Heavy Metal Tolerance Genes Research Articles

Genomic characterization of a clonal emergent Salmonella Minnesota lineage in Brazil reveals the presence of a novel megaplasmid of resistance and virulence.

Salmonella Minnesota has emerged in Brazil as the predominant serovar in poultry and poultry products, along with Salmonella Heidelberg. To understand the emergence of Salmonella Minnesota over the last few years in Brazil, we performed a comparative analysis between 69 selected S. Minnesota genomes from Pathogen Detection database and 65 clonal emergent genomes isolated from Brazil. We demonstrate the presence of multidrug resistance genes against tetracycline [tet(A)], sulfonamide (sul2), and AmpC beta-lactamase (blaCMY-2) in emergent genomes, along with the carriage of a megaplasmid of resistance and virulence (~210 kb), designated pESM (plasmid for emergent Salmonella Minnesota). pESM is an IncC/A2 plasmid predicted to increase S. Minnesota environmental tolerance to mercury (mer operon) and provide resistance to tetracycline and ampicillin due to the presence of tet(A) and blaCMY-2, respectively. Moreover, pESM carries the yersiniabactin siderophore (high-pathogenicity island of Yersinia) related to the iron uptake. The temporal inference demonstrated that the most recent common ancestor dated from ~1978 and that the clonal emergent genomes carrying the pESM belong to a completely different lineage of S. Minnesota. Our results indicate that the presence of pESM likely contributes to the emergence of S. Minnesota and is precisely related to the successful spread of this particular clonal lineage in Brazil.IMPORTANCESalmonella Minnesota has emerged in Brazil as one of the leading serovars related to human and animal infection, presenting high virulence and antibiotic resistance to drugs classified as the highest priority for clinical treatment in humans. This study performed whole-genome sequencing, temporal analysis, and phylogenetics to understand the genetic insights related to the emergence of Salmonella Minnesota in Brazil. Long-read sequencing has led to the identification and characterization of a unique megaplasmid carrying virulence, antibiotic resistance, and heavy-metal tolerance genes, which may play a central role in S. Minnesota's successful emergence in Brazil and possibly worldwide. The potentially high transmissibility of this plasmid between clones and serovars represents a risk to public health since its acquisition may increase Salmonella's fitness, virulence, resistance, and persistence. Understanding the genetic aspects related to the emergence of serovars can help devise measures to mitigate the spread of hazardous multidrug-resistant strains.

Antimicrobial resistance, virulence potential and genomic epidemiology of global genomes of the rare Salmonella enterica serovar Orion.

Our aim is to characterize through whole-genome sequencing (WGS) the antimicrobial resistance (AMR) and heavy metal tolerance (HMT) genes content, plasmid presence, virulence potential and genomic diversity of the rare non-typhoid Salmonella enterica serovar Orion (S. Orion) from 19 countries of the African, American, Eastern Mediterranean, European, Southeastern Asia and Western Pacific regions. Totally 324 S. Orion genomes were screened for AMR, HMT and virulence genes, plasmids and Salmonella Pathogenicity Islands (SPIs). Genomic diversity was investigated using Multi-Locus Sequence Typing (MLST) and core-genome MLST (cgMLST). Efflux pump encoding genes mdsA and mdsB were present in all genomes analysed, while quinolone chromosomal point mutations and aminoglycoside, beta-lactam, colistin, lincosamide, macrolide, phenicol, sulphonamide, trimethoprim, tetracycline and disinfectant resistance genes were found in 0.3%-5.9%. A total of 17 genomes (5.2%) from Canada, the United Kingdom, the USA and Tanzania showed a potential multi-drug resistance profile. Gold tolerance genes golS and golT were detected in all genomes analysed, while arsenic, copper, mercury, silver and tellurium tolerance genes were found in 0.3%-35.5%. Col(MGD2) was the most frequently detected plasmid, in 15.4% of the genomes. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, serum resistance, stress adaptation, type III secretion systems and six SPIs (1, 2, 3, 4, 5, 9, 12, 13, 14 and C63PI) were detected. ST639 was assigned to 89.2% of the S. Orion genomes, while cgMLST showed core-genome STs and clusters of strains specific by countries. The high virulence factor frequencies, the genomic similarity among some non-clinical and clinical strains circulating worldwide and the presence of a strain carrying a resistance gene against a last resource antimicrobial like colistin, highlight the potential risk of S. Orion strains for public health and food safety and reinforce the importance to not underestimate the potential hazard of rare non-typhoid Salmonella serovars.

