Mercury Resistance Research Articles

Characterization of blaCMY-2-carrying IncC and rmtB-carrying IncI1/ST136 plasmids in an avian Escherichia coli ST224 strain

To analyze characteristics and underlying evolutionary processes of IncC and IncI1 plasmids in a multidrug-resistant avian E. coli strain, antibiotic susceptibility testing, PCR, conjugation assays, and next-generation sequencing were performed. The type 1 IncC plasmid pEC009.1 harbored three antimicrobial resistance regions including ISEcp1-blaCMY-2-blc-sugE, ARI-B resistance island, and ARI-A island that was a mosaic multidrug resistance region (MRR) comprised of a class 1 integron with cassette array |aac(6′)-II(aacA7)|qacE∆1|sul1|, IS26-mphR(A)-mrx-mph(A)-IS26, IS26-fosA3-IS26, and mercury resistance cluster merRTPABDE. It is the first report of three different size circular forms derived from IS26-mphR(A)-mrx-mph(A)-IS26-fosA3-IS26 in ARI-A of type 1 IncC plasmid. In IncI1/ST136 pEC009.2, the truncated transposon Tn1722 carrying blaTEM-1b, rmtB, aac(3)-IId(aacC2d), and a class 1 integron with cassette array |dfrA12|orfF|aadA2|, inserted into the plasmid backbone generating 5-bp direct repeats (DRs, TATAA) at the boundaries of the region, which was highly similar to that of other IncI1 plasmids, and differed by the arrangements of resistance determinants. Comparison among two epidemic plasmid lineages showed complex MRRs respectively located in the specific position in type 1 IncC and IncI1/ST136 plasmids with conserved backbones, and these have evolved via multiple events involved in mobile elements-mediated loss and gain of resistance genes and accessory genes. Strains harboring these plasmids may serve as a reservoir for antibiotic resistance genes, thereby contributing to the rapid spread of resistance genes and posing a public health threat.

Description of Salinimonas profundi sp. nov., a deep-sea bacterium harboring a transposon Tn6333.

A Gram-staining-negative bacterium, strain HHU 13199T, was isolated from a marine sediment sample collected from South China Sea (119°19.896'E, 19°41.569'N) at a depth of 2918m. The 16S rRNA gene sequence analysis indicated that strain HHU 13199T represents a member of the genus Salinimonas with the highest sequence similarity (99.8%) to the type strain S. iocasae KX18D6T. However, the average nucleotide identity values and digital DNA-DNA hybridization between strain HHU 13199T and closely related members of the genus Salinimonas were all below the cut-off level (95-96 % and 70%, respectively) for species delineation. This strain grew with sea salt of 0.5-18% (w/v) (optimum, 2-5%), but no growth observed when using NaCl instead. The major fatty acids are C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The predominant isoprenoid quinone is ubiquinone-8. The polar lipids mainly consist of phosphatidylethanolamine, and phosphatidylglycerol. Genomic characterization revealed that strain HHU 13199T harbors a distinct type I-F CRISPR-Cas system and plenty of genes associated with heavy metal resistance, including a transposon (Tn6333) conferring mercury resistance. In addition, a phylogenetic tree based on the bac120 core genes suggested that the genus Salinimonas should be a subclade within Alteromonas. On the basis of the phenotypic, phylogenetic and chemotaxonomic characterizations, strain HHU 13199T represents a novel species of the genus Salinimonas, for which the name Salinimonas profundi sp. nov. is proposed. The type strain is HHU 13199T (= KCTC 72837T = MCCC 1K04127T).

