J. Vaun McArthur

Spatial Patterns in Antibiotic Resistance among Stream Bacteria: Effects of Industrial Pollution

ABSTRACT 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 (r2 = 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.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities

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.

THE EFFECT OF INVERTEBRATE PREDATORS ON LEAF LITTER PROCESSING IN AN ALPINE STREAM

The effect of the predators Megarcys signata (Plecoptera: Perlodidae) and Rhyacophila sp. (Trichoptera: Rhyacophilidae) on the abundance of macroinvertebrates and on the rates of leaf processing in artificial leaf packs was studied by using manipulative field experiments. Predators were confined within artificially constructed leaf packs in an alpine stream in Utah. Both predators signif- icantly reduced the rate of breakdown of leaves in the fall, but had no effect in late winter, when the most important shredder, Zapada cinctipes, emerges. Predation on shredders caused a reduction in breakdown rates and an increase in the residence time of the leaf litter in the stream. In experimental treatments where predators significantly reduced the numbers of shredders and decreased the rate of leaf processing, leaf pack half-life increased an average of 10.3 d. These experiments demonstrate that invertebrate shredders can contribute substantially to the rate of leaf processing in streams and suggest that one of the factors limiting the abundance of natural shredder populations is predation.

Interspecific leaf interactions during decomposition in aquatic and floodplain ecosystems

An experiment was designed to test the importance of the potential interaction (inhibition or enhancement) between slow and fast decaying leaf species on processing rates in a stream and its floodplain. The decomposition of water oak (Quercus nigra) and sweetgum (Liquidambar styraciflua) in single-species packs was compared with water oak plus sweetgum in mixed-species packs within three habitats (stream snags, floodplain pools, and elevated floodplain surfaces) at three sites in coastal plain streams. Fast-decaying sweetgum leaves did not enhance the rate of oak decomposition. Sweetgum leaves in mixed packs decomposed more slowly than single species packs in seven out of nine comparisons. Increases in bacterial density on leaves were depressed in mixed-species packs relative to single-species sweetgum packs. Fungal hyphae could not be observed in mixed or single-species packs. The effect of oak leaves on sweetgum leaves was affected by frequency and period of inundation. Macroinvertebrate shredders were rare or absent from most leaf packs collected from stream snags and floodplain pools. Over 40% of leaf packs placed in the stream contained no shredders, while another 28% contained <0.001 g shredders/g leaf dry weight. Therefore, shredders were too rare to influence overall leaf processing rates. These studies suggest that microbial processing accounts for most leaf decomposition and oak leaf leachate is shown to be inhibitory to microbial processing of sweetgum leaves.

An exploration of factors influencing lotic insect species richness.

An understanding of factors that influence species richness of lotic insects is generally lacking. We present comparative data on aquatic insect species richness from several North American and other streams. Factors such as large sample numbers and drainage area (species area relationships) are not significant predictors of species richness across the streams we examined. We explore several hypotheses regarding the origins and maintenance of species richness using Upper Three Runs Creek (UTR), South Carolina, USA, as a reference stream. UTR has the highest species richness of any stream in the Western Hemisphere. Hypotheses examined included historical, regional and local processes such as: (1) Evolutionary time, (2) disturbance regime/environmental variability, (3) temperature/evolutionary-speed, (4) productivity, and (5) habitat heterogeneity. Of these hypotheses, we suggest that productivity and habitat heterogeneity appear to contribute most to the high species richness found in UTR. We believe that multidisciplinary analysis of other streams is necessary because without this crucial information our knowledge of, and desire to protect biodiversity in streams will be wanting.

Genetic Diversity of Bacteria along a Stream Continuum

Adaptation is a fundamental concept in ecology and evolution. Determining whether organisms are adapted requires genetic analysis of the organism. We characterized the genetic structure of isolates of two species of aquatic bacteria (Pseudomonas cepacia and P. pickettii) using starch gel electrophoresis to determine the relationship between genetic diversity and stream location. Genetic diversity in these species of bacteria did not change longitudinally. However, each location in the stream had genetically unique bacteria. Genetic distance among isolates was found to be related to geographical distance, i.e., isolates farther apart in the stream were more genetically dissimilar. The results suggest localized adaptation. Soil forms of P. cepacia collected from the same catchment were very different genetically from the aquatic forms of the same species. Analyzing patterns of allozymes may be of value in determining the source of bacteria in aquatic ecosystems.

Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

ABSTRACT Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.

The Effect of Leaf Pack Composition on Processing: A Comparison of Mixed and Single Species Packs

The effect of leaf species composition on decomposition patterns was examined in a coastal plain stream. Red maple leaves (Acer rubrum) decomposed at the same rate separately or when mixed with cypress leaves (Taxodium distichum). Cypress addition increased structural integrity but its effects differed between sites with different hydrologic regimes. Invertebrate communities varied slightly between mixed and single species packs, however invertebrates did not appear to be the primary agent of decomposition. Mixed species packs may be an alternative method to fine mesh bags for studying processing of small, narrow leaves in a more realistic manner.

Information spiraling: Movement of bacteria and their genes in streams

Bacteria in transport in streams are largely derived from other parts of the ecosystem. Here we review factors that influence transport of bacteria and their movement between habitats (such as sediment, water column, rocks, wood, and leaves) and consider the role of these movements in ecosystem processes. Bacteria enter the water column by sloughing, scouring, as a consequence of changes in morphology or hydrophobicity, or dislodgment by invertebrates and fish or other aquatic vertebrates. Transported cells (which may be planktonic or particle-associated) that colonize surfaces may establish new gene pools through cell division (vertical transfer) or genetic exchange (lateral transfer). Genetic information is also transported in streams as free or protected DNA or in bacteriophages. Movement of these vectors causes genetic information to spiral along a stream in a manner analogous to that of nutrients and organic carbon. Spiraling refers to the pattern of transport, uptake or attachment, and release of a molecule or cell. The flow of water in streams causes this cycle of attachment and release to be displaced downstream resulting in a spiral rather than a closed, stationary loop.

Spatial Analysis of Antibiotic Resistance Along Metal Contaminated Streams

The spatial pattern of antibiotic resistance in culturable sediment bacteria from four freshwater streams was examined. Previous research suggests that the prevalence of antibiotic resistance may increase in populations via indirect or coselection from heavy metal contamination. Sample bacteria from each stream were grown in media containing one of four antibiotics—tetracycline, chloramphenicol, kanamycin, and streptomycin—at concentrations greater than the minimum inhibitory concentration, plus a control. Bacteria showed high susceptibilities to the former two antibiotics. We summarized the latter two more prevalent (aminoglycoside) resistance responses and ten metals concentrations per sediment sample, by Principal Components Analysis. Respectively, 63 and 58% of the variability was explained in the first principal component of each variable set. We used these multivariate summary metrics [i.e., first principal component (PC) scores] as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream sampled. Results show a significant and negative correlation between metals PC scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently in sediments than in the water column. Our most important finding comes from geostatistical cross-variogram analysis, which shows that increasing metal concentration scores are spatially associated with decreasing aminoglycoside resistance scores—a negative correlation, but holds for contaminated streams only. We suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or “cocktail effect” from complex combinations of pollution mediated selection agents.