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Abstract Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, β-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.

Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, β-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment. [ABSTRACT FROM AUTHOR]

Understanding the role of foods in the emergence and spread of antimicrobial resistance necessitates the initial documentation of antibiotic resistance genes within bacterial species found in foods. Here, the NCBI Pathogen Detection database was used to query antimicrobial resistance gene prevalence in foodborne and human clinical bacterial isolates. Of the 1,843,630 sequence entries, 639,087 (34.7%) were assigned to foodborne or human clinical sources with 147,788 (23.14%) from food and 427,614 (76.88%) from humans. The majority of foodborne isolates were either Salmonella (47.88%), Campylobacter (23.03%), Escherichia (11.79%), or Listeria (11.3%), and the remaining 6% belonged to 20 other genera. Most foodborne isolates were from meat/poultry (95,251 or 64.45%), followed by multi-product mixed food sources (29,892 or 20.23%) and fish/seafood (6503 or 4.4%); however, the most prominent isolation source varied depending on the genus/species. Resistance gene carriage also varied depending on isolation source and genus/species. Of note, Klebsiella pneumoniae and Enterobacter spp. carried larger proportions of the quinolone resistance gene qnrS and some clinically relevant beta-lactam resistance genes in comparison to Salmonella and Escherichia coli. The prevalence of mec in S. aureus did not significantly differ between meat/poultry and multi-product sources relative to clinical sources, whereas this resistance was rare in isolates from dairy sources. The proportion of biocide resistance in Bacillus and Escherichia was significantly higher in clinical isolates compared to many foodborne sources but significantly lower in clinical Listeria compared to foodborne Listeria. This work exposes the gaps in current publicly available sequence data repositories, which are largely composed of clinical isolates and are biased towards specific highly abundant pathogenic species. We also highlight the importance of requiring and curating metadata on sequence submission to not only ensure correct information and data interpretation but also foster efficient analysis, sharing, and collaboration. To effectively monitor resistance carriage in food production, additional work on sequencing and characterizing AMR carriage in common commensal foodborne bacteria is critical. [ABSTRACT FROM AUTHOR]

Present study was performed with the aim to isolate Heavy metal Tolerant- PGPB (HMT-PGPB) from metal-contaminated site and use them for Cr bioremediation. Six different bacterial strains were obtained from the endosphere of Solanum virginicum L. roots and cultured using nutrient agar media amended with 20 mg/L of Cr(VI). The ability of these Cr(VI) tolerant bacterial isolates were assessed for PGP traits like producing siderophores, indole-3-acetic acid (IAA), and phosphate solubilization. The findings indicated that all of the isolates could produce exopolymeric substances and IAA, five of them could produce siderophores, and three could solubilize phosphate. Furthermore, the minimum inhibitory concentrations of these strains werealso determined. These strains were identified as Bacillus licheniformis SxR1, B. tequilensis SxR2, B. subtilis SxR3, B. velezensis SxR4, B. amyloliquefaciens SxR6, and B. stercoris SxR8. To validate the findings, it is crucial to comprehend how Cr(VI) affects Bacillus sp. SxR1 cells to determine the course of uptake and bacterial cell alteration, which was assessed via Fourier Transform-Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM).

Global concerns about the preservation and restoration of aquatic environments are rising and pollution related to heavy metals is one of the main worries. Indeed, this issue has been a challenge for the metallurgical industry and other activities associated with metal contamination. Thus, over time, several physical and chemical methods have been developed and applied to remove metals from water. However, these methods can be associated with high costs, and bioremediation using plants, fungi, and bacteria is considered a viable alternative. This paper reports experiments on lead removal from an aqueous medium using active and inactive Serratia marcescens CCMA 1010 and a study on the effect of lead in this bacterial strain regarding the expression of the zntR gene, which produces the zntR protein known to have a role as a regulator of the ZntA efflux pump of metals (Pb2+, Cd2+, Zn2+). The amount of removed Pb2+ by active biomass remained below ∼25 mg/L for the initial concentrations tested up to 120 mg/L but increased to removals of ∼70 and ∼167 mg/L for the tested concentrations of 220 and 300 mg/L, respectively. On the other hand, the removal of Pb2+ by inactive biomass increased in direct relation to the initial tested concentration, with removed percentages around 25 %. Interestingly, in cultures with 15 and 60 mg/L Pb2+ the expression of zntR was 27 to 74 and 87 to 177 times lower (respectively) than in cultures without Pb2+, while in cultures with 120 mg/L Pb2+, the zntR expression was just 1.3 to 9.3 times lower than in the absence of Pb2+. The results confirm the potential of S. marcescens CCMA 1010 for Pb2+ biosorption, the presence of Pb2+ resistance mechanisms in this strain, and contribute to a better understanding of the ZntA transmembrane protein regulation.

Management of tailings at metal mine smelter sites can reduce the potential hazards associated with exposure to toxic metal(loid)s and residual organic flotation reagents. In addition, microbes in the tailings harboring multi-resistance genes (e.g., tolerance to multiple antimicrobial agents) can cause high rates of morbidity and global economic problems. The potential co-selection mechanisms of antibiotic resistance genes (ARGs) and metal(loid) resistance genes (MRGs) during tailings sulfate-reducing bacteria (SRB) treatment have been poorly investigated. Samples were collected from a nonferrous metal mine tailing site treated with an established SRB protocol and were analyzed for selected geochemical properties and high throughput sequencing of 16S rRNA gene barcoding. Based on the shotgun metagenomic analysis, the bacterial domain was dominant in nonferrous metal(loid)-rich tailings treated with SRB for 12 months. KEGGs related to ARGs and MRGs were detected. Thiobacillus and Sphingomonas were the main genera carrying the bacA and mexEF resistance operons, along with Sulfuricella which were also found as the main genera carrying MRGs. The SRB treatment may mediate the distribution of numerous resistance genes. KOs based on the metagenomic database indicated that ARGs (mexNW, merD, sul, and bla) and MRGs (czcABCR and copRS genes) were found on the same contig. The SRB strains (Desulfosporosinus and Desulfotomaculum), and the acidophilic strain Acidiphilium significantly contributed to the distribution of sul genes. The functional metabolic pathways related to siderophores metabolism were largely from anaerobic genera of Streptomyces and Microbacterium. The presence of arsenate reductase, metal efflux pump, and Fe transport genes indicated that SRB treatment plays a key role in the metal(loid)s transformation. Overall, our findings show that bio-treatment is an effective tool for managing ARGs/MRGs and metals in tailings that contain numerous metal(loid) contaminants. [ABSTRACT FROM AUTHOR]

