subsurface geological formations, the Middendorf and the Cape Fear formations, underlying the southeastern coastal plain of South Carolina. The Middendorf formation in this region consists of permeable, saturated, sandy sediments; the Cape Fear formation consists mainly of less permeable sediments. The core segments were separated by vertical distances ranging from several centimeters to 48 m. Aerobic chemoheterotrophic bacteria were enumerated on a dilute medium, and populations ranged from 3.1 to 6.4 log CFU g of sediment^-1 in the Middendorf cores and from below detection to 4.3 log CFU g^-1 in the Cape Fear cores. A total of 198 morphologically distinct colony types were isolated, purified, and subjected to 108 different physiological measurements. The isolates from the two formations were distinct (i.e., they produced substantially different response patterns to the various physiological measurements), as were those in different core samples from the same formation. Cluster analysis revealed 21 different biotypes based on similarities of 75% or higher in response patterns to 21 physiological assays. One biotype contained 57 (29%) of the subsurface isolates, 10 biotypes contained 5 or more isolates, and the remainder had 4 or fewer. The organic compounds that were most commonly metabolized by the subsurface bacteria included Tween 40 (85%) and β-hydroxybutyric acid (60%). Organic acids, in general, were also commonly metabolized by the subsurface bacteria. Isolates from the Cape Fear core segments were capable of metabolizing a higher percentage of the substrates than were bacteria isolated from the Middendorf formation. Although the heterogeneous distributions of bacteria in deep subsurface sediments may make it difficult to use aquifer microcosms to predict in situ biotransformation rates, the diversity of the physiological properties of these organisms offers promise for in situ remediation of contaminants.     

Occurrence and Multiple Antibiotic Resistance Profiles of Non-fermentative Gram-Negative Microflora in Five Brands of Non-carbonated French Bottled Spring Water

Abstract Five brands of French bottled mineral water were analyzed by heterotrophic plate counts (HPC) and for the presence of multiple antibiotic resistant bacteria. HPC at 22°C were around 10⁴ colony forming units ml^-1 on R2A medium. Enumeration on PCA/10, MH, and especially PCA and King B media was less efficient. At 37°C, HPC were two to three orders of magnitude less than at 22°C. Moreover, phenotypic diversity (7 to 15 phenotypes) was optimal on R2A incubated at 22°C. All isolates were identified as non-fermentative Gram-negative rods and 75% were non-identifiable with the API 20NE system. Stenotrophomonas maltophilia and fluorescent Pseudomonas were isolated on VIA and CFC selective agar media, respectively. Burkholderia cepacia strains were not isolated on BCSA medium. The species S. maltophilia was found in 33%, 28%, and 11% of sample from springs A, D, and E, respectively. Independent of brand, isolates from HPC media were less efficient to achieve confluent growth in 18 h on MH at 30 or 37°C (0 to 40%) than isolates from selective media (28 to 63%). Seventy percent of the total isolates from dominant microflora (1–5 2 10³ CFU ml^-1 on HPC media) were resistant against two or four antibiotics. The antibiotics concerned were principally aztreonam, ampicillin, and nalidixic acid. The remaining dominant bacteria showed a 6–9 multiple antibiotic resistant (MAR) pattern. All isolates were susceptible to newer antimicrobial agents. Owing to their low nutrient and temperature requirements, these isolates are unlikely to cause concern to public heath. Fifty percent of strains isolated from selective media (non-dominant microflora, 4–40 CFU l^-1) showed a 10–18 MAR pattern and 33%, identified as S. maltophilia, a 20–27 MAR pattern. However, minocycline was effective against all isolates. Owing to its low concentration, colonization of human intestine by MAR S. maltophilia is unlikely.     

Cultivable Bacterial Diversity of Alkaline Lonar Lake, India

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 10²–10⁶ cfu g⁻¹ and 10²–10⁴ cfu ml⁻¹, respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to γ-Proteobacteria, Alcaligenes to β-Proteobacteria and Paracoccus to α-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.