Virulence and Antibiotic Resistance Characteristics of Vibrio Isolates From Rustic Environmental Freshwaters.

The study investigated the occurrence of antimicrobial resistance genes and virulence determinants in Vibrio species recovered from different freshwater sheds in rustic milieu. A total of 118 Vibrio isolates comprising Vibrio fluvialis (n=41), Vibrio mimicus (n=40) and V. vulnificus (n=37) was identified by amplification of ToxR, vmh and hsp60 genes. The amplification of virulence genes indicated that V. mimicus (toxR, zot, ctx, VPI, and ompU) genes were detected in 12.5%, 32.5%, 45%, 37.5% and 10% respectively. V. fluvialis genes (stn, hupO and vfh) were harboured in 48.8%, 14.6% and 19.5% isolates congruently. The other virulence genes that include vcgC and vcgE were observed in 63.1% and 29% of isolates belonging to V. vulnificus. With the exceptions of imipenem, meropenem and ciprofloxacin, most isolates exhibited more than 50% resistance to antibiotics. The antimicrobial resistance was more prevalent for polymyxin B (100%), azithromycin (100%) and least in ciprofloxacin (16.1%). Multiple antibiotic resistance index range was 0.3 and 0.8 with most isolates showing MARI of 0.8. The blaTEM, AmpC, blaGES, blaIMP, blaOXA-48 and blaKPC genes were detected in 53.3%, 42%, 29.6%, 16.6%, 15%, 11.3% and 5.6% of the isolates. Non-beta lactamases such as streptomycin resistance (aadA and strA), gentamicin resistance (aphA1) and quinolone resistance gene (qnrVC) were found in 5.2%, 44.3%, 26% and 2.8%. Chloramphenicol resistance genes (cmlA1 and catII) were found in 5.2% and 44.3% among the isolates. Our findings reveal the presence of antimicrobial resistance genes and virulent Vibrio species in aquatic environment which can have potential risk to human and animal's health.

Relationships Between Organic Matter Contents and Bacterial Hydrolytic Enzyme Activities in Soils: Comparisons Between Seasons.

Microbial assemblages in terrestrial environments, such as soils, utilize hydrolytic enzymes to function biologically in various environments including for the degradation of organic carbon compounds and cycling of nutrients that eventually contributes to the ecological and agricultural productivity of such environments. In this study, 3 soil types (i.e., sandy, loamy and clayey) with varying characteristics were collected within the premises of Albion College in Michigan, with the goal of comparing the occurrences of indigenous bacterial populations and their respective hydrolytic enzyme activities in the soils. The soils were examined for their organic matter content (% OM), while bacterial abundance was determined by combinations of viable counts and nucleic acid staining, and enzymatic activities measured using fluorescein diacetate (FDA) analysis. Results from the study showed loamy soil to have a significantly higher % OM at 30% on average as compared to 2.5% and 6.6% recorded in the sandy and clayey soils. Comparatively, bacterial numbers (both viable and total counts) were also significantly higher in loamy soils than the other two soils. The same trend was observed for FDA analysis with higher fluorescein released in the loamy soil relative to the two other soils. Overall, clear differences were observed in the relationships between % OM and bacterial numbers and hydrolytic enzyme activities among the three soil types and between the two seasons examined. The results suggest that % OM strongly influences both bacterial abundance and hydrolytic enzyme activities in loamy soil and less so in both sandy and clayey soils examined in the study. This study in conclusion revealed potential strong relationships between soil organic carbon and indigenous bacterial populations as well as their FDA activities in various soil types.

Effects of iron and copper treatments on the bacterioplankton assemblages from surface waters along the north branch of the Kalamazoo River, Michigan, USA.

The effects of varying concentrations (ranging from 0 to 10 µM) of two different metals that is, iron (Fe) and copper (Cu) on indigenous bacterial populations and their hydrolytic enzyme activities within the bacterioplankton assemblages from the surface waters of the Kalamazoo River were examined under controlled microcosm conditions. The two metals were added to water samples collected from the Kalamazoo River and examined for bacterial abundance and leucine aminopeptidase activities at various time intervals over a 48 h incubation period in the dark. Results revealed no concentration effects on the bacterial populations in the presence of both Fe and Cu, although the bacterial numbers varied significantly over time in both microcosms. Conversely, leucine aminopeptidase activities based on post-hoc tests using Bonferroni correction revealed significant differences to increasing concentrations of both metals over the study period. These results further validate previous knowledge regarding the importance of various metal ions in regulating bacterial community structures and also suggest that aminopeptidase have the potential of effectively functioning using diverse trace and heavy metals as extracellular peptidase cofactors in aquatic systems.

Community Composition and Diversity of Coastal Bacterioplankton Assemblages in Lakes Michigan, Erie, and Huron.

The Laurentian Great Lakes, including Lakes Superior, Michigan, Huron, Erie, and Ontario, located in the eastern part of North America are considered the largest of freshwater lakes in the world; however, very little is known about the diversity and distribution of indigenous microbial assemblages within these vast bodies of freshwater systems. Therefore, to delineate the microbial structure and community composition in these aquatic environments, combinations of high-throughput sequencing and fluorescent in situ hybridization (FISH) approaches were utilized to quantitatively characterize the occurrence, diversity, and distribution of bacterioplankton assemblages in six different sites located along the coastal regions of Lakes Michigan, Huron, and Erie. Phylogenetic examination showed a diverse bacterial community belonging to 11 different taxonomic groups. Pyrosequencing results revealed that the majority of the sequences were clustered into four main groups, i.e., Proteobacteria, Bacteriodetes, Actinobacteria, and Cyanobacteria, while fluorescent in situ hybridization also showed the numerical dominance of members of the Gammaproteobacteria and the Cytophaga-Flavobacterium in the six lake sites examined. Overall, the assemblages were shown to be quite diverse in distribution among the lake sites examined, comprising mostly of various heterotrophic populations, with the exception of the Lake Erie-Sandusky Bay site with more than 50% domination by autotrophic Cyanobacteria. This indicates that combinations of factors including water chemistry and various anthropogenic disturbances as well as the lake morphometric characteristics are probably influencing the community structure and diversity of the bacterial assemblages within the systems.

Influence of zinc on bacterial populations and their proteolytic enzyme activities in freshwater environments: a cross-site comparison.

Temporal responses of indigenous bacterial populations and proteolytic enzyme (i.e., aminopeptidase) activities in the bacterioplankton assemblages from 3 separate freshwater environments were examined after exposure to various zinc (Zn) concentrations under controlled microcosm conditions. Zn concentrations (ranging from 0 to 10 µmol/L) were added to water samples collected from the Kalamazoo River, Rice Creek, and Huron River and examined for bacterial abundance and aminopeptidase activities at various time intervals over a 48 h incubation period in the dark. The results showed that the Zn concentrations did not significantly influence total bacterial counts directly; however, aminopeptidase activities varied significantly to increasing zinc treatments over time. Also, analysis of variance and linear regression analyses revealed significant positive relationships between bacterial numbers and their hydrolytic enzyme activities, suggesting that both probably co-vary with increasing Zn concentrations in aquatic systems. The results from this study serve as additional evidence of the ecological role of Zn as an extracellular peptidase cofactor on the dynamics of bacterial assemblages in aquatic environments.

Isolation, characterization and community diversity of indigenous putative toluene-degrading bacterial populations with catechol-2,3-dioxygenase genes in contaminated soils.

Indigenous bacterial assemblages with putative hydrocarbon-degrading capabilities were isolated, characterized and screened for the presence of the catechol-2,3-dioxygenase (C23O) gene after exposure to toluene in two different (i.e., pristine and conditioned) soil communities. The indigenous bacterial populations were exposed to the hydrocarbon substrate by the addition of toluene concentrations, ranging from 0.5 % to 10 % V/W in 10 g of each soil and incubated at 30 °C for upwards of 12 days. In total, 25 isolates (11 in pristine soil and 14 in conditioned soil) were phenotypically characterized according to standard microbiological methods and also screened for the 238-bp C23O gene fragment. Additionally, 16S rRNA analysis of the isolates identified some of them as belonging to the genera Bacillus, Exiguobacterium, Enterobacter, Pseudomonas and Stenotrophomonas. Furthermore, the two clone libraries that were constructed from these toluene-contaminated soils also revealed somewhat disparate phylotypes (i.e., 70 % Actinobacteria and Firmicutes to 30 % Proteobacteria in conditioned soil, whereas in pristine soil: 66 % Actinobacteria and Firmicutes; 21 % Proteobacteria and 13 % Bacteroidetes). The differences observed in bacterial phylotypes between these two soil communities may probably be associated with previous exposure to hydrocarbon sources by indigenous populations in the conditioned soil as compared to the pristine soil.