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from postharvest chicken samples, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella in preharvest and in particular their makeup of antimicrobial resistance genes and virulence factors. We used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella enterica isolates (n = 55) recovered from the litter of commercial broiler chicken raised in four colocated broiler houses of one integrated farm over three consecutive flocks. The chicken were raised under a newly adopted “No Antibiotics Ever” program, and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several virulence factors on plasmids. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-associated metal resistance and virulence genes, which could play a role in their persistence in litter.

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al3+, the oxyanion CrO42-, and the oxycation UO22+. Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al3+ binds intracellular arginine, CrO42- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO22+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. Here, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment. IMPORTANCE Studying microbial interactions with their environment can lead to a deeper understanding of biological molecular mechanisms. In this study, two global techniques, RB-TnSeq and activity metabolomics, were successfully used to probe the interactions between a metal-resistant microorganism, Pantoea sp. strain MT58, and metals contaminating a site where the organism can be located. A number of novel metal-microbe interactions were uncovered, including Al3+ toxicity targeting arginine synthesis, which could lead to a deeper understanding of the impact Al3+ contamination has on microbial communities as well as its impact on higher-level organisms, including plants for whom Al3+ contamination is an issue. Using multiomic approaches like the one described here is a way to further our understanding of microbial interactions and their impacts on the environment overall.

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.

Viruses are ubiquitous microbiome components, shaping ecosystems via strain-specific predation, horizontal gene transfer and redistribution of nutrients through host lysis. Viral impacts are important in groundwater ecosystems, where microbes drive many nutrient fluxes and metabolic processes; however, little is known about the diversity of viruses in these environments. We analyzed four groundwater plasmidomes (the entire plasmid content of an environment) and identified 200 viral sequences, which clustered into 41 genus-level viral clusters (approximately equivalent to viral genera) including 9 known and 32 putative new genera. We used publicly available bacterial whole-genome sequences (WGS) and WGS from 261 bacterial isolates from this groundwater environment to identify potential viral hosts. We linked 76 of the 200 viral sequences to a range of bacterial phyla, the majority associated with Proteobacteria, followed by Firmicutes, Bacteroidetes, and Actinobacteria. The publicly available WGS enabled mapping bacterial hosts to several viral sequences. The WGS of groundwater isolates increased the depth of host prediction by allowing host identification at the strain level. The latter included 4 viruses that were almost entirely (>99% query coverage, >99% identity) identified as integrated in the genomes of Pseudomonas, Acidovorax, and Castellaniella strains, resulting in high-confidence host assignments. Lastly, 21 of these viruses carried putative auxiliary metabolite genes for metal and antibiotic resistance, which might drive their infection cycles and/or provide selective advantage to infected hosts. Exploring the groundwater virome provides a necessary foundation for integration of viruses into ecosystem models where they are key players in microbial adaption to environmental stress. IMPORTANCE To our knowledge, this is the first study to identify the bacteriophage distribution in a groundwater ecosystem shedding light on their prevalence and distribution across metal-contaminated and background sites. Our study is uniquely based on selective sequencing of solely the extrachromosomal elements of a microbiome followed by analysis for viral signatures, thus establishing a more focused approach for phage identifications. Using this method, we detected several novel phage genera along with those previously established. Our approach of using the whole-genome sequences of hundreds of bacterial isolates from the same site enabled us to make host assignments with high confidence, several at strain levels. Certain phage genes suggest that they provide an environment-specific selective advantage to their bacterial hosts. Our study lays the foundation for future research on directed phage isolations using specific bacterial host strains to further characterize groundwater phages, their life cycles, and their effects on groundwater microbiome and biogeochemistry.

The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment.IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, a leading cause of poultry mortality worldwide. It is crucial to control APEC in broiler breeders as it is vertically transferred to progeny via eggs. However, there is only limited knowledge on the current APEC population in breeders. This study characterized 28 APEC strains isolated from broiler breeders with colibacillosis. The genotypic-virulence characteristics as well as antimicrobial and heavy-metal resistance patterns of the isolates were determined. Results showed that O88 is the most prevalent serogroup and B2 is the predominant phylogenetic group. Among virulence genes, genes for iron acquisition (iroN and iutA), protectins (iss and ompT), and toxin production (hlyF) exhibited the highest prevalence. Further, 93% of the isolates carried at least one antimicrobial resistance gene with highest prevalence for tetracycline gene tetA. Among the isolates, 10.71% exhibited multidrug resistance. All isolates carried at least one heavy-metal resistance gene with the highest prevalence for arsenic gene arsC and the highest resistance towards silver. Our findings provide insight into the characteristics of current APEC populations in broiler breeders in Mississippi. This will help future research on the pathogenesis of APEC and the development of effective prevention and control strategies against APEC in broiler breeders.

ABSTRACT Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.