Different survival strategies of the phosphate-mineralizing bacterium Enterobacter sp. PMB-5 in response to cadmium stress: Biomineralization, biosorption, and bioaccumulation

The phosphate-mineralizing bacteria (PMBs) has shown great potential as a sustainable solution to support pollution remediation through its induced mineralization capacity. However, few studies have been conducted on the mechanism of cadmium (Cd) tolerance in PMBs. In this study, a PMB strain, Enterobacter sp. PMB-5, screened from Cd-contaminated rhizosphere soil, has high resistance to Cd (540 - 1220 mg/L) and solubilized phosphate (232.08 mg/L). The removal experiments showed that the strain PMB-5 removed 71.69–98.24% and 34.83–76.36% of Cd with and without biomineralization, respectively. The characterization result of SEM, EDS, TEM, XPS and XRD revealed that PMB-5 induced Cd to form amorphous phosphate precipitation through biomineralization and adopted different survival strategies, including biomineralization, bioaccumulation, and biosorption to resistance Cd in the microbial induced phosphate precipitation (MIPP) system and the non-MIPP system, respectively. Moreover, the results of whole genome sequencing and qRT-PCR indicated that phosphorus metabolism genes such as pst, pit, phn, ugp, ppk, etc. and heavy metal tolerance genes (including ion transport, ion efflux, redox, antioxidant stress), such as czcD, zntA, mgtA, mgtC, katE, SOD2, dsbA, cysM, etc. were molecular for the PMB-5 mineralization and Cd tolerance of PMB-5. Together, our findings suggested Enterobacter sp. PMB-5 is a potential target for developing more effective bioinoculants for Cd contamination remediation.

Whole genome analyses of toxicants tolerance genes of Apis mellifera gut-derived Enterococcus faecium strains

BackgroundBecause of its social nature, the honeybee is regularly exposed to environmental toxicants such as heavy metals and xenobiotics. These toxicants are known to exert strong selective pressure on the gut microbiome’s structure and diversity. For example, resistant microbial members are more likely to dominate in maintaining a stable microbiome, which is critical for bee health. Therefore, the aim of this study was to examine the Enterococcus faecium strains isolated from bee guts for their in vitro growth and tolerability to diverse heavy metals and xenobiotics. An additional aim was to analyze the genomes of E. faecium isolates to assess the molecular bases of resistance and compare them with E. faecium species isolated from other environmental sources.ResultsThe E. faecium bee isolates were able to tolerate high levels (up to 200 mg/L) of toxicants, including cadmium, zinc, benzoate, phenol and hexane. Moreover, the isolates could tolerate toluene and copper at up to 100 mg/L. The genome of E. faecium Am5, isolated from the larval stage of Apis mellifera gut, was about 2.7 Mb in size, had a GC content of 37.9% and 2,827 predicted coding sequences. Overall, the Am5 genome features were comparable with previously sequenced bee-gut isolates, E. faecium Am1, Bee9, SM21, and H7. The genomes of the bee isolates provided insight into the observed heavy metal tolerance. For example, heavy metal tolerance and/or regulation genes were present, including czcD (cobalt/zinc/cadmium resistance), cadA (exporting ATPase), cutC (cytoplasmic copper homeostasis) and zur (zinc uptake regulation). Additionally, genes associated with nine KEGG xenobiotic biodegradation pathways were detected, including γ-hexachlorocyclohexane, benzoate, biphenyl, bisphenol A, tetrachloroethene, 1,4-dichlorobenzene, ethylbenzene, trinitrotoluene and caprolactam. Interestingly, a comparative genomics study demonstrated the conservation of toxicant resistance genes across a variety of E. faecium counterparts isolated from other environmental sources such as non-human mammals, humans, avians, and marine animals.ConclusionsHoneybee gut-derived E. faecium strains can tolerate a variety of heavy metals. Moreover, their genomes encode many xenobiotic biodegradation pathways. Further research is required to examine E. faecium strains potential to boost host resistance to environmental toxins.

