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Made available in DSpace on 2022-04-29T08:31:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Sulfate-reducing bacteria (SRB) can be used to remove metals from wastewater, sewage, and contaminated areas. However, metals can be toxic to this group of bacteria. Sediments from port areas present abundance of SRB and also metal contamination. Their microbial community has been exposed to metals and can be a good inoculum for isolation of metal-resistant SRB. The objective of the study was to analyze how metals influence activity and composition of sulfate-reducing bacteria. Enrichment cultures were prepared with a different metal (Zn, Cr, Cu, and Cd) range concentration tracking activity of SRB and 16S rRNA sequencing in order to access the community. The SRB activity decreased when there was an increase in the concentration of the metals tested. The highest concentration of metals precipitated were 0.2 mM of Cd, 5.4 mM of Zn, 4.5 mM of Cu, and 9.6 mM of Cr. The more toxic metals were Cd and Cu and had a greater community similarity with less SRB and more fermenters (e.g., Citrobacter and Clostridium). Meanwhile, the enrichments with less toxic metals (Cr and Zn) had more sequences affiliated to SRB genera (mainly Desulfovibrio). A new Desulfovibrio species was isolated. This type of study can be useful to understand the effects of metals in SRB communities and help to optimize wastewater treatment processes contaminated by metals. The new Desulfovibrio species may be important in future studies on bioremediation of neutral pH effluents contaminated by metals. Department of Biochemistry and Microbiology Biosciences Institute São Paulo State University – Rio Claro Campus (UNESP IB/RC)), Av. 24 A, 1515, Jardim Vila Bela, Rio Claro Biological Processes Laboratory São Carlos School of Engineering University of São Paulo, Av. João Dagnone, 1100, Santa Angelina, São Carlos Biosciences Institute São Paulo State University – São Paulo State´s Coast Campus (UNESP IB/CLP), Praça Infante Dom Henrique, s/n - Parque Bitaru Department of Agrotechnology and Food Sciences Wageningen University and Research, Stippeneng 4, 6708WE School of Technological Sciences Federal University of Alfenas (UNIFAL-MG) Minas Gerais, Rodovia Aurélio Vilela, n 11.999 Cidade Universitária, Minas Gerais Department of Biochemistry and Microbiology Biosciences Institute São Paulo State University – Rio Claro Campus (UNESP IB/RC)), Av. 24 A, 1515, Jardim Vila Bela, Rio Claro Biosciences Institute São Paulo State University – São Paulo State´s Coast Campus (UNESP IB/CLP), Praça Infante Dom Henrique, s/n - Parque Bitaru CAPES: 88882.434109/2019-01

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host-microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C.elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host's response to chronic Cu stress.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Various industrial, agricultural and military operations have released huge amounts of toxic heavy metals into the environment with deleterious effects on soils, water and air. Under metal stress, soil microorganisms including plant growth promoting bacteria (PGPB) have developed many strategies to evade the toxicity generated by the various heavy metals. Such metal resistant PGPB, when used as bioinoculant or biofertilizers, significantly improved the growth of plants in heavy metal contaminated/stressed soils. Application of bacteria possessing metal detoxifying traits along with plant-beneficial properties is a cost effective and environmental friendly metal bioremediation approach. This review highlights the different mechanisms of metal resistance and plant growth promotion of metal resistant PGPB as well as the recent development in exploitation of these bacteria in bioremediation of heavy metals in different agroecosystems. Keywords: Bioremediation, Bioinoculant, Heavy metals, Plant growth promoting bacteria, Metal resistance, Rhizobacteria

The uncontrolled and widespread use of (nano)silver compounds has led to the increased release of these compounds into the environment, raising concerns about their negative impact on ecosystems. Concomitantly, silver resistance determinants are widely spread among environmental and clinically relevant bacteria although the underlying mechanisms are not yet fully understood. We show that Cupriavidus metallidurans is able to adapt to toxic silver concentrations. However, none of the known silver resistance determinants present in C. metallidurans are involved in the adaptative response. Instead, increased silver resistance is achieved by the concerted action of a two-component system AgrR–AgrS, previously not associated with metal resistance, and two periplasmic proteins PrsQ1 and PrsQ2. Both proteins belong to an unique group of small, uncharacterized, secreted proteins restricted to the genera Cupriavidus and Ralstonia. This system gives C. metallidurans the ability to withstand much higher silver concentrations. The latter could be facilitated by the accumulation of silver ions and the formation of silver nanoparticles.