Plant-associated bacterial populations on native and invasive plant species: comparisons between 2 freshwater environments.

Plant-microbial interactions have been well studied because of the ecological importance of such relationships in aquatic systems. However, general knowledge regarding the composition of these biofilm communities is still evolving, partly as a result of several confounding factors that are attributable to plant host properties and to hydrodynamic conditions in aquatic environments. In this study, the occurrences of various bacterial phylogenetic taxa on 2 native plants, i.e., mayapple (Podophyllum peltatum L.) and cow parsnip (Heracleum maximum Bartram), and on an invasive species, i.e., garlic mustard (Alliaria petiolata (M. Bieb.) Cavara & Grande), were quantitatively examined using nucleic acid staining and fluorescence in situ hybridization. The plants were incubated in triplicates for about a week within the Kalamazoo River and Pierce Cedar Creek as well as in microcosms. The bacterial groups targeted for enumeration are known to globally occur in relatively high abundance and are also ubiquitously distributed in freshwater environments. Fluorescence in situ hybridization analyses of the bacterioplankton assemblages revealed that the majority of bacterial cells that hybridized with the different probes were similar between the 2 sites. In contrast, the plant-associated populations while similar on the 3 plants incubated in Kalamazoo River, their representations were highest on the 2 native plants relative to the invasive species in Pierce Cedar Creek. Overall, our results further suggested that epiphytic bacterial assemblages are probably under the influences of and probably subsequently respond to multiple variables and conditions in aquatic milieus.

Diel fluctuations in the abundance and community diversity of coastal bacterioplankton assemblages over a tidal cycle.

The diel change in abundance and community diversity of the bacterioplankton assemblages within the Pacific Ocean at a fixed location in Monterey Bay, California (USA) were examined with several culture-independent (i.e., nucleic acid staining, fluorescence in situ hybridization {FISH}, and 16S ribosomal RNA gene libraries) approaches over a tidal cycle. FISH analyses revealed the quantitative predominance of bacterial members belonging to the Cytophaga-Flavobacterium cluster as well as two Proteobacteria (α- and γ-) subclasses within the bacterioplankton assemblages, especially during high tide (HT) and outgoing tide (OT) than the other tidal events. While the clone libraries showed that majority of the sequences were similar to the 16S rRNA gene sequences of unknown bacteria (32% to 73%), however, the operational taxonomic units from members of the α-Proteobacteria, Bacteroidetes, Firmicutes, and Cyanobacteria were also well represented during the four tidal events examined. Comparatively, sequence diversity was highest in OT, lowest in low tide, and very similar between HT and incoming tide. The results indicate that the dynamics of bacterial occurrence and diversity appeared to be more pronounced during HT and OT, further indicative of the ecological importance of several environmental variables including temperature, light intensity, and nutrient availability that are also concurrently fluctuating during these tidal events in marine systems.

Molecular analyses of the diversity in marine bacterioplankton assemblages along the coastline of the northeastern Gulf of Mexico.

Bacterial community diversity in marine bacterioplankton assemblages were examined in 3 coastal locations along the northeastern Gulf of Mexico (GOM) using 16S rRNA gene libraries and fluorescence in situ hybridization approaches. The majority of the sequences (30%-60%) were similar to the 16S rRNA gene sequences of unknown bacteria; however, the operational taxonomic units from members of the Cyanobacteria, Proteobacteria, and Bacteroidetes were also present at the 3 GOM sites. Overall, sequence diversity was more similar between the Gulf sites of Carrabelle and Ochlockonee than between either of the Gulf sites and Apalachicola Bay. Fluorescence in situ hybridization analyses revealed the quantitative predominance of members of the Alphaproteobacteria subclass and the Cytophaga-Flavobacterium cluster within the bacterioplankton assemblages. In general, the study further reveals the presence of many bacterial taxa that have been previously found to be dominant in coastal marine environments. Differences observed in the representation of the various bacterial phylogenetic groups among the GOM coastal sites could be partly attributed to dynamic variations in several site-specific conditions, including intermittent tidal events, nutrient availability, and anthropogenic influences.

Comparison of fecal indicator bacterial populations in surface waters of the Kalamazoo River, USA.

Surface waters along the Kalamazoo River, USA, were examined for occurrence and population trends of fecal indicator bacteria (FIB) with culture-based and culture-independent methods. The two methods recorded discrepancies in FIB counts, with the culture-independent method revealing more consistent numbers between the river sites. FIB cells that hybridized with the ECO1482 probe were highest in the downstream site, while the upstream site recorded higher ENF343 hybridized cells. Spatial and temporal differences in FIB populations were probably attributable to contrasting fecal pollution influences, vegetation type, varying environmental conditions as well as several in-stream factors between the two river sites.