The rare Salmonella enterica serovar Isangi: genomic characterization of the antimicrobial resistance, virulence potential and epidemiology of Brazilian strains in comparison to global isolates.

Introduction. Salmonella enterica serovar Isangi (S. Isangi) is a rare non-typhoidal serovar, related to invasive nosocomial infections in various countries and to increasing antimicrobial resistance rates.Gap statement. Despite existing reports on S. Isangi, there is a lack of information of specific traits regarding this serovar, which could be improved through genomic analyses.Aim. Our goals were to characterize the antimicrobial resistance, virulence potential and genomic relatedness of 11 S. Isangi strains from Brazil in comparison to 185 genomes of global isolates using whole-genome sequencing (WGS) data.Methodology. Phenotypic resistance was determined by disc-diffusion. The search for resistance genes, plasmids, prophages, Salmonella pathogenicity islands (SPIs) and virulence genes, plus multi-locus sequence typing (MLST) and core-genome MLST (cgMLST) were performed using WGS.Results. Brazilian S. Isangi strains showed phenotypic resistance to nalidixic acid, ciprofloxacin and streptomycin, and harboured antimicrobial resistance [qnrB19, aac(6')-Iaa, mdsAB] and heavy metal tolerance (arsD, golST) genes. Col(pHAD28) and IncFII(S) plasmids, virulence genes related to adherence, macrophage induction, magnesium uptake, regulation and type III secretion systems, 12 SPIs and eight prophages were detected. The 185 additional global genomes analysed harboured resistance genes against 11 classes of antimicrobial compounds, 22 types of plasmids, 32 prophages, 14 SPIs, and additional virulence genes related to serum resistance, stress adaptation and toxins. Sequence type (ST)216 was assigned to genomes from Brazil and other countries, while ST335 was the most frequent ST, especially among South African genomes. cgMLST showed that Brazilian genomes were more closely related to genomes from European and African countries, the USA and Taiwan, while the majority of South African genomes were more closely related among each other.Conclusion. The presence of S. Isangi strains from Brazil and different countries showing a close genomic correlation, antimicrobial resistance profiles to drugs used in human therapy and a large number of virulence determinants reinforced the need for stronger initiatives to monitor rare non-typhoidal Salmonella serovars such as S. Isangi in order to prevent its dissemination among human and non-human sources.

Genotypic and Phenotypic Characterization of Antimicrobial and Heavy Metal Tolerance in Salmonella enterica and Escherichia coli Isolates from Swine Feed Mills

Antimicrobials and heavy metals are commonly used in the animal feed industry. The role of in-feed antimicrobials on the evolution and persistence of resistance in enteric bacteria is not well described. Whole-Genome Sequencing (WGS) is widely used for genetic characterizations of bacterial isolates, including antimicrobial resistance, heavy metal tolerance, virulence factors, and relatedness to other sequenced isolates. The goals of this study were to i) use WGS to characterize Salmonella enterica (n = 33) and Escherichia coli (n = 30) isolated from swine feed and feed mill environments; and ii) investigate their genotypic and phenotypic antimicrobial and heavy metal tolerance. Salmonella isolates belonged to 10 serovars, the most common being Cubana, Senftenberg, and Tennessee. E. coli isolates were grouped into 22 O groups. Phenotypic resistance to at least one antimicrobial was observed in 19 Salmonella (57.6%) and 17 E. coli (56.7%) isolates, whereas multidrug resistance (resistant to ≥3 antimicrobial classes) was observed in four Salmonella (12%) and two E. coli (7%) isolates. Antimicrobial resistance genes were identified in 17 Salmonella (51%) and 29 E. coli (97%), with 11 and 29 isolates possessing genes conferring resistance to multiple antimicrobial classes. Phenotypically, 53% Salmonella and 58% E. coli presented resistance to copper and arsenic. All isolates that possessed the copper resistance operon were resistant to the highest concentration tested (40 mM). Heavy metal tolerance genes to copper and silver were present in 26 Salmonella isolates. Our study showed a strong agreement between predicted and measured resistances when comparing genotypic and phenotypic data for antimicrobial resistance, with an overall concordance of 99% and 98.3% for Salmonella and E. coli, respectively.