1442. Acquisition and Transferability Mechanisms of Mercury Resistance Genes in Latin-American Staphylococcus aureus Strains

BackgroundLatin-American (LA) countries are among the largest mercury (Hg) polluters in the world. Fittingly, a significant high frequency (>50%) of Hg resistance genes (MRG) has been observed in LA MRSA genomes, including USA300-LV clone, which contains the genomic element COMER, encoding for copper and Hg resistance genes adjacent to SCCmecIVc/E. Co-selection of MRG and antibiotic resistance genes may be facilitated by shared transferable genetic elements, nevertheless, analyses of the genetic MRG context in strains other than USA300-LV are lacking. In this study, we aimed to characterize possible mechanisms of acquisition and transfer of MRG in LA S. aureus.MethodsWe sequenced 6 MRSA and 2 MSSA clinical isolates harboring MRG from Colombia, Ecuador, Peru and Chile using short-read (Illumina) and long-read (ONT) sequencing. Hybrid assemblies were constructed using Flye and iterative polishing with Medaka and Racon. Identification of insertion sequences, rearrangements and assessment of the genomic context was investigated using ISfinder, MAUVE, PlasmidFinder and SnapGene.ResultsHighly contiguous genome assemblies allowed us to identify the localization and genetic background of MRG. For MRSA belonging to USA300-LV (SCCmecIVc/E) and Brazilian (SCCmecIII) clones, we confirmed the presence of MRG within SCCmec. In contrast, for the 4 MRSA belonging to Chilean/Cordobes clone (SCCmecI), collected from Colombia, Chile and Peru, MRG were located on ~30kbp plasmids genetically related that also contained the blaZ beta-lactamase and cadmium/arsenic resistance genes. In MSSA strains, we observed both plasmidic and chromosomal localizations of MGR. Interestingly, in one of the MSSA, MRG were inserted downstream of orfX, along with repA, suggesting a plasmidic origin. In all these cases, MRG were flanked by IS6 family elements.ConclusionGenomic architecture of SCCmec types IVc/E and III might facilitate MRG transferability, whereas for the highly prevalent Chilean/Cordobes clone (SCCmecI) MRG acquisition occurs through plasmids. Our findings underscore the mechanisms of MRG transference in LA S. aureus likely related to antibiotic resistance co-selection.Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Characterizing and Evaluating the Diverse Microbial Communities and Their Mercury Resistance Potential from Hot Spring Sites Representing Gradients in Temperature and pH in Yellowstone National Park

Yellowstone National Park is home to many different hot springs, lakes, geysers, pools, and basins that range in pH, chemical composition, and temperature. These different environmental variations provide a broad range of conditions that select and grow diverse communities of microorganisms. In this study, we collected samples from geochemically diverse lakes and springs to characterize the microbial communities present through 16S rRNA metagenomic analysis. This information was then used to observe how various microorganisms survive in high mercury environments. The results show the presence of microorganisms that have been studied in previous literature. The results also depict gradients of microorganisms including thermophilic bacteria and archaea that exist in these extreme environments. In addition, beta diversity analyses of the sequence data showed site clustering based primarily on temperature instead of pH or sample site, suggesting that while pH, temperature, and sample site were all shown to be significant, temperature is the strongest factor driving microorganism community development. While it is important to characterize the microorganism community present, it is also important to understand how this community functions as a result of its selection. Along with looking at community composition, genomic material was tested to see if it contained mercury methylating (hgcA) or mercury reducing (merA) genes. Out of 22 samples, three of them were observed to have merA genes, while no samples had hgcA genes. These results indicate that microorganisms in Mustard and Nymph Springs may use mercury reduction. Understanding how microorganisms survive in environments with high concentrations of toxic pollutants is crucial because it can be used as a model to better understand mechanisms of resistance and the biogeochemical cycle, as well as for bioremediation and other solutions to anthropogenic problems.