This project was funded by SCK CEN (Belgium) through the PhD project of Tom Rogiers., Cupriavidus metallidurans is a model bacterium to study molecular metal resistance mechanisms and its use for the bioremediation of several metals has been shown. However, its mechanisms for radionuclide resistance are unexplored. We investigated the interaction with uranium and associated cellular response to uranium for Cupriavidus metallidurans NA4. Strain NA4 actively captured 98 ± 1% of the uranium in its biomass after growing 24 h in the presence of 100 μM uranyl nitrate. TEM HAADF-EDX microscopy confirmed intracellular uraniumphosphate precipitates that were mainly associated with polyhydroxybutyrate. Furthermore, whole transcriptome sequencing indicated a complex transcriptional response with upregulation of genes encoding general stress-related proteins and several genes involved in metal resistance. More in particular, gene clusters known to be involved in copper and silver resistance were differentially expressed. This study provides further insights into bacterial interactions with and their response to uranium. Our results could be promising for uranium bioremediation purposes with the multi-metal resistant bacterium C. metallidurans NA4., SCK CEN (Belgium)

Alternative antimicrobials, such as metals, are one of the methods currently used to help mitigate antibiotic resistance. Metal-based antimicrobials such as copper and silver are used currently both to prevent and to treat infections. Although the efficacy of these antimicrobials has been determined in single-species culture, bacteria rarely exist in a single-species group in the environment. Both Pseudomonas aeruginosa and Staphylococcus aureus are often found associated with each other in severe chronic infections displaying increased virulence and antibiotic tolerance. In this study, we determined that multiple compounds secreted by P. aeruginosa are able to increase the tolerance of S. aureus to both copper and silver. This work demonstrates the expansive chemical communication occurring in polymicrobial infections between bacteria., Metal-based antimicrobials have been used for thousands of years to treat and prevent bacterial infections. Currently, both silver and copper are used in health care and industry to prevent and treat the spread of harmful bacteria. However, like most antimicrobial agents, their efficacy against polymicrobial infections has not been fully elucidated. Coinfection with Pseudomonas aeruginosa and Staphylococcus aureus and the resulting interactions have been implicated in higher virulence, antibiotic resistance, and increased chronic infections. Here, the influence of secreted compounds from P. aeruginosa on metal antimicrobial tolerance in S. aureus was examined. This study determined that multiple compounds from P. aeruginosa increase the tolerance of S. aureus to copper and/or silver when cultured in simulated wound fluid. The presence of these secreted compounds from P. aeruginosa during exposure of S. aureus to copper or silver increased the MIC from 500 μM to 2,000 μM for copper and 16 to 63 μM for silver. The contribution of specific compounds to S. aureus tolerance was determined using gene deletion and disruption mutants, and metabolite analysis. Compounds identified as potential contributors were then individually added to S. aureus during metal exposure. Copper tolerance in S. aureus was found to be increased by amino acids and dihydroaeruginoate (Dha) secreted by P. aeruginosa. The silver tolerance provided to S. aureus was influenced only by two amino acids, serine and threonine, as well as the Pseudomonas quinolone signal (PQS) molecules from P. aeruginosa. IMPORTANCE Alternative antimicrobials, such as metals, are one of the methods currently used to help mitigate antibiotic resistance. Metal-based antimicrobials such as copper and silver are used currently both to prevent and to treat infections. Although the efficacy of these antimicrobials has been determined in single-species culture, bacteria rarely exist in a single-species group in the environment. Both Pseudomonas aeruginosa and Staphylococcus aureus are often found associated with each other in severe chronic infections displaying increased virulence and antibiotic tolerance. In this study, we determined that multiple compounds secreted by P. aeruginosa are able to increase the tolerance of S. aureus to both copper and silver. This work demonstrates the expansive chemical communication occurring in polymicrobial infections between bacteria.