Comparison of cell size, chlorophyll fluorescence and cadmium (Cd2+) bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to Cd2+

Background: Heavy metal contamination presents a constant threat to biological systems. Simultaneously, heavy metals have become one of the major contaminants in the aquatic ecosystems. In this regard, the investigation of heavy metal-tolerance genes in algae is relevant. Chlamydomonas reinhardtii is a unicellular green alga, and an excellent model organism used in heavy metal studies. In C. reinhardtii, a novel gene designated as Cia7, was hypothesized to play a role in heavy metal homeostasis due to CIA7’s conserved cysteine-residue motif. This study compared two strains of C. reinhardtii, cc4425, the wild-type with the functional CIA7 protein and cc5013, the mutant strain with the disrupted cia7- gene. The hypothesis was that the expression of Cia7 contributes to an increased cadmium (Cd)-tolerance in C. reinhardtii. The Cd-tolerance would be described by physiological markers of microalgae health, and by intracellular accumulation of the metal. Methods: The objectives of this study were (1) to compare chlorophyll fluorescence and cell size in cc4425 and cc5013 exposed to Cd2+, and (2) to compare Cd2+ bioaccumulation in cc4425 and cc5013 strains in different growth media. Flow cytometry, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis were performed. Results: There was no significant statistical difference in Cd2+ bioaccumulation between the two strains, cc4425 and cc5013, regardless of growth media. However, a statistically significant difference in Cd2+ bioaccumulation (p<0.0001) was determined between the media (with acetate and without acetate). The cia7- mutant, cc5013 was found to be more susceptible to a Cd2+-induced decrease in chlorophyll fluorescence and had a reduced cell size compared to cc4425, the wild-type strain. Conclusions: These observed differences between the strains suggest that CIA7’s biological activity could play a direct or indirect role in increasing Cd tolerance in C. reinhardtii.

Prevalence of efflux pump and heavy metal tolerance encoding genes among Salmonella enterica serovar Infantis strains from diverse sources in Brazil.

Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) is a non-typhoid, zoonotic and foodborne serovar with worldwide distribution, and often associated with increasing antimicrobial resistance. Efflux pumps are antimicrobial resistance mechanisms able to promote and increase resistance levels to multiple distinct drug classes. Heavy metal tolerance genes have been demonstrated to promote resistance against these compounds and act in the co-selection of antimicrobial resistant strains. Despite the relevance of S. Infantis in clinical and non-clinical fields, few studies worldwide have investigated the occurrence of such genes in strains from diverse sources. Therefore, the present study aimed at determining the prevalence of antimicrobial efflux pump and heavy metal tolerance genes and their genomic relatedness through core-genome multi-locus sequence typing (cgMLST) of 80 S. Infantis strains isolated from food, environmental, human and animal sources from 2013 to 2018 in Brazil. Twenty efflux pump encoding genes were detected, with 17 of these (acrA, acrB, baeR, crp, emrB, emrR, hns, kdpE, kpnF, marA, marR, mdtK, msbA, rsmA, sdiA, soxR and soxS) detected in all strains studied, golS in 98.75%, mdfA in 58.75% and tet(A) in 37.5%. Tolerance genes to arsenic (arsR) were detected in 100% of the strains, gold (golS and golT) in 98.75%, silver (silABCDEFPRS) in 36.25% and mercury (merR and merT) in 1.25%. cgMLST demonstrated a closer genetic relationship among strains harboring similar profiles of heavy metal and efflux pump encoding genes, despite their origin. In conclusion, the high prevalence of some efflux pump and heavy metal tolerance encoding genes alert us about the importance of strong surveillance measures to monitor resistance and the transmission of S. Infantis among diverse sources in Brazil.