Transcriptomic analyses reveal the pathways associated with the volatilization and resistance of mercury(II) in the fungus Lecythophora sp. DC-F1

The biotic enzymatic reduction of mercury II [Hg(II)] to elemental Hg [Hg(0)] is an important pathway for Hg detoxification in natural ecosystems. However, the mechanisms of Hg(II) volatilization and resistance in fungi have not been understood completely. In the present study, we investigated the mechanisms of Hg(II) volatilization and resistance in the fungus Lecythophora sp. DC-F1. Hg(II) volatilization occurred during the investigation via the reduction of Hg(II) to Hg(0) in DC-F1. Comparative transcriptome analyses of DC-F1 revealed 3439 differentially expressed genes under 10 mg/L Hg(II) stress, among which 2770 were up-regulated and 669 were down-regulated. Functional enrichment analyses of genes and pathways further suggested that the Hg(II) resistance of DC-F1 is a multisystem collaborative process with three important transcriptional responses to Hg(II) stress: a mer-mediated Hg detoxification system, a thiol compound metabolism, and a cell reactive oxygen species stress response system. The phylogenetic analysis of merA protein homologs suggests that the Hg(II) reduction by merA is widely distributed in fungi. Overall, this study provides evidence for the reduction of Hg(II) to Hg(0) in fungi via the mer-mediated Hg detoxification system and offers a comprehensive explanation for its role within Hg biogeochemical cycling. These findings offer a strong theoretical basis for the application of fungi in the bioremediation of Hg-contaminated envionments.

The Impact of Mercury Selection and Conjugative Genetic Elements on Community Structure and Resistance Gene Transfer.

Carriage of resistance genes can underpin bacterial survival, and by spreading these genes between species, mobile genetic elements (MGEs) can potentially protect diversity within microbial communities. The spread of MGEs could be affected by environmental factors such as selection for resistance, and biological factors such as plasmid host range, with consequences for individual species and for community structure. Here we cultured a focal bacterial strain, Pseudomonas fluorescens SBW25, embedded within a soil microbial community, with and without mercury selection, and with and without mercury resistance plasmids (pQBR57 or pQBR103), to investigate the effects of selection and resistance gene introduction on (1) the focal species; (2) the community as a whole; (3) the spread of the introduced mer resistance operon. We found that P. fluorescens SBW25 only escaped competitive exclusion by other members of community under mercury selection, even when it did not begin with a mercury resistance plasmid, due to its propensity to acquire resistance from the community by horizontal gene transfer. Mercury pollution had a significant effect on community structure, decreasing alpha diversity within communities while increasing beta diversity between communities, a pattern that was not affected by the introduction of mercury resistance plasmids by P. fluorescens SBW25. Nevertheless, the introduced merA gene spread to a phylogenetically diverse set of recipients over the 5 weeks of the experiment, as assessed by epicPCR. Our data demonstrates how the effects of MGEs can be experimentally assessed for individual lineages, the wider community, and for the spread of adaptive traits.

Distribution of heavy metal resistance elements in Canadian Salmonella 4,[5],12:i:- populations and association with the monophasic genotypes and phenotype.

Salmonella 4,[5],12:i:- are monophasic S. Typhimurium variants incapable of producing the second-phase flagellar antigen. They have emerged since the mid-1990s to become one of the most prevalent Salmonella serotypes causing human disease world-wide. Multiple genetic events associated with different genetic elements can result in the monophasic phenotype. Several jurisdictions have reported the emergence of a Salmonella 4,[5],12:i:- clone with SGI-4 and a genetic element (MREL) encoding a mercury resistance operon and antibiotic resistance loci that disrupts the second phase antigen region near the iroB locus in the Salmonella genome. We have sequenced 810 human and animal Canadian Salmonella 4,[5],12:i:- isolates and determined that isolates with SGI-4 and the mercury resistance element (MREL; also known as RR1&RR2) constitute several global clades containing various proportions of Canadian, US, and European isolates. Detailed analysis of the data provides a clearer picture of how these heavy metal elements interact with bacteria within the Salmonella population to produce the monophasic phenotype. Insertion of the MREL near iroB is associated with several deletions and rearrangements of the adjacent flaAB hin region, which may be useful for defining human case clusters that could represent outbreaks. Plasmids carrying genes encoding silver, copