Shell Condition and Survival of Puget Sound Pteropods Are Impaired by Ocean Acidification Conditions

We tested whether the thecosome pteropod Limacina helicina from Puget Sound, an urbanized estuary in the northwest continental US, experiences shell dissolution and altered mortality rates when exposed to the high CO₂, low aragonite saturation state (Ω_a) conditions that occur in Puget Sound and the northeast Pacific Ocean. Five, week-long experiments were conducted in which we incubated pteropods collected from Puget Sound in four carbon chemistry conditions: current summer surface (∼460–500 µatm CO₂, Ω_a≈1.59), current deep water or surface conditions during upwelling (∼760 and ∼1600–1700 µatm CO₂, Ω_a≈1.17 and 0.56), and future deep water or surface conditions during upwelling (∼2800–3400 µatm CO₂, Ω_a≈0.28). We measured shell condition using a scoring regime of five shell characteristics that capture different aspects of shell dissolution. We characterized carbon chemistry conditions in statistical analyses with Ω_a, and conducted analyses considering Ω_a both as a continuous dataset and as discrete treatments. Shell dissolution increased linearly as aragonite saturation state decreased. Discrete treatment comparisons indicate that shell dissolution was greater in undersaturated treatments compared to oversaturated treatments. Survival increased linearly with aragonite saturation state, though discrete treatment comparisons indicated that survival was similar in all but the lowest saturation state treatment. These results indicate that, under starvation conditions, pteropod survival may not be greatly affected by current and expected near-future aragonite saturation state in the NE Pacific, but shell dissolution may. Given that subsurface waters in Puget Sound’s main basin are undersaturated with respect to aragonite in the winter and can be undersaturated in the summer, the condition and persistence of the species in this estuary warrants further study.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Signals and Responses: Choreographing the Complex Interaction between Legumes and α- and β-Rhizobia

The nitrogen-fixing symbiosis between bacteria in the family Rhizobiaceae and members of the legume family (Fabaceae) has been well studied, particularly from the perspective of the early signaling and recognition events. Recent studies of non-nodulating legume mutants have resulted in the identification of a number of genes that are responsive to signal molecules from the bacteria. However, a second group of nodule-forming bacteria, completely unrelated to the Rhizobiaceae, which are α-Proteobacteria, has been discovered. These bacteria belong to the β-Proteobacteria and have been designated β-rhizobia to distinguish them from the better-known α-rhizobia. Here, we review what is known in this economically important symbiosis about the interaction between legumes and α-rhizobia, and we incorporate information, where known, about the β-rhizobia.Key Words: biological nitrogen fixation, recognition, specificity, α-rhizobia, β-rhizobia     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.     

The functional and taxonomic richness of wastewater treatment plant microbial communities are associated with each other and with ambient nitrogen and carbon availability

The number of functional traits of a wastewater treatment plant (WWTP) microbial community (i.e. functional richness) is thought to be an important determinant of its overall functional performance, but the ecological factors that determine functional richness remain unclear. The number of taxa within a community (i.e. taxonomic richness) is one ecological factor that might be important. Communities that contain more taxa are more likely to have more functional traits, and a positive association is therefore expected between functional and taxonomic richness. Empirical tests for this positive association among WWTP communities, however, are lacking. We address this knowledge gap by measuring the functional and taxonomic richness of 10 independent WWTP communities. We demonstrate that functional and taxonomic richness are positively associated with each other. We further demonstrate that functional and taxonomic richness are negatively associated with the effluent NH₄‐N and BOD₅ concentrations. This led us to hypothesize that correlated variation in functional and taxonomic richness is likely related to variation in ambient nitrogen and carbon availability. We finally demonstrate that this hypothesis is consistent with the functional and taxonomic attributes of the WWTP communities. Together, our results improve our basic understanding of the ecology and functioning of WWTP communities.     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

Quantifying Nonspecific TEM β-Lactamase (blaTEM) Genes in a Wastewater Stream

To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are β-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by β-lactamases. Of the β-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM β-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla_TEM genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla_TEM are more likely to survive the treatment process. Thus, β-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.     

Spatial and Temporal Patterns in Macrofaunal Diversity Components Relative to Sea Floor Landscape Structure

We examined temporal changes in macrofaunal α- and β-diversity over several spatial scales (within patches, among patches, across landscapes and across regions) in Long Island Sound on the northeast USA coast. Regional ε-diversity was estimated at 144 taxa, however γ-diversity fluctuated over time as did α- and β-diversity components. Based on additive partitioning, patch- and region-scale β-diversity components generally had the highest contributions to γ-diversity; lower percentages were found at within-patch and landscape scales. Multiplicative diversity partitioning indicated highest species turnover at within- and among patch scales. For all partition results, within-patch and patch-scale β-diversity increased sharply when hypoxia impacted benthic communities. Spatial variation in diversity components can be attributed to the collection of different patch types at varying spatial scales and their associated habitats across the benthic landscapes, as well as gradients in depth and other estuarine-scale characteristics. Temporal variation in diversity components across spatial scales may be related to seasonal changes in habitat heterogeneity, species population dynamics, and seasonal disturbances. Rare species were significant and temporally consistent components of macrofaunal diversity patterns over different spatial scales. Our findings agree with other marine and terrestrial studies that show diversity components vary significantly over different spatial scales and the importance of habitat/landscape heterogeneity in supporting diversity. However, our results indicate that the relative contributions of scale-specific β-diversity components can also change significantly over time. Thus, studies of diversity patterns across patches and landscapes based on data collected at one time, or assembled into a single data set from different times, may not capture the full suite of diversity patterns that occur over varying spatial scales and any time-specific determinants of those patterns. Many factors that shape and maintain sedimentary communities vary temporally, and appear to play an important role in determining and maintaining macrofaunal diversity over different spatial scales.     

Spatial and Temporal Biogeography of Soil Microbial Communities in Arid and Semiarid Regions

Microbial communities in soils may change in accordance with distance, season, climate, soil texture and other environmental parameters. Microbial diversity patterns have been extensively surveyed in temperate regions, but few such studies attempted to address them with respect to spatial and temporal scales and their correlations to environmental factors, especially in arid ecosystems. In order to fill this gap on a regional scale, the molecular fingerprints and abundance of three taxonomic groups – Bacteria, α-Proteobacteria and Actinobacteria – were sampled from soils 0.5–100 km apart in arid, semi-arid, dry Mediterranean and shoreline Mediterranean regions in Israel. Additionally, on a local scale, the molecular fingerprints of three taxonomic groups – Bacteria, Archaea and Fungi – were sampled from soils 1 cm–500 m apart in the semi-arid region, in both summer and winter. Fingerprints of the Bacteria differentiated between all regions (P<0.02), while those of the α-Proteobacteria differentiated between some of the regions (0.01<P<0.09), and actinobacterial fingerprints were similar among all regions (P>0.05). Locally, fingerprints of archaea and fungi did not display distance-decay relationships (P>0.13), that is, the dissimilarity between communities did not increase with geographic distance. Neither was this phenomenon evident in bacterial samples in summer (P>0.24); in winter, however, differences between bacterial communities significantly increased as the geographic distances between them grew (P<0.01). Microbial community structures, as well as microbial abundance, were both significantly correlated to precipitation and soil characteristics: texture, organic matter and water content (R²>0.60, P<0.01). We conclude that on the whole, microbial biogeography in arid and semi-arid soils in Israel is determined more by specific environmental factors than geographic distances and spatial distribution patterns.     

Complete Genome Sequence and Comparative Genomics of a Novel Myxobacterium Myxococcus hansupus

Myxobacteria, a group of Gram-negative aerobes, belong to the class δ-proteobacteria and order Myxococcales. Unlike anaerobic δ-proteobacteria, they exhibit several unusual physiogenomic properties like gliding motility, desiccation-resistant myxospores and large genomes with high coding density. Here we report a 9.5 Mbp complete genome of Myxococcus hansupus that encodes 7,753 proteins. Phylogenomic and genome-genome distance based analysis suggest that Myxococcus hansupus is a novel member of the genus Myxococcus. Comparative genome analysis with other members of the genus Myxococcus was performed to explore their genome diversity. The variation in number of unique proteins observed across different species is suggestive of diversity at the genus level while the overrepresentation of several Pfam families indicates the extent and mode of genome expansion as compared to non-Myxococcales δ-proteobacteria.     

Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10⁶ CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.     

北极海洋沉积物中锰细菌的分离与系统发育

对中国第二次北极科学考察采集的北极海洋沉积物中的锰细菌进行了筛选、分离和系统发育分析。根据其在筛选平板上菌落的形态学特征,分别从站位P11和S11采集的沉积物中分离到了21株和19株锰细菌。系统发育分析表明,两个站位的锰细菌群落组成有着明显的差别。站位P11分离的可培养锰细菌主要由细菌域（Bacteria）中变形杆菌门的γ-变形杆菌纲（γ-Proteobacteria）和放线菌纲（Actinobacteria）组成,二者分别占86％和14％;γ-变形杆菌纲主要包括嗜冷杆菌属（Psychrobacter）、希瓦氏菌属（Shewanella）、假交替单胞菌属（Pseudoaheromonas）、不动杆菌属（Acinetobacter）、海杆菌属（Marinobacter）,其中以嗜冷杆菌属为主,其比例可达67％。从站位S11分离到的可培养锰细菌主要包括细菌域中变形杆菌门的α-变形杆菌纲（α-Proteobacteria）和γ-变形杆菌纲以及拟杆菌门（Bacteroides）中的黄杆菌纲（Flavobaeteria）;γ-变形杆菌纲主要包括希瓦氏菌属、海单胞菌属（Marinomonas）和交替单胞菌属（Aheromonas）,α-变形杆菌纲主要为鞘氨醇单胞菌属（Sphingomonas）。实验菌株均对Mn^2＋有着较强的抗性,其中以菌株Marinomonas sp．S11-S-4耐受性最高。     