Aim. To investigate the cultural, morphological, physiological and biochemical properties of the isolated chemoorganotrophic heavy metal resistant isolates of bacteria from different samples obtained during the Ukrainian Antarctic expedition in 2019 for further selection of the most resistant to heavy metal compounds and biochemically active substances. Methods. Pure cultures of bacteria were isolated using tryptone soy agar and nutrient agar. Obtained isolates were sown on agar media containing Cu(II) (0.3, 0.1, 1.5, 8, 78 mM), Pb(II) (0.00009, 0.0005, 0.005, 0.05, 0.5 mM), Cr(VI) (0.00096, 0.0096, 0.96, 9.6 mM). The morphological properties of the bacteria were studied using an Axio Lab.A1 Carl Zeiss binocular microscope, an Olympus IX73 inverted microscope with DP-74 digital camera and transmission electron microscopy. Endospores were detected using the Peshkov-Trujillo method. Catalase, oxidase, amylase, lipolytic, protease activities, ability to fix nitrogen were determined. The ability of microorganisms to metabolize organic carbon sources was determined during growth on Hiss medium with different carbohydrates and alcohols. The determination of the physiological properties of obtained isolates was performed using the Remel RapID™ ANA II system. Results. 92 isolates of psychrophilic microorganisms that grew at temperatures 2 °C, 6 °C and 20 °C were isolated from investigated samples. Among the isolated microorganisms, 64 grew on media containing 0.3– 1.6 mM Cu(II), or 0.00009–0.004 mM Pb(II) or 0.01–0.9 mM Cr(VI). 9 isolates of psychrophilic bacteria were resistant to Cu(II) (1.5–78 mM), Pb(II) (0.5 mM), Cr(VI) (0.96–9.6 mM). Morphological, physiological and biochemical properties of 9 multiresistant isolates were described. Conclusions. The physiological and biochemical properties of obtained isolates of An tarctic microorganisms that are resistant to Cu(II) (1.5–78 mM), Pb(II) (0.5 mM), Cr(VI) (0.96–9.6 mM) are determined. Selected isolates of microorganisms are able to use monosaccharides, disaccharides, alcohols as carbon sources; possess urease, protease, lipase, aminopeptidase activities. Selected heavy metal resistant isolates can be used for further investigation and development of technologies for bioremediation of environment.

Improvement of industrial productions through more environment-friendly processes is a hot topic. In particular, land and marine environment pollution is a main concern, considering that recalcitrant compounds can be spread and persist for a long time. In this context, an efficient and cost-effective treatment of wastewater derived from industrial applications is crucial. Phytodepuration has been considered as a possible solution and it is based on the use of plants and their associated microorganisms to remove and/or transform pollutants. In this work we investigated the culturable microbiota of Phragmites australis roots, sampled from the constructed wetlands (CWs) pilot plant in the G.I.D.A. SpA wastewater treatment plant (WWTP) of Calice (Prato, Tuscany, Italy) before and after the CW activation in order to check how the influx of wastewater might affect the resident bacterial community. P. australis specimens were sampled and a panel of 294 culturable bacteria were isolated and characterized. This allowed to identify the dynamics of the microbiota composition triggered by the presence of wastewater. 27 out of 37 bacterial genera detected were exclusively associated to wastewater, and Pseudomonas was constantly the most represented genus. Moreover, isolates were assayed for their resistance against eight different antibiotics and synthetic wastewater (SWW). Data obtained revealed the presence of resistant phenotypes, including multi-drug resistant bacteria, and a general trend regarding the temporal evolution of resistance patterns: indeed, a direct correlation linking the appearance of antibiotic- and SWW-resistance with the time of exposure to wastewater was observed. In particular, nine isolates showed an interesting behavior since their growth was positively affected by the highest concentrations of SWW. Noteworthy, this study is among the few investigating the P. australis microbiota prior to the plant activation.

Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing problem worldwide. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. Here, nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized, and their distribution across both noncontaminated and contaminated areas at ORR was examined. While the seven strains have various pH ranges for growth, carbon source preferences, and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were identified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis, and all four were found in both noncontaminated and contaminated areas. By using environmentally relevant metal concentrations, we successfully isolated multiple organisms from both ORR noncontaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination. IMPORTANCE Nitrate contamination is a global issue that affects groundwater quality. In some cases, cocontamination of groundwater with nitrate and mixtures of heavy metals could decrease microbially mediated nitrate removal, thereby increasing the duration of nitrate contamination. Here, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located in pristine as well as contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the effect of nitrate and metal contamination on groundwater microbial communities.

Background Direct and indirect selection pressures imposed by antibiotics and co-selective agents and horizontal gene transfer are fundamental drivers of the evolution and spread of antibiotic resistance. Therefore, effective environmental monitoring tools should ideally capture not only antibiotic resistance genes (ARGs), but also mobile genetic elements (MGEs) and indicators of co-selective forces, such as metal resistance genes (MRGs). A major challenge towards characterizing the potential human health risk of antibiotic resistance is the ability to identify ARG-carrying microorganisms, of which human pathogens are arguably of greatest risk. Historically, short reads produced by next-generation sequencing technologies have hampered confidence in assemblies for achieving these purposes. Results Here, we introduce NanoARG, an online computational resource that takes advantage of the long reads produced by nanopore sequencing technology. Specifically, long nanopore reads enable identification of ARGs in the context of relevant neighboring genes, thus providing valuable insight into mobility, co-selection, and pathogenicity. NanoARG was applied to study a variety of nanopore sequencing data to demonstrate its functionality. NanoARG was further validated through characterizing its ability to correctly identify ARGs in sequences of varying lengths and a range of sequencing error rates. Conclusions NanoARG allows users to upload sequence data online and provides various means to analyze and visualize the data, including quantitative and simultaneous profiling of ARGs, MRGs, MGEs, and putative pathogens. A user-friendly interface allows users the analysis of long DNA sequences (including assembled contigs), facilitating data processing, analysis, and visualization. NanoARG is publicly available and freely accessible at https://bench.cs.vt.edu/nanoarg . Published version

Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains., Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.

Landfill leachate is a persistent contamination threat for terrestrial waters. Microbial metabolism in landfills transforms contaminants and contributes to greenhouse gas emissions. A better understanding of landfill-associated microbial communities will inform bioremediation of solid waste environments and improve pathogen monitoring. We leveraged shotgun metagenomics to investigate the microbial communities of the Riverton City dump and the adjoining Duhaney River near Kingston City, Jamaica. We identified no overlap between the microbial communities inhabiting the Riverton City dump leachate and the Duhaney River. Both communities are predicted to degrade aromatic compounds, which are ubiquitous environmental pollutants. Adversely, microbes in both environments are predicted to withstand widely used antibiotics, antiseptics, and metal contamination. The absence of evidence for microbial transfer from the leachate to the river is encouraging; however, the Duhaney River contained several organisms with predicted pathogenic lifestyles, indicating that the river represents a human health risk regardless of impact from the dump., The Riverton City dump is Jamaica’s largest solid waste disposal site, but it lacks engineered protection for leachate containment and treatment. Shotgun metagenomics was used to survey the microbial communities in the Riverton City dump leachate and in surface waters of the Duhaney River, an urban waterway abutting the dump. The community within the leachate pond was taxonomically distinct from that found in the surface waters of the Duhaney River. Higher microbial diversity was observed within the dump leachate, with members of the Bacteroidetes, Firmicutes, Gammaproteobacteria, Deltaproteobacteria, and Tenericutes being the most abundant, while the river community was dominated by Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The microbial communities exhibit similar functional potential profiles, including chemoorganoheterotrophy as the dominant metabolism, and the potential to degrade aromatic compounds. From reconstruction of metagenome-assembled genomes (MAGs), organisms within both environments are predicted to survive in the presence of multiple antibiotics, antiseptics, biocides, and metals. Strong virulence potential coincided with the most diverse multiple resistance profiles in 1 of 5 leachate MAGs and 5 of 33 river MAGs. Unexpectedly, the microbial resistance profiles were more varied and widespread in the river populations, where we had expected the chemical composition of the leachate to select and enrich for resistance characteristics. This study provides valuable insights into the total functional potential of a landfill leachate microbial community and identifies possible human health hazards within the Duhaney River and Riverton City dump, urban environments with the potential to impact human populations. IMPORTANCE Landfill leachate is a persistent contamination threat for terrestrial waters. Microbial metabolism in landfills transforms contaminants and contributes to greenhouse gas emissions. A better understanding of landfill-associated microbial communities will inform bioremediation of solid waste environments and improve pathogen monitoring. We leveraged shotgun metagenomics to investigate the microbial communities of the Riverton City dump and the adjoining Duhaney River near Kingston City, Jamaica. We identified no overlap between the microbial communities inhabiting the Riverton City dump leachate and the Duhaney River. Both communities are predicted to degrade aromatic compounds, which are ubiquitous environmental pollutants. Adversely, microbes in both environments are predicted to withstand widely used antibiotics, antiseptics, and metal contamination. The absence of evidence for microbial transfer from the leachate to the river is encouraging; however, the Duhaney River contained several organisms with predicted pathogenic lifestyles, indicating that the river represents a human health risk regardless of impact from the dump.