Some archaea from the genus Sulfolobus are important for bioleaching of copper, where metal resistant microorganisms are required. Biofilm generation is one of the ways microorganisms cope with some stimuli in nature, including heavy metals. The response to external factors, particularly in the biofilm form of life, is still underexplored in archaea. To explore how model thermoacidophilic archaeon Saccharolobus solfataricus faces copper stress during this lifestyle, changes in biofilms were studied using crystal violet staining, confocal fluorescence microscopy, and qPCR approaches. It was found that biofilm formation reached a maximum at 0.5 mM Cu, before starting to decrease at higher metal concentrations. The morphology of biofilms at 0.5 mM Cu was observed to be different, displaying lower thickness, different sugar patterns, and higher amounts of cells compared to standard growing conditions. Furthermore, copA, which is responsive to intracellular Cu concentration, was downregulated in biofilm cells when compared with planktonic cells exposed to the same metal concentration. The latest results suggests that cells in biofilms are less exposed to Cu than those in planktonic culture. In a PolyP-deficient strain, Cu was not able to induce biofilm formation at 0.5 mM. In summary, the findings reported here suggest that the biofilm form of life confers S. solfataricus advantages to face stress caused by Cu.Biofilm formation remains a relatively unexplored topic in archaeal research. Therefore, this knowledge in model organisms such as S. solfataricus, and how they use it to face stress, could be of great importance to engineer organisms with improved capabilities to be applied in biotechnological processes, such as bioleaching of metals. [ABSTRACT FROM AUTHOR]

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

Plasmid-mediated horizontal gene transfer (HGT) is a major driver of genetic diversity in bacteria. We experimentally validated the function of a putative mercury resistance operon present on an abundant 8-kbp native plasmid found in groundwater samples without detectable levels of mercury. Phylogenetic analyses of the plasmid-encoded mercury reductases from the studied groundwater site show them to be distinct from those reported in proximal metal-contaminated sites. We synthesized the entire native plasmid and demonstrated that the plasmid was sufficient to confer functional mercury resistance in Escherichia coli Given the possibility that natural transformation is a prevalent HGT mechanism in the low-cell-density environments of groundwaters, we also assayed bacterial strains from this environment for competence. We used the native plasmid-encoded metal resistance to design a screen and identified 17 strains positive for natural transformation. We selected 2 of the positive strains along with a model bacterium to fully confirm HGT via natural transformation. From an ecological perspective, the role of the native plasmid population in providing advantageous traits combined with the microbiome's capacity to take up environmental DNA enables rapid adaptation to environmental stresses.IMPORTANCE Horizontal transfer of mobile genetic elements via natural transformation has been poorly understood in environmental microbes. Here, we confirm the functionality of a native plasmid-encoded mercury resistance operon in a model microbe and then query for the dissemination of this resistance trait via natural transformation into environmental bacterial isolates. We identified 17 strains including Gram-positive and Gram-negative bacteria to be naturally competent. These strains were able to successfully take up the plasmid DNA and obtain a clear growth advantage in the presence of mercury. Our study provides important insights into gene dissemination via natural transformation enabling rapid adaptation to dynamic stresses in groundwater environments.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and “free” iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+- export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, a leading cause of poultry mortality worldwide. It is crucial to control APEC in broiler breeders as it is vertically transferred to progeny via eggs. However, there is only limited knowledge on the current APEC population in breeders. This study characterized 28 APEC strains isolated from broiler breeders with colibacillosis. The genotypic-virulence characteristics as well as antimicrobial and heavy-metal resistance patterns of the isolates were determined. Results showed that O88 is the most prevalent serogroup and B2 is the predominant phylogenetic group. Among virulence genes, genes for iron acquisition (iroN and iutA), protectins (iss and ompT), and toxin production (hlyF) exhibited the highest prevalence. Further, 93% of the isolates carried at least one antimicrobial resistance gene with highest prevalence for tetracycline gene tetA. Among the isolates, 10.71% exhibited multidrug resistance. All isolates carried at least one heavy-metal resistance gene with the highest prevalence for arsenic gene arsC and the highest resistance towards silver. Our findings provide insight into the characteristics of current APEC populations in broiler breeders in Mississippi. This will help future research on the pathogenesis of APEC and the development of effective prevention and control strategies against APEC in broiler breeders. [ABSTRACT FROM AUTHOR]

Metal-resistant bacteria are recommended for metal removal applications due to their rapid multiplication and growth rates. To ensure safety replenishment in contaminated areas frequently hampered by heavy metal toxicity, it is crucial to comprehend their coping mechanisms under heavy metal stress. This study primarily examines the role of EPS (exopolysaccharide) in Stenotrophomonas maltophilia, a Gram-negative, aerobic, and rod-shaped bacteria, in response to Cd, as well as the binding behavior and biosorption mechanism between EPS and Cd, using SEM and FTIR. The studies showed that Stenotrophomonas maltophilia, can resist up to 150 μM of Cd due to the binding of Cd to EPS. SEM analysis showed significant morphological changes and FTIR was to identify main structural groups like carboxyl and hydroxyl which confirms the presence of EPS. The study will also describe the mechanism of cross-reactivity between exopolysaccharide and siderophore production in metal-tolerant Stenotrophomonas maltophilia. This study proved that siderophore-mediated metal detoxification and effective absorption have been linked to metal chelation. [ABSTRACT FROM AUTHOR]