Bioremediation of Multiple Heavy Metals Mediated by Antarctic Marine Isolated Dietzia psychralcaliphila JI1D

Extreme environments host numerous microorganisms perfectly adapted to survive in such harsh conditions. In recent years, many bacteria isolated from these inhospitable environments have shown interesting biotechnological applications, including the bioremediation of polluted sites by hydrocarbons and heavy metals. In this work, we present Dietzia psychralcaliphila JI1D, a psychrophilic bacterium, isolated from Deception Island, Antarctica, which is able to resist high concentrations (up to 1000 ppm) of heavy metals and to favor their removal from polluted water systems. In detail, D. psychralcaliphila JI1D can actively promote the sequestration of arsenic, copper, and zinc from the medium up to a maximum of 31.6%, 49.4%, and 38.9%, respectively. Moreover, genome analysis allowed for the identification of heavy metal tolerance genes, thus shedding light on the mechanisms underlying the detoxification ability of the bacterium. Other than the demonstrated ability of D. psychralcaliphila JI1D to degrade polycyclic aromatic hydrocarbons, this study indicates the possibility of using this bacterium in the bioremediation of contaminated matrices, for example, those containing inorganic pollutants.

Metagenomic and genomic characterization of heavy metal tolerance and resistance genes in the rhizosphere microbiome of Avicennia germinans in a semi-arid mangrove forest in the tropics.

Mangroves are often exposed to heavy metals that accumulate in the food chain, generate toxicity to mangrove plants and affect microbial diversity. This study determined the abundance of genes associated with resistance and tolerance to heavy metals in the rhizosphere microbiome of Avicennia germinans from a semi-arid mangrove of La Guajira-Colombia by metagenomics and genomics approach. Twenty-eight genes associated with tolerance and 49 genes related to resistance to heavy metals were detected. Genes associated with tolerance and resistance to Cu, especially cusA and copA, were the most abundant. The highest number of genes for tolerance and resistance were for Zn and Co, respectively. The isolate Vibrio fluvialis showed the ability to tolerate Cu, Ni, Zn, and Cd. This work used a complementary approach of metagenomics and genomics to characterize the potential of mangrove microorganisms to tolerate and resist heavy metals and the influence of salinity on their abundance.

Role of Plasmid in Pesticide Degradation and Metal Tolerance in Two Plant Growth-Promoting Rhizobacteria Bacillus cereus (NCIM 5557) and Bacillus safensis (NCIM 5558).

Disha A (Bacillus cereus) and Disha B (Bacillus safensis) were isolated from pesticide-infested agricultural field and showed tolerance against pesticides, heavy metals, and antibiotics. The isolates exhibited PGPR activities in vitro as well as in field conditions in lentil (Lens culinaris) and cow pea (Vigna unguiculata). Both the Bacillus species could not be grown in mineral salt medium but efficiently grown in the media supplemented with pesticide (imidacloprid/carbendazim) demonstrating the utilization of pesticide as carbon/nitrogen source. The HPLC studies exhibited the pesticide (imidacloprid/carbendazim) degradation by both the bacteria. B. safensis showed better degradation of carbendazim (88.93%) and imidacloprid (82.48%) than that of B. cereus 78.07% and 49.12%, respectively. The bacterial isolates showed high concentration of heavy metal tolerance viz. lead, molybdenum, cadmium, copper, cobalt, and zinc, except mercury. Both the bacteria possessed single plasmid. The plasmid-cured isolates of B. cereus did not tolerate any pesticide, whereas that of B. safensis tolerated all the pesticides, like wild strains. The plasmid curing experiments did not affect the heavy metal tolerance ability of both the bacteria indicating the genomic nature of heavy metal tolerance genes, whereas pesticide resistance genes are plasmid-dependent in B. cereus but genomic in B. safensis.