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system ( cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae , the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu 2+ to Cu 1+ , which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae . IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae , remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu 2+ to Cu 1+ . This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

Cellulose-degrading bacterial strains, KC3 and 5, capable also of degrading both low and high molecular weight PAHs were isolated from koala faeces for the first time. KC3 and 5 belong to the genera Bacillus and Pseudomonas, respectively. These strains completely degraded the model 3-(phenanthrene), 4-(pyrene) and 5-(benzo-a-pyrene) ring PAHs at 6, 7 and 40–50 days, correspondingly. They were shown to be able to produce a rhamnolipid type of biosurfactant during PAH degradation. The biosurfactants produced from both the strains showed good pH (2–12) as well as thermal (up to 80 °C) stability and were able to tolerate up to 20 g L−1 salinity. The strains also had resistance towards heavy metals, attributed to the amount of biosurfactant produced. The Bacillus strain in particular showed excellent metal resistance; the minimum inhibitory concentrations were 5 (Cd2+, Cu2+) and 7 (Pb2+, Zn2+) mg L−1 of relatively bioavailable metal ions, but >15 mg L−1 metal concentrations were lethal to the microbe. Additionally, both strains possessed activity of more than one extracellular enzyme (cellulase, lipase and protease). The limiting factors in PAH biodegradation are low PAH bioavailability and microbial intolerance towards HMW PAHs and co-contaminants (heavy metals). The novel strains identified thus had (a) potential to biodegrade both LMW and HMW PAHs, (b) pH, thermal and saline-tolerant biosurfactant production that aids PAH solubility enhancement, and importantly, (c) heavy metal resistance. Both Bacillus and Pseudomonas strains are appropriate candidates in field-scale PAH bioremediation at mixed contamination sites and for several industrial applications due to their enzymatic activities. Refereed/Peer-reviewed

Uranium contaminated sites harbor viable and metabolically active microorganisms, capable of interacting with uranium and impacting its distribution. This study wants to contribute to a better understanding of the microbe-uranium interactions in the Urgeirica mine (Portugal) by enumerating, identifying and characterizing U resistant strains. Rhodanobacter genus included a major group of U (VI) resistant isolates up to 2 mM U (VI). The analysis suggests that different species belonging to this genus have different resistance profiles. The recovery of these bacteria as a major group of isolates indicate that these microorganisms might have an important ecological role in these environments.

Record-breaking floods in September 2013 caused massive damage to homes and infrastructure across the Colorado Front Range and heavily impacted the Cache La Poudre River watershed. Given the unique nature of this watershed as a test-bed for tracking environmental pathways of antibiotic resistance gene (ARG) dissemination, we sought to determine the impact of extreme flooding on ARG reservoirs in river water and sediment. We utilized high-throughput DNA sequencing to obtain metagenomic profiles of ARGs before and after flooding, and investigated 23 antibiotics and 14 metals as putative selective agents during post-flood recovery. With 277 ARG subtypes identified across samples, total bulk water ARGs decreased following the flood but recovered to near pre-flood abundances by ten months post-flood at both a pristine site and at a site historically heavily influenced by wastewater treatment plants and animal feeding operations. Network analysis of de novo assembled sequencing reads into 52,556 scaffolds identified ARGs likely located on mobile genetic elements, with up to 11 ARGs per plasmid-associated scaffold. Bulk water bacterial phylogeny correlated with ARG profiles while sediment phylogeny varied along the river’s anthropogenic gradient. This rare flood afforded the opportunity to gain deeper insight into factors influencing the spread of ARGs in watersheds.