Influence of phylogenetic scale on the relationships of taxonomic and phylogenetic turnovers with environment for angiosperms in China

We aim to assess the influence of phylogenetic scale on the relationships of taxonomic and phylogenetic turnovers with environment for angiosperms in China. Specifically, we quantify the effects of contemporary climate on β‐diversity at different phylogenetic scales representing different evolutionary depths of angiosperms. We sampled a latitudinal gradient and a longitudinal gradient of angiosperm assemblages across China (each ≥3400 km). Species composition in each assemblage was documented. Three metrics of β‐diversity (β_sim.tax measuring taxonomic β‐diversity; β_sim.phy and D_pw measuring tip‐ and basal‐weighted phylogenetic β‐diversity, respectively) were quantified among assemblages at sequential depths in the evolutionary history of angiosperms from the tips to deeper branches. This was done by slicing the angiosperm phylogenetic tree at six evolutionary depths (0, 15, 30, 45, 60, and 75 million years ago). β‐diversity at each evolutionary depth was related to geographic and climatic distances between assemblages. In general, the relationship between β‐diversity and climatic distance decreased from shallow to deep evolutionary time slice for all the three metrics. The slopes of the decreasing trends for β_sim.tax and β_sim.phy were much steeper for the latitudinal gradient than for the longitudinal gradient. The decreasing trend of the strength of the relationship was monotonic in all cases except for D_pw across the longitudinal gradient. Geographic distance between assemblages explained little variation in β‐diversity that was not explained by climatic distance. Our study shows that the strength of the relationship between β‐diversity and climatic distance decreases conspicuously from shallow to deep evolutionary depth for the latitudinal gradient, but this decreasing trend is less steep for the longitudinal gradient than for the latitudinal gradient, which likely reflects the influence of historical processes (e.g., the collision of the Indian plate with the Eurasian plate) on β‐diversity.     

Variations within Class-A β-Lactamase Physiochemical Properties Reflect Evolutionary and Environmental Patterns,but not Antibiotic Specificity

The bacterial enzyme β-lactamase hydrolyzes the β-lactam ring of penicillin and chemically related antibiotics, rendering them ineffective. Due to rampant antibiotic overuse, the enzyme is evolving new resistance activities at an alarming rate. Related, the enzyme''s global physiochemical properties exhibit various amounts of conservation and variability across the family. To that end, we characterize the extent of property conservation within twelve different class-A β-lactamases, and conclusively establish that the systematic variations therein parallel their evolutionary history. Large and systematic differences within electrostatic potential maps and pairwise residue-to-residue couplings are observed across the protein, which robustly reflect phylogenetic outgroups. Other properties are more conserved (such as residue pK_a values, electrostatic networks, and backbone flexibility), yet they also have systematic variations that parallel the phylogeny in a statistically significant way. Similarly, the above properties also parallel the environmental condition of the bacteria they are from in a statistically significant way. However, it is interesting and surprising that the only one of the global properties (protein charge) parallels the functional specificity patterns; meaning antibiotic resistance activities are not significantly constraining the global physiochemical properties. Rather, extended spectrum activities can emerge from the background of nearly any set of electrostatic and dynamic properties.     

Classification of Beta-Lactamases and Penicillin Binding Proteins Using Ligand-Centric Network Models

β-lactamase mediated antibiotic resistance is an important health issue and the discovery of new β-lactam type antibiotics or β-lactamase inhibitors is an area of intense research. Today, there are about a thousand β-lactamases due to the evolutionary pressure exerted by these ligands. While β-lactamases hydrolyse the β-lactam ring of antibiotics, rendering them ineffective, Penicillin-Binding Proteins (PBPs), which share high structural similarity with β-lactamases, also confer antibiotic resistance to their host organism by acquiring mutations that allow them to continue their participation in cell wall biosynthesis. In this paper, we propose a novel approach to include ligand sharing information for classifying and clustering β-lactamases and PBPs in an effort to elucidate the ligand induced evolution of these β-lactam binding proteins. We first present a detailed summary of the β-lactamase and PBP families in the Protein Data Bank, as well as the compounds they bind to. Then, we build two different types of networks in which the proteins are represented as nodes, and two proteins are connected by an edge with a weight that depends on the number of shared identical or similar ligands. These models are analyzed under three different edge weight settings, namely unweighted, weighted, and normalized weighted. A detailed comparison of these six networks showed that the use of ligand sharing information to cluster proteins resulted in modules comprising proteins with not only sequence similarity but also functional similarity. Consideration of ligand similarity highlighted some interactions that were not detected in the identical ligand network. Analysing the β-lactamases and PBPs using ligand-centric network models enabled the identification of novel relationships, suggesting that these models can be used to examine other protein families to obtain information on their ligand induced evolutionary paths.     

Factors That Affect Transfer of the IncI1 β-Lactam Resistance Plasmid pESBL-283 between E. coli Strains

The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Novel Method for Rapid Assessment of Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.