Influence of dissolved organic matter and inorganic nutrients on the biofilm bacterial community on artificial substrates in a northeastern Ohio, USA, stream.

Stream bacteria may be influenced by the composition and availability of dissolved organic matter (DOM) and inorganic nutrients, but knowledge about how individual phylogenetic groups in biofilm are affected is still limited. In this study, the influence of DOM and inorganic nutrients on stream biofilm bacteria was examined. Biofilms were developed on artificial substrates (unglazed ceramic tiles) for 21 days in a northeastern Ohio (USA) stream for five consecutive seasons. Then, the developed biofilm assemblages were exposed, in the laboratory, to DOM (glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate, phosphate, and nitrate and phosphate in combination) amendments for 6 days. Bacterial numbers in the biofilms were generally higher in response to the DOM treatments than to the inorganic nutrient treatments. There were also apparent seasonal variations in the response patterns of the individual bacterial taxa to the nutrient treatments; an indication that limiting resources to bacteria in stream biofilms may change over time. Overall, in contrast to the other treatments, bacterial abundance was generally highest in response to the low-molecular-weight DOM (i.e., glucose) treatment. These results further suggest that there are interactions among the different bacterial groups in biofilms that are impacted by the associated nutrient dynamics among seasons in stream ecosystems.

Microbial communities and fecal indicator bacteria associated with Cladophora mats on beach sites along Lake Michigan shores.

A high biomasses of Cladophora, a filamentous green alga, is found mainly during the summer along the shores of Lake Michigan. In this study, the abundance and persistence of the fecal indicator bacterium Escherichia coli and sulfate-reducing bacteria (SRB) on Cladophora mats collected at Lake Michigan beaches were evaluated using both culture-based and molecular analyses. Additionally, 16S rRNA gene cloning and sequencing were used to examine the bacterial community composition. Overall, E. coli was detected in all 63 samples obtained from 11 sites, and the average levels at most beaches ranged from 2,700 CFU/100 g (wet weight) of Cladophora to 7,500 CFU/100 g of Cladophora. However, three beaches were found to have site average E. coli densities of 12,800, 21,130, and 27,950 CFU/100 g of Cladophora. The E. coli levels in the lake water collected at the same time from these three sites were less than the recommended U.S. Environmental Protection Agency limit, 235 CFU/100 ml. E. coli also persisted on Cladophora mats in microcosms at room temperature for more than 7 days, and in some experiments it persisted for as long as 28 days. The SRB densities on Cladophora mats were relatively high, ranging from 4.4x10(6) cells/g (6.64 log CFU/g) to 5.73x10(6) cells/g (6.76 log CFU/g) and accounting for between 20% and 27% of the total bacterial counts. Partial sequences of the 16S rRNA gene clones revealed a phylogenetically diverse community, in which the Cytophaga-Flavobacterium-Bacteroides cluster and the low-G+C-content gram-positive bacteria were the dominant organisms, accounting for 40% and 12.8%, respectively, of the total clone library. These results further reveal the potential public health and ecological significance of Cladophora mats that are commonly found along the shoreline of Lake Michigan, especially with regard to the potential to harbor microorganisms associated with fecal pollution and odor-causing bacteria.

Seasonal response of stream biofilm communities to dissolved organic matter and nutrient enrichments.

Dissolved organic matter (DOM) and inorganic nutrients may affect microbial communities in streams, but little is known about the impact of these factors on specific taxa within bacterial assemblages in biofilms. In this study, nutrient diffusing artificial substrates were used to examine bacterial responses to DOM (i.e., glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate and phosphate singly and in combination). Artificial substrates were deployed for five seasons, from summer 2002 to summer 2003, in a northeastern Ohio stream. Differences were observed in the responses of bacterial taxa examined to various DOM and inorganic nutrient treatments, and the response patterns varied seasonally, indicating that resources that limit the bacterial communities change over time. Overall, the greatest responses were to labile, low-molecular-weight DOM (i.e., glucose) at times when chlorophyll a concentrations were low due to scouring during significant storm events. Different types of DOM and inorganic nutrients induced various responses among bacterial taxa in the biofilms examined, and these responses would not have been apparent if they were examined at the community level or if seasonal changes were not taken into account.