Urban sewage sludge (USS) is increasingly being used as an alternative organic amendment in agriculture. Because USS originates mostly from human excreta, partially metabolized pharmaceuticals have also been considered in risk assessment studies after reuse. In this regard, we investigated the cumulative effect of five annual USS applications on the spread of antibiotic-resistant bacteria (ARB) and their subsequent resistance to toxic metals in two unvegetated soils. Eventually, USS contained bacterial strains resistant to all addressed antibiotics with indices of resistance varying between 0.25 for gentamicin to 38% for ampicillin and azithromycin. Sludge-amended soils showed also the emergence of resistome for all tested antibiotics compared to non-treated controls. In this regard, the increase of sludge dose generally correlated with ARB counts, while soil texture had no influence. On the other hand, the multi-antibiotic resistance (MAR) of 52 isolates selected from USS and different soil treatments was investigated for 10 most prescribed antibiotics. Nine isolates showed significant MAR index (≥ 0.3) and co-resistance to Cd, As and Be as well. However, events including an extreme flash flood and the termination of USS applications significantly disrupted ARB communities in all soil treatments. In any case, this study highlighted the risks of ARB spread in sludge-amended soils and a greater concern with the recent exacerbation of antibiotic overuse following COVID-19 outbreak. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing of pathogenic bacteria Stenotrophomonas maltophilia from a less polluted environment of permafrost can help understand the intrinsic resistome of both antibiotics and metals. This study aimed to examine the maximum minimum inhibitory concentration (MIC) of both antibiotics and metals, as well as antibiotic resistance genes and metal resistance genes annotated from whole-genome sequences. The permafrost S. maltophilia was sensitive to ciprofloxacin, tetracycline, streptomycin, and bacitracin, and resistant to chloramphenicol, trimethoprim-sulfamethoxazole, erythromycin, Zn2+, Ni2+, Cu2+, and Cr6+, with a lower maximum MIC, compared with clinical S. maltophilia. The former strain belonged to the lower antibiotic resistance gene (ARG) and metal resistance gene (MRG) clusters compared with the latter ones. The permafrost strain contained no or only one kind of ARG or MRG on a single genomic island, which explained the aforementioned lower maximum MIC and less diversity of ARGs or MRGs. The result indicated that the co-occurrence of antibiotic and metal resistance was due to a certain innate ability of S. maltophilia. The continuous human use of antibiotics or metals induced selective pressure, resulting in higher MIC and more diverse ARGs and MRGs in human-impacted environments. [ABSTRACT FROM AUTHOR]

Introduction: The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. Methods: To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. Results and Discussion: In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes. [ABSTRACT FROM AUTHOR]

The bacterial strain GCP5 was isolated from the gut of a bottom-dwelling fish Lepidocephalichthys guntea , that lives in the Magurmari River near North Bengal University in Siliguri, India. GCP5 was phylogenetically assigned to the Shigella genus using whole genome-based trees, k-mer analysis, the multilocus species tree (MLST), and single nucleotide polymorphism (SNP)-based trees, and the genetic makeup of the isolate was determined following assembly of the genome sequences and genome annotation with several bioinformatics tools. The presence of a complete package of general-secretory-pathway (gsp) genes, grouped in an operon identical to a well-characterized type II secretion system (T2SS), was confirmed by genome mining of Shigella sp. GCP5. The operon's gsp genes shared the most homology with Escherichia coli gsp genes. A few more high-pathogenicity islands (HPIs) in the GCP5 genome were validated using the pan-genomes analysis pipeline (PGAP) and island viewer. Several antibiotic-resistance genes were found in this genome, as well as the existence of key antibiotic efflux pump families, allowing for the creation of a gene network of several antibiotic efflux transporters. In addition, the genome contained genes specific for nickel transport, the nik ABCD system, and the RND family transporter cus CFBA, which confers resistance to copper and silver by effluxing out Cu+ and Ag+ ions. [ABSTRACT FROM AUTHOR]

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.

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.IMPORTANCE 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.

Trace elements (TEs) availability, biochemical activity and functional gene diversity was studied in a Cu-contaminated soil, revegetated for six years with a mixed stand of willow, black poplar, and false indigo-bush, and amended or not with compost plus dolomitic limestone (OMDL). The OMDL amendment significantly reduced Cu and As availability and soil toxicity, and increased the biochemical activity and microbial functional diversity assessed with the GEOCHIP technique, as compared to the unamended soil (Unt). The OMDL soil showed significantly higher abundance of 25 functional genes involved in decomposition organic compounds, and 11, 3 and 11 functional genes involved in the N, P and S biogeochemical cycles. Functional gene abundance was positively correlated with nutrient contents but negatively correlated with Cu availability and soil toxicity. The abundance of microbial functional genes encoding for resistance to various TEs also increased, possibly due to the microbial proliferation and lower Cu exposure in the presence of high total soil Cu concentration. Genes encoding for antibiotic resistance due to the co-occurrence of TEs and antibiotic resistant genes on genetic mobile elements. Overall, phytomanagement confirmed its potential to restore the biological fertility and diversity of a severely Cu-contaminated soil, but the increase of TEs and antibiotic resistant gene abundances deserve attention in future studies.

Environmental reservoir of antibiotic and other resistance genes are an emerging public health concern, and especially so in food production sites. This study aimed to explore the co-occurrence of antibiotic resistance genes (ARG) and resistance genes to other compounds, including heavy metals and biocides, in urban vegetable garden soils. Total soil DNA was extracted and subjected to metagenomic analysis on ARG, metal resistance genes (MRG), biocide resistance genes (BRG), multi-compound resistance genes (MCRG), and mobile genetic elements (MGE). The relative abundance of ARGs and MRGs were 2.00–2.90 × 10−1 and 4.22–6.00 × 10−2 copies per 16S rRNA gene, respectively. The most abundant ARGs included multidrug, vancomycin, bacitracin, fosmidomycin, and macrolide-lincosamide-streptogramin (MLS) resistance genes. Common MRGs consisted of genes with resistance to copper (dnaK, copA, cueA), arsenic (arsM), multi-metal (recG_BAC0356, ruvB, cueA), iron (acn), and chromium (recG). BRGs identified were those conferring resistance to multi-biocide (smrA, mexW, tbtB, and emhB), peroxide (sodA, sodB), and phenolic compound (triC). MCRGs were found with resistance to biocide-metal (dpsA, ruvB, actP, ideR), drug-biocide-metal (mdtB), or drug-biocide (mexF, ceoB, sme, mexK, sdeB). Multidrug resistance genes (mexF, mexW, multidrug transporter gene), MLS resistance gene (macB), and MCRG (ruvB) showed most correlations with ARGs and resistance genes to other compounds. The data demonstrate extensive co-occurrence of ARGs, MRGs, BRGs, and MCRGs in urban agricultural environments and suggest the great potential of soil contaminants as co-selection agents for antibiotic resistance.

