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Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobiumgroup, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly publishedMethylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal-or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (P < 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (r 2 ‫؍‬ 0.54, P ‫؍‬ 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired t test, P < 0.05) but not for kanamycin resistance (P > 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.Stream ecosystems are usually connected from headwaters to mouth, and this connectivity provides a means for dissemination and colonization of species. Most streams validate Vannote et al.'s (22) river continuum concept with serial replacement of diverse plants and animals (19,20,23). The river continuum concept provides a theoretical basis for the distribution of organisms and biogeochemical transformations along river systems. Interestingly, microbes are mentioned in this concept but no meaningful predictions of their distributions are presented. Since bacteria are important components of all river systems, it is important to know if they further validate the continuum concept. McArthur et al. (12) demonstrated genetic changes among populations of Burkholderia cepacia and Pseudomonas pickettii along a stream continuum, although both species were persistent at all sampling sites. Wise et al. (25,26) further validated these results. Since they reported that specific genotypes were repeatedly associated with restricted local stream conditions, selection among genotypes was inferred.Since most bacteria cannot be cultured, molecular technologies have been used to detect them or their genes in natural environments (3). Using some of these techniques, Leff et al. (8) reported the nptII gene to occur nonrandomly in a stream. nptII abundance was greater in bank sediments than in channel sediments or on submerged leaf surfaces. The results were similar at each sampling site. The nptII gene, from transposon Tn5, encodes the neomycin phosphotransferase responsible for resistance to kanamycin and neomycin; thus, nptII distribution is useful in studying genetic adaptations of bacteria under natural conditions. Selection for resistance to antibiotics by bacteria in natural and modified stream environments, e.g., below sewage treatment plants (16,17), below feedlots where feed for livestock has been supplemented with antibiotics (7), and even in streams assumed to be pristine (8), may be important to managing streams for human health. However, it is unclear what the selective advantage of resist...

Genetic diversity in natural populations of the bacterium Pseudomonas cepacia was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains.

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