Comparison of cell size, chlorophyll fluorescence and cadmium (Cd2+) bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to Cd2

Background: Heavy metal contamination presents a constant threat to biological systems. Simultaneously, heavy metals have become one of the major contaminants in the aquatic ecosystems. In this regard, the investigation of heavy metal-tolerance genes in algae is relevant. Chlamydomonas reinhardtii is a unicellular green alga, and an excellent model organism used in heavy metal studies. In C. reinhardtii, a novel gene designated as Cia7, was hypothesized to play a role in heavy metal homeostasis due to CIA7’s conserved cysteine-residue motif. This study compared two strains of C. reinhardtii, cc4425, the wild-type with the functional CIA7 protein and cc5013, the mutant strain with the disrupted cia7- gene. The hypothesis was that the expression of Cia7 contributes to an increased cadmium (Cd)-tolerance in C. reinhardtii. The Cd-tolerance would be described by physiological markers of microalgae health, and by intracellular accumulation of the metal. Methods: The objectives of this study were (1) to compare chlorophyll fluorescence and cell size in cc4425 and cc5013 exposed to Cd2+, and (2) to compare Cd2+ bioaccumulation in cc4425 and cc5013 strains in different growth media. Flow cytometry, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis were performed. Results: There was no significant statistical difference in Cd2+ bioaccumulation between the two strains, cc4425 and cc5013, regardless of growth media. However, a statistically significant difference in Cd2+ bioaccumulation (p<0.0001) was determined between the media (with acetate and without acetate). The cia7- mutant, cc5013 was found to be more susceptible to a Cd2+-induced decrease in chlorophyll fluorescence and had a reduced cell size compared to cc4425, the wild-type strain. Conclusions: These observed differences between the strains suggest that CIA7’s biological activity could play a direct or indirect role in increasing Cd tolerance in C. reinhardtii.

Heavy Metal Tolerance Genes Associated With Contaminated Sediments From an E-Waste Recycling River in Southern China.

Heavy metal pollution that results from electronic waste (e-waste) recycling activities has severe ecological environmental toxicity impacts on recycling areas. The distribution of heavy metals and the impact on the bacteria in these areas have received much attention. However, the diversity and composition of the microbial communities and the characteristics of heavy metal resistance genes (HMRGs) in the river sediments after long-term e-waste contamination still remain unclear. In this study, eight river sediment samples along a river in a recycling area were studied for the heavy metal concentration and the microbial community composition. The microbial community consisted of 13 phyla including Firmicutes (ranging from 10.45 to 36.63%), Proteobacteria (11.76 to 32.59%), Actinobacteria (14.81 to 27.45%), and unclassified bacteria. The abundance of Firmicutes increased along with the level of contaminants, while Actinobacteria decreased. A canonical correspondence analysis (CCA) showed that the concentration of mercury was significantly correlated with the microbial community and species distribution, which agreed with an analysis of the potential ecological risk index. Moreover, manually curated HMRGs were established, and the HMRG analysis results according to Illumina high-throughput sequencing showed that the abundance of HMRGs was positively related to the level of contamination, demonstrating a variety of resistance mechanisms to adapt, accommodate, and live under heavy metal-contaminated conditions. These findings increase the understanding of the changes in microbial communities in e-waste recycling areas and extend our knowledge of the HMRGs involved in the recovery of the ecological environment.

Identification of novel heavy metal detoxification proteins in Solanum tuberosum: Insights to improve food security protection from metal ion stress.

With increasingly serious environmental pollution problems, research has focused on identifying functional genes within plants that can help ensure food security and soil governance. In particular, plants seem to have been able to evolve specific functional genes to respond to environmental changes by losing partial gene functions, thereby representing a novel adaptation mechanism. Herein, a new category of functional genes was identified and investigated, providing new directions for understanding heavy metal detoxification mechanisms. Interestingly, this category of proteins appears to exhibit specific complexing functions for heavy metals. Further, a new approach was established to evaluate ATP-binding cassette (ABC) transporter family functions using microRNA targeted inhibition. Moreover, mutant and functional genes were identified for future research targets. Expression profiling under five heavy metal stress treatments provided an important framework to further study defense responses of plants to metal exposure. In conclusion, the new insights identified here provide a theoretical basis and reference to better understand the mechanisms of heavy metal tolerance in potato plants. Further, these new data provide additional directions and foundations for mining gene resources for heavy metal tolerance genes to improve safe, green crop production and plant treatment of heavy metal soil pollution.

The plasmidome of multidrug-resistant emergent Salmonella serovars isolated from poultry

The rapid emergence of resistant bacteria is occurring worldwide. The understanding of the dissemination of antimicrobial resistance using high-throughput sequencing and bioinformatics approaches is providing valuable insights into the genetic basis of the horizontal gene transfer and the emergence of the antibiotic resistance threat. This ultimately can offer vital clues to the development of coordinated efforts to implement new policies to continue fighting against bacterial infections. The poultry microbiota is characterized as a potential reservoir of resistance genes, mostly derived from the Enterobacteriaceae which have become increasingly important in human and animal infections. In this work, complete genome sequences were achieved for four multidrug-resistant Salmonella spp. isolated from poultry from different farms in Brazil. We identified highly similar IncHI2-ST2 megaplasmids (larger than 275.000bp) in all Salmonella isolates studied. These megaplasmids carry a resistome comprised of eleven different resistance genes (aac(6')-Iaa, aadA1b, aph(4)-Ia, aph(6)-Id, aph(3″)-Ib, aph(3')-Ia, aac(3)-Iva, sul1, tetA, tetB and dfrA1b) and four heavy metal tolerance operons (telluride, mercury, silver and copper). In conclusion, the multidrug-resistant plasmids identified in S. enterica serovar Schwarzengrund and Newport isolated from poultry show a variety of antibiotic resistance and heavy metal tolerance genes, providing advantages for the bacteria to survive under extremely unfavorable conditions.

Peeling the Layers Away: The Genomic Characterization of Bacillus pumilus 64-1, an Isolate With Antimicrobial Activity From the Marine Sponge Plakina cyanorosea (Porifera, Homoscleromorpha)

Bacillus pumilus 64-1, a bacterial strain isolated from the marine sponge Plakina cyanorosea, which exhibits antimicrobial activity against both pathogenic and drug-resistant Gram-positive and Gram-negative bacteria. This study aimed to conduct an in-depth genomic analysis of this bioactive sponge-derived strain. The nearly complete genome of strain 64-1 consists of 3.6 Mbp (41.5% GC), which includes 3,705 coding sequences (CDS). An open pangenome was observed when limiting to the type strains of the B. pumilus group and aquatic-derived B. pumilus representatives. The genome appears to encode for at least 12 potential biosynthetic gene clusters (BGCs), including both types I and III polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), and one NRPS-T1PKS hybrid, among others. In particular, bacilysin and other bacteriocin-coding genes were found and may be associated with the detected antimicrobial activity. Strain 64-1 also appears to possess a broad repertoire of genes encoding for plant cell wall-degrading carbohydrate-active enzymes (CAZymes). A myriad of genes which may be involved in various process required by the strain in its marine habitat, such as those encoding for osmoprotectory transport systems and the biosynthesis of compatible solutes were also present. Several heavy metal tolerance genes are also present, together with various mobile elements including a region encoding for a type III-B Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region, four prophage segments and transposase elements. This is the first report on the genomic characterization of a cultivable bacterial member of the Plakina cyanorosea holobiont.

High occurrence of heavy metal tolerance genes in bacteria isolated from wastewater: A new concern?

Some heavy metals have antimicrobial activity and are considered as potential alternatives to traditional antibiotic therapy. However, heavy metal tolerance genes (HMTG) have been already detected and coding different tolerance mechanisms. Considering that certain metals are promising for antimicrobial therapy, evaluation of HMTG dissemination in bacteria from sewage is essential to understand the evolution of these bacteria and to predict antimicrobial use and control. The present study aimed to evaluate the occurrence of bacteria carrying HMTG in samples of hospital wastewater and from urban wastewater treatment plant (WWTP). The acquired HMTG were investigated by PCR in bacterial collection previously characterized for antibiotic resistant genes (ARGs). HMTG searched include arsB (arsenic efflux pump), czcA (cadmium, zinc and cobalt efflux pump), merA (mercuric reductase), pcoD (copper efflux pump), silA (silver efflux pump) and terF (tellurite resistance protein). Among 45 isolates, 82% of them carried at last one HMTG, in which the silA and pcoD tolerance genes were the most prevalent. A very strong positive correlation was found between these genes (r = 0.91, p < 0.0001). Tolerance genes merA, arsB, czcA and terF were detected in 47%, 13%, 13% and 7% of the isolates, respectively. It was found that 15 isolates co-harbored ARGs (β-lactamase encoding genes). HMTG are probably more dispersed than ARGs in bacteria, representing a new concern for heavy metals use as effective antimicrobials. To the best of our knowledge, this is the first study on the HMTG searched in Hafnia alvei, Serratia fonticola and Serratia liquefaciens. Hospital wastewater treatment implementation and additional technologies for treatment in WWTP can reduce the impacts on water resources and HMTG spread, ensureing the environmental and human health safety.

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs.

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems.

Comparative Study of Gut Microbiota in Wild and Captive Giant Pandas (Ailuropoda melanoleuca).

Captive breeding has been used as an effective approach to protecting endangered animals but its effect on the gut microbiome and the conservation status of these species is largely unknown. The giant panda is a flagship species for the conservation of wildlife. With integrated efforts including captive breeding, this species has been recently upgraded from “endangered” to “vulnerable” (IUCN 2016). Since a large proportion (21.8%) of their global population is still captive, it is critical to understand how captivity changes the gut microbiome of these pandas and how such alterations to the microbiome might affect their future fitness and potential impact on the ecosystem after release into the wild. Here, we use 16S rRNA (ribosomal RNA) marker gene sequencing and shotgun metagenomics sequencing to demonstrate that the fecal microbiomes differ substantially between wild and captive giant pandas. Fecal microbiome diversity was significantly lower in captive pandas, as was the diversity of functional genes. Additionally, captive pandas have reduced functional potential for cellulose degradation but enriched metabolic pathways for starch metabolism, indicating that they may not adapt to a wild diet after being released into the wild since a major component of their diet in the wild will be bamboo. Most significantly, we observed a significantly higher level of amylase activity but a lower level of cellulase activity in captive giant panda feces than those of wild giant pandas, shown by an in vitro experimental assay. Furthermore, antibiotic resistance genes and virulence factors, as well as heavy metal tolerance genes were enriched in the microbiomes of captive pandas, which raises a great concern of spreading these genes to other wild animals and ecosystems when they are released into a wild environment. Our results clearly show that captivity has altered the giant panda microbiome, which could have unintended negative consequences on their adaptability and the ecosystem during the reintroduction of giant pandas into the wild.

Evaluation of heavy metal tolerance genes in plasmids harbored in multidrug-resistant Salmonella enterica and Escherichia coli isolated from poultry in Brazil

The emergence and spread of bacteria tolerant to heavy metal ions used in disinfectants have become a new threat to antimicrobial therapies. In this study, metal tolerance genes were analyzed in multidrug-resistant (MDR) Enterobacteriaceae isolated from chickens in Brazil. Different tolerance genes were found disseminated among the MDR isolates.