Richa Singh,1 Priyanka Wagh,1 Sweety Wadhwani,1 Sharvari Gaidhani,2 Avinash Kumbhar,3 Jayesh Bellare,4 Balu Ananda Chopade1 1Department of Microbiology, University of Pune, Pune, Maharashtra, India; 2Institute of Bioinformatics and Biotechnology, University of Pune, Pune, Maharashtra, India; 3Department of Chemistry, University of Pune, Pune, Maharashtra, India; 4Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra, India Background: The development of nontoxic methods of synthesizing nanoparticles is a major step in nanotechnology to allow their application in nanomedicine. The present study aims to biosynthesize silver nanoparticles (AgNPs) using a cell-free extract of Acinetobacter spp. and evaluate their antibacterial activity. Methods: Eighteen strains of Acinetobacter were screened for AgNP synthesis. AgNPs were characterized using various techniques. Reaction parameters were optimized, and their effect on the morphology of AgNPs was studied. The synergistic potential of AgNPs on 14 antibiotics against seven pathogens was determined by disc-diffusion, broth-microdilution, and minimum bactericidal concentration assays. The efficacy of AgNPs was evaluated as per the minimum inhibitory concentration (MIC) breakpoints of the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Only A. calcoaceticus LRVP54 produced AgNPs within 24 hours. Monodisperse spherical nanoparticles of 8–12 nm were obtained with 0.7 mM silver nitrate at 70°C. During optimization, a blue-shift in ultraviolet-visible spectra was seen. X-ray diffraction data and lattice fringes (d =0.23 nm) observed under high-resolution transmission electron microscope confirmed the crystallinity of AgNPs. These AgNPs were found to be more effective against Gram-negative compared with Gram-positive microorganisms. Overall, AgNPs showed the highest synergy with vancomycin in the disc-diffusion assay. For Enterobacter aerogenes, a 3.8-fold increase in inhibition zone area was observed after the addition of AgNPs with vancomycin. Reduction in MIC and minimum bactericidal concentration was observed on exposure of AgNPs with antibiotics. Interestingly, multidrug-resistant A. baumannii was highly sensitized in the presence of AgNPs and became susceptible to antibiotics except cephalosporins. Similarly, the vancomycin-resistant strain of Streptococcus mutans was also found to be susceptible to antibiotic treatment when AgNPs were added. These biogenic AgNPs showed significant synergistic activity on the β-lactam class of antibiotics. Conclusion: This is the first report of synthesis of AgNPs using A. calcoaceticus LRVP54 and their significant synergistic activity with antibiotics resulting in increased susceptibility of multidrug-resistant bacteria evaluated as per MIC breakpoints of the CLSI standard. Keywords: Ag nanoparticles, lattice fringes, disc-diffusion, minimum inhibitory concentration, synergistic activity

Copper homeostasis in bacteria is challenged by periodic elevation of copper levels in the environment, arising from both natural sources and human inputs. Several mechanisms have evolved to efflux copper from bacterial cells, including the cus (copper sensing copper efflux system), and pco (plasmid-borne copper resistance system) systems. The genes belonging to these two systems can be physically clustered in a Copper Homeostasis and Silver Resistance Island (CHASRI) on both plasmids and chromosomes in Enterobacteria. Increasing use of copper in agricultural and industrial applications raises questions about the role of human activity in the evolution of novel copper resistance mechanisms. Here we present evidence that CHASRI emerged and diversified in response to copper deposition across aerobic and anaerobic environments. An analysis of diversification rates and a molecular clock model suggest that CHASRI experienced repeated episodes of elevated diversification that could correspond to peaks in human copper production. Phylogenetic analyses suggest that CHASRI originated in a relative of Enterobacter cloacae as the ultimate product of sequential assembly of several pre-existing two-gene modules. Once assembled, CHASRI dispersed via horizontal gene transfer within Enterobacteriaceae and also to certain members of Shewanellaceae, where the original pco module was replaced by a divergent pco homolog. Analyses of copper stress mitigation suggest that CHASRI confers increased resistance aerobically, anaerobically, and during shifts between aerobic and anaerobic environments, which could explain its persistence in facultative anaerobes and emergent enteric pathogens.