IntroductionThe antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk.MethodsTo further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences.Results and DiscussionIn silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

Bacterial antimicrobial resistance (AMR) has emerged as a significant concern worldwide. The microbial community profile and potential AMR level in aquaculture ponds are often undervalued and attract less attention than other aquatic environments. We used amplicon and metagenomic shotgun sequencing to study microbial communities and AMR in six freshwater polyculture ponds in rural and urban areas of Bangladesh. Amplicon sequencing revealed different community structures between rural and urban ponds, with urban ponds having a higher bacterial diversity and opportunistic pathogens including Streptococcus , Staphylococcus , and Corynebacterium. Despite proteobacterial dominance, Firmicutes was the most interactive in the community network, especially in the urban ponds. Metagenomes showed that drug resistance was the most common type of AMR found, while metal resistance was only observed in urban ponds. AMR and metal resistance genes were found mainly in beta and gamma-proteobacteria in urban ponds, while AMR was found primarily in alpha-proteobacteria in rural ponds. We identified potential pathogens with a high profile of AMR and metal resistance in urban aquaculture ponds. As these ponds provide a significant source of protein for humans, our results raise significant concerns for the environmental sustainability of this food source and the dissemination of AMR into the food chain. [Display omitted] • Bacterial diversity and antimicrobial resistance were investigated in rural and urban aquaculture ponds. • Higher bacterial diversity and more opportunistic pathogens were observed in urban ponds. • Metal resistance classes were observed only in urban ponds. • Beta and gamma-proteobacteria were involved in the majority of resistance. [ABSTRACT FROM AUTHOR]

The study’s primary purposes were establishing the number of microorganisms that exhibit hydrolytic activity in Antarctic soil and mosses samples, isolation of metal-resistant strains of bacteria, and description of their physiological and biochemical properties. Samples collected during the XXIII Ukrainian Antarctic Expedition in 2019 were used. The number of colony-forming units of microorganisms exhibiting proteolytic, amylolytic, cellulase, lipolytic activity was studied. Pure bacterial cultures were isolated using standard microbiological methods. Determination of resistance of isolates to heavy metals was estimated after their cultivation during ten days on agar plates with different concentrations of CdCl2 ⋅ 2.5H2O, CoCl2 ⋅ 6H2O, K2Cr2O7, FeSO4 ⋅ 7H2O, CuCl2 ⋅ 2H2O. Identification of strains was based on the sequencing of the 16S rRNA gene, morphological, physiological, and biochemical properties. Among the 23 isolates, nine metal-resistant strains were selected, four of which were identified as Pseudomonas yamanorum ІМV B-7916 and 79_102, and as P. arsenicoxidans 5A_1N_24, and 89_1T_89. Among the selected strains, the most resistant to heavy metals was P. yamanorum 79_102. All studied strains synthesize lipases during growth on medium with tween-20, which contains 0.5–1 mM of ferrous sulfate and copper (II) chloride. The studied strains produce exopolysaccharides during growth at 6 and 22 °C. The most effective among these strains exopolysaccharides are synthesized by P. arsenicoxidans 5A_1N_24 — 768 mg/g of dry weight. Our results expand the knowledge about the diversity of microorganisms of extreme biotopes, their properties, resistance to heavy metal compounds.

Bacterial metal metabolism and resistance genes are important indicators for microbial ecology, biogeochemistry, and biotechnology. Their investigation enables understanding how bacteria influence the geochemical cycles of elements and how bacteria can be employed in the bioremediation of polluted areas. For environmental screening, the polymerase chain reaction (PCR) technique has been used as the method of choice for the detection of metal metabolism and resistance genes, and subsequently, many different PCR primer pairs have been developed for such screening. The aim of this study was the development of a database of PCR primers suitable for the screening of metal metabolism and resistance genes in environmental samples. We conducted an in silico benchmark of 291 previously published PCR primer pairs designed to amplify genes involved in bacterial metabolism and resistance to arsenic, cadmium, chromium, cobalt, copper, gold, iron, lead, mercury, nickel, silver, and zinc. Our analysis showed that only 16 PCR primer pairs can be considered well designed and suitable for reliable screening in an environmental setup. Furthermore, 55 primer pairs delivered ambiguous results, while the remaining 220 pairs showed no in silico PCR product within their expected product size range. Based on the obtained results, we developed a ranked database (LCPDb-MET; http://lcpdb.ddlemb.com/met/) of primers suitable for the screening of bacterial metal metabolism and resistance genes in various environmental settings. Moreover, since in the course of the study we recognized a serious problem with the classification of metal metabolism and resistance reference sequences, we developed also the METGeneDb database (http://lcpdb.ddlemb.com/downloads). This database covers 106 genes with 7,624 unique sequences divided into 137 potential gene subclusters and it can be used as the reference database in the genomic and metagenomic studies.