International audience; The aim of this study was to test whether silver nanoparticles (Ag-NPs) released into estuarine environments result in increased antibiotic resistance amongst the natural bacterial population in estuarine sediments. A 50-day microcosm exposure experiment was carried out to investigate the effects of Ag-NPs (50 nm average diameter) on the antibiotic resistance of bacteria in sediments from an estuary in southwest England. Experimental microcosms were constructed using 3.5 kg sediment cores with 20 l of overlaying seawater treated with (final) Ag-NPs concentrations of 0, 50 or 2000 μg l (n = 3). Sediment samples were screened at the end of the exposure period for the presence of bacteria resistant to eight different antibiotics. Multivariate statistical analyses showed that there was no increase in antibiotic resistance amongst the bacterial population in the sediment due to the dosing of the microcosms with Ag-NPs. This study indicates that, under the tested conditions, Ag-NPs released into the coastal marine environment do not increase antibiotic resistance among naturally occurring bacteria in estuarine sediments. These results contrast previous findings where antimicrobial effects of Ag-NPs on key bacterial species in laboratory experiments have been demonstrated, and reasons for this are discussed. The negligible effects demonstrated on bacterial populations under the selected estuarine conditions, provide important information on no observed effect concentrations (NOECs) for environmental regulation.

Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution. Published version

The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated metal resistance may explain the observed characteristics of the new Sphingobium isolate.

Strain desinganted R22 and R25, isolated from Sali River sediments, Argentina, were highly resistant to chromium. these strains were shown by 16S rRNA sequencing studies to be Streptomyces spp.; this affiliation was consistent with morphological and chemical characteristics. Growth of R22 and R25 in medium containing 100 mg/l chromate was reduced by only 23% and 34%, respectively, compared with growth in medium without added chromium. Streptomyces sp. strains R22 and R25 both accumulated chromium with yields of 10.0 and 5.6 mg Cr/g of dry weight, respectively, and chromate concentration of 50 mg/ml. Cell fractionation studies with strain R22 showed that the great majority of the chromium were associated with the cell wall fraction. Streptomyces R22 and R25 may have applications in bioremediation of chromium contamination. Fil: Amoroso, Maria Julia del R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; Argentina Fil: Castro, Guillermo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Duran, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial ; Argentina Fil: Peraud, O.. University of Maryland; Estados Unidos Fil: Oliver, G.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; Argentina Fil: Hill, R. T.. University of Maryland; Estados Unidos

Cadmium (Cd) is a major environmental hazard, which usually is detected in its ionic form of Cd(2+). It also causes adverse toxic effects on human health and other living organisms. Cd-resistant bacteria were isolated from Cd-contaminated soils. One isolate, TAK1, was highly resistance level to Cd toxicity. TAK1 was isolated from soil contaminated with a high Cd concentration (204.1 mg.kg(-1)). The result of 16S rDNA sequence analysis found that the TAK1 showed the similarity to Ralstonia sp. Physiological adaptive and cross-protective responses to Cd and Zn killing were investigated in Ralstonia sp.TAK1. Exposure to a low concentration of Cd induced adaptive resistance to higher concentrations of Cd. In addition, pretreatment of Ralstonia sp.TAK1 with an inducing concentration of Cd conferred cross-protective response against subsequent exposure to the lethal concentrations of Zn. The induced adaptive and cross-protective response Ralstonia sp.TAK1 required newly synthesized protein(s). Cd-induced adaptive and cross-protective responses against Cd and Zn toxicity are the important mechanisms used by Ralstonia sp.TAK1 to survive in the heavy metal-contaminated environments. These findings might lead to the use of Ralstonia sp.TAK1 for microbial based remediation in Cd and Zn-contaminated soils.

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.