ABSTRACT Bacterial pathogens and their hosts engage in intense competition for critical nutrients during infection, including metals such as iron, copper, and zinc. Some metals are limited by the host, and some are deployed by the host as antimicrobials. To counter metal limitation, pathogens deploy high-affinity metal acquisition systems, best exemplified by siderophores to acquire iron. Although pathogen strategies to resist the toxic effects of high Cu have been elucidated, the role of Cu starvation and the existence of Cu acquisition systems are less well characterized. In this study, we examined the role of diisonitrile chalkophores of pathogenic mycobacteria, synthesized by the enzymes encoded by the virulence-associated nrp gene cluster, in metal acquisition. nrp gene cluster expression is strongly induced by starvation or chelation of Cu but not starvation of Zn or excess Cu. Mycobacterium tuberculosis and Mycobacterium marinum strains lacking the nrp-encoded nonribosomal peptide sythetase, the fadD10 adenylate-forming enzyme, or the uncharacterized upstream gene ppe1 are all sensitized to Cu, but not Zn, starvation. This low Cu sensitivity is rescued by genetic complementation or by provision of a synthetic diisonitrile chalkophore. These data demonstrate that diisonitrile lipopeptides in mycobacteria are chalkophores that facilitate survival under Cu-limiting conditions and suggest that Cu starvation is a relevant stress for M. tuberculosis in the host. IMPORTANCE Bacterial pathogens and their hosts engage in intense competition for nutrients, including metals. Mycobacterium tuberculosis, the cause of tuberculosis, lives within host macrophages and is subject to diverse stresses, including metal excess and metal limitation. In this study, we demonstrated that the nrp gene cluster, required for M. tuberculosis virulence and which directs synthesis of diisonitrile lipopeptides, mediates copper acquisition. Copper, but not zinc, deprivation strongly induces diisonitrile biosynthesis, and M. tuberculosis strains lacking the nrp gene, or the associated genes fadD10 or ppe1, are all sensitized to copper chelation or copper deprivation. These results establish a copper binding, or chalkophore, system in M. tuberculosis and indicate that resistance to copper restriction plays an important role in the ability of this global pathogen to cause infection.

ABSTRACT The environmental deterioration produced by heavy metals derived from anthropogenic activities has gradually increased. The worldwide dissemination of toxic metals in crop soils represents a threat for sustainability and biosafety in agriculture and requires strategies for the recovery of metal-polluted crop soils. The biorestoration of metal-polluted soils using technologies that combine plants and microorganisms has gained attention in recent decades due to the beneficial and synergistic effects produced by its biotic interactions. In this context, native and heavy metal-resistant plant growth-promoting bacteria (PGPB) play a crucial role in the development of strategies for sustainable biorestoration of metal-contaminated soils. In this study, we present a genomic analysis and characterization of the rhizospheric bacterium Bacillus megaterium HgT21 isolated from metal-polluted soil from Zacatecas, Mexico. The results reveal that this autochthonous bacterium contains an important set of genes related to a variety of operons associated with mercury, arsenic, copper, cobalt, cadmium, zinc and aluminum resistance. Additionally, halotolerance-, beta-lactam resistance-, phosphate solubilization-, and plant growth-promotion-related genes were identified. The analysis of resistance to metal ions revealed resistance to mercury (HgII+), arsenate [AsO4]³–, cobalt (Co2+), zinc (Zn2+), and copper (Cu2+). Moreover, the ability of the HgT21 strain to produce indole acetic acid (a phytohormone) and promote the growth of Arabidopsis thaliana seedlings in vitro was also demonstrated. The genotype and phenotype of Bacillus megaterium HgT21 reveal its potential to be used as a model of both plant growth-promoting and metal multiresistant bacteria. IMPORTANCE Metal-polluted environments are natural sources of a wide variety of PGPB adapted to cope with toxic metal concentrations. In this work, the bacterial strain Bacillus megaterium HgT21 was isolated from metal-contaminated soil and is proposed as a model for the study of metal multiresistance in spore-forming Gram-positive bacteria due to the presence of a variety of metal resistance-associated genes similar to those encountered in the metal multiresistant Gram-negative Cupriavidus metallidurans CH34. The ability of B. megaterium HgT21 to promote the growth of plants also makes it suitable for the study of plant-bacteria interactions in metal-polluted environments, which is key for the development of techniques for the biorestoration of metal-contaminated soils used for agriculture.

ABSTRACT: Objectives: The aim of this study was to characterise the whole genome sequence of multidrug resistance (MDR) Salmonella Rissen strain SCSW714 of swine origin. Methods: The whole genome of SCSW714 was sequenced using the Illumina Hiseq platform combined with the Nanopore PromethION platform and assembled by software Unicycler. NCBI Prokaryotic Genome Annotation Pipeline was used to annotate the genome of SCSW714. The sequence type (ST) as well as antimicrobial resistance genes were determined by MLST 2.0 and ResFinder 4.1, respectively. Results: The chromosome of SCSW714 was 4 928 262 bp in size with a GC content of 52.1%. Strain SCSW714 contained a total of 4759 genes, including 4531 protein-coding sequences, 108 pseudogenes and 120 RNAs. It belonged to ST469 and carried six resistance genes including tet(A), dfrA12, sul3, aadA2, aadA1 and blaTEM-1b. All of the six resistance genes were carried by a novel MDR Tn7-pco-sil transposon designated as Tn6777. Tn6777 was stable in S. Rissen and could be excised from S. Rissen chromosome. Conclusion: We report a complete genome sequence of S. Rissen and characterised a novel MDR Tn7-like pco- and sil-containing transposon for the first time. The excision of Tn6777 suggests that Tn6777 has functional activity and may promote the co-spreading of metal and antimicrobial resistance genes. The complete genome sequence of S. Rissen strain SCSW714 provides valuable information for tracing the potential spread from swine to humans.

The spread of antibiotic resistance is becoming a serious global health concern. Numerous studies have been done to investigate the dynamics of antibiotic resistance genes (ARGs) in both indoor and outdoor environments. Nonetheless, few studies are available about the dynamics of the antibiotic resistome (total content of ARGs in the microbial cultures or communities) under stress in outer space environments. In this study, we aimed to experimentally investigate the dynamics of ARGs and metal resistance genes (MRGs) in Kombucha Mutualistic Community (KMC) samples exposed to Mars-like conditions simulated during the BIOMEX experiment outside the International Space Station with analysis of the metagenomics data previously produced. Thus, we compared them with those of the respective non-exposed KMC samples. The antibiotic resistome responded to the Mars-like conditions by enriching its diversity with ARGs after exposure, which were not found in non-exposed samples (i.e., tet and van genes against tetracycline and vancomycin, respectively). Furthermore, ARGs and MRGs were correlated; therefore, their co-selection could be assumed as a mechanism for maintaining antibiotic resistance in Mars-like environments. Overall, these results highlight the high plasticity of the antibiotic resistome in response to extraterrestrial conditions and in the absence of anthropogenic stresses. [ABSTRACT FROM AUTHOR]

Bacteriophage GiJojo is a myovirus isolated from soil that infects Streptomyces mirabilis NRRL B-2400, with a genome length of 115,161 bp containing 180 genes and 29 tRNAs. Of those genes, 59 have been assigned functions. GiJojo is a member of the BS cluster of actinobacteriophages.

ABSTRACT: Objectives: The aim of this study was to describe the genome sequences of 38 antibiotic-resistant Escherichia coli isolated from veal calves. Methods: The isolates were recovered in 2015 from nine veal farms in the eastern USA and were screened for antibiotic susceptibility using an automated microdilution procedure. The draft genomes were sequenced on an Illumina NextSeq 500 platform and were assembled using SPAdes. Results: In total, 294 resistance genes, categorised into 42 unique genes, conferring resistance to seven different antibiotic classes were detected. Extended-spectrum β-lactamase (ESBL) genes (blaCTX-M and blaCMY) and the azithromycin resistance gene mph(A) were detected in multiple genomes. Furthermore, mutations in gyrA, parC and parE conferring resistance to fluoroquinolones were detected, as were mutations in the ampC promoter responsible for hyperproduction of β-lactamases. We identified 25 unique sequence types (STs), including STs that are associated with extraintestinal infections. Conclusion: The results of this study indicate a high level of diversity among multidrug-resistant E. coli isolates from veal operations. The identification of multiple isolates encoding resistance to β-lactams, macrolides and fluoroquinolones as well as virulence factors responsible for human infections warrants more study on the ecology of antibiotic resistance in veal operations.

ABSTRACT Development of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli is one the greatest threats faced by mankind. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants (i.e., sheep and goats) in China. The aim of this study was to identify and characterize the resistance profiles, resistomes, and sequence features of 67 ESBL-producing E. coli isolates from sheep in northwest China. The findings showed that blaCTX-M and blaTEM were the most prevalent. Interestingly, we found that the resistance gene mcr-1 was widespread in sheep merely from Shaanxi areas, accounting for 19.2% (5/26). The highly prevalent serotypes and FumC-FimH (CH) typing isolates were O8 and C4H32, respectively. High-risk E. coli clones, such as sequence type 10 (ST10), ST23, ST44, and ST58, were also found in China’s sheep population. A total of 67 ESBL-producing isolates were divided into five phylogenetic groups, namely, B1 (n = 47, 70.1%), B2 (n = 1, 1.5%), C (n = 14, 20.9%), E (n = 1, 1.5%), and F (n = 1, 1.5%), with the phylogenetic groups for 3 isolates (4.5%) remaining unknown. Moreover, ESBL-producing E. coli isolates were also characterized by the abundance and diversity of biocide/metal resistance genes and insert sequences. We found that in ESBL-producing E. coli isolates, there were two different types of isolates, those containing ESBL genes or not, which led to large discrepancies between resistance phenotypes and resistomes. In summary, our study provides a comprehensive overview of resistance profiles and genome sequence features in ESBL-producing E. coli and highlights the possible role of sheep as antibiotic resistance gene disseminators into humans. IMPORTANCE Antimicrobial resistance (AMR), especially the simultaneous resistance to several antibiotics (multidrug resistance [MDR]), is one of the greatest threats to global public health in the 21st century. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants in China. This study is the largest and most comprehensive analysis of ESBL-producing E. coli isolates from sheep, including antibiotic resistance profiles, phylogenetic groups, serotypes, multilocus sequence types (MLST), insert sequences (IS), antibiotic resistance genes, disinfectant resistance genes, and heavy metal resistance genes. We recommend extending the surveillance of AMR of sheep-origin E. coli to prevent future public health risks.

Some archaea from the genus Sulfolobus are important for bioleaching of copper, where metal resistant microorganisms are required. Biofilm generation is one of the ways microorganisms cope with some stimuli in nature, including heavy metals. The response to external factors, particularly in the biofilm form of life, is still underexplored in archaea. To explore how model thermoacidophilic archaeon Saccharolobus solfataricus faces copper stress during this lifestyle, changes in biofilms were studied using crystal violet staining, confocal fluorescence microscopy, and qPCR approaches. It was found that biofilm formation reached a maximum at 0.5 mM Cu, before starting to decrease at higher metal concentrations. The morphology of biofilms at 0.5 mM Cu was observed to be different, displaying lower thickness, different sugar patterns, and higher amounts of cells compared to standard growing conditions. Furthermore, copA, which is responsive to intracellular Cu concentration, was downregulated in biofilm cells when compared with planktonic cells exposed to the same metal concentration. The latest results suggests that cells in biofilms are less exposed to Cu than those in planktonic culture. In a PolyP-deficient strain, Cu was not able to induce biofilm formation at 0.5 mM. In summary, the findings reported here suggest that the biofilm form of life confers S. solfataricus advantages to face stress caused by Cu.Biofilm formation remains a relatively unexplored topic in archaeal research. Therefore, this knowledge in model organisms such as S. solfataricus, and how they use it to face stress, could be of great importance to engineer organisms with improved capabilities to be applied in biotechnological processes, such as bioleaching of metals.

Growing concerns about the adverse effects of Pb(II) on public health have drawn much attention to the development of efficient and environmentally friendly treatment methods. Here we report the isolation of a novel Pb(II)-resistant Methylobacterium sp. MTS1 strain from abandoned mine soil that has potential as a biosorbent for the removal of Pb(II). The isolated MTS1 strain not only showed high resistance even at 1,000 mg/L, but also showed excellent performance in removing Pb(II) through biosorption. The maximum removal capacity and removal efficiency of Pb(II) were found to be 56.55±6.2 mg/g and 98.6±0.6%, respectively, under optimized conditions (pH 7; biomass, 1 g/L; contact time, 60 min). Hydroxyl, amide, carboxyl, phosphate, nitro compound, and disulfide groups as well as various functional groups such as C, O, and P were identified as key factors for Pb(II) removal. In addition, equilibrium data obtained by biosorption and adsorption kinetic model were in agreement with Langmuir isotherm and pseudo-second-order models, indicating that the biosorption process involved monolayer chemisorption at uniformly distributed active sites on the surface of the MTS1 strain. [ABSTRACT FROM AUTHOR]

Abstract Background The use of metal-containing and antimicrobial compounds is a common practice in aquaculture operations in Nigeria. Only a few studies have focused on the impact of these chemicals on metal and antibiotic resistance development in the bacterial flora of aquaculture. This study therefore sets out to fill this gap. Results Five metals, namely copper (Cu), zinc (Zn), lead (Pb), nickel (Ni) and chromium (Cr), were detected at different concentrations in the pond water samples, with most values obtained exceeding the maximum recommended national standard for fisheries and recreation water quality criteria set by the National Environmental Standards and Regulations Enforcement Agency (NESREA) in Nigeria. A total of 121 Vibrio spp. were recovered from the aquaculture pond water with Vibrio cholerae having the highest frequency (37) followed by Vibrio parahaemolyticus (36), Vibrio vulnificus (36) and Vibrio alginolyticus (12). Seventy-five isolates were recovered from ponds with history of antibiotic use, while the ponds with no recognized history of antibiotic use had forty-six isolates. The percentage resistance to antibiotics in the isolates was: ampicillin (88.5%), amoxicillin–clavulanate (44.3%), tetracycline (41%), trimethoprim-sulfamethoxazole (22.1%), chloramphenicol (15.6%), ciprofloxacin (11.5%), imipenem (5.7%), ceftazidime (4.1%) and gentamicin (0%). All the isolates tolerated all the metals to 400 µg/mL concentration while only the trio of Pb, Ni and Cr were tolerated beyond the concentration. Zn and Cu appeared to be the most toxic metals on Vibrio spp., with no isolate surviving beyond the 400 µg/mL concentration mark, while 57 of the 121 isolates survived Cr at the highest concentration (3200 µg/mL), making it the most tolerated of the metals. Conclusion This study reports a high level of metal contamination in the aquaculture ponds studied. The concentration of the metals exceeded the maximum permissible limits in Nigeria. Also, there was coexistence of metal and antibiotic resistance in the bacteria recovered from the aquaculture ponds. There is a need to regulate the use of chemicals, especially antibiotics and some biocidal compounds, to clamp down on the introduction of metals and antibiotics into the aquaculture systems, as these chemicals could facilitate the development of resistance traits in aquaculture bacteria by creating a selective pressure in the aquaculture environment.

ABSTRACT The genome of the metal-resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans contains a large number of horizontally acquired plasmids and genomic islands that were integrated into its chromosome or chromid. For the C. metallidurans CH34 wild-type strain growing under nonchallenging conditions, 5,763 transcriptional starting sequences (TSSs) were determined. Using a custom-built motif discovery software based on hidden Markov models, patterns upstream of the TSSs were identified. The pattern TTGACA, −35.6 ± 1.6 bp upstream of the TSSs, in combination with a TATAAT sequence 15.8 ± 1.4 bp upstream occurred frequently, especially upstream of the TSSs for 48 housekeeping genes, and these were assigned to promoters used by RNA polymerase containing the main housekeeping sigma factor RpoD. From patterns upstream of the housekeeping genes, a score for RpoD-dependent promoters in C. metallidurans was derived and applied to all 5,763 TSSs. Among these, 2,572 TSSs could be associated with RpoD with high probability, 373 with low probability, and 2,818 with no probability. In a detailed analysis of horizontally acquired genes involved in metal resistance and not involved in this process, the TSSs responsible for the expression of these genes under nonchallenging conditions were assigned to RpoD- or non-RpoD-dependent promoters. RpoD-dependent promoters occurred frequently in horizontally acquired metal resistance and other determinants, which should allow their initial expression in a new host. However, other sigma factors and sense/antisense effects also contribute—maybe to mold in subsequent adaptation steps the assimilated gene into the regulatory network of the cell. IMPORTANCE In their natural environment, bacteria are constantly acquiring genes by horizontal gene transfer. To be of any benefit, these genes should be expressed. We show here that the main housekeeping sigma factor RpoD plays an important role in the expression of horizontally acquired genes in the metal-resistant hydrogen-oxidizing bacterium C. metallidurans. By conservation of the RpoD recognition consensus sequence, a newly arriving gene has a high probability to be expressed in the new host cell. In addition to integrons and genes travelling together with that for their sigma factor, conservation of the RpoD consensus sequence may be an important contributor to the overall evolutionary success of horizontal gene transfer in bacteria. Using C. metallidurans as an example, this publication sheds some light on the fate and function of horizontally acquired genes in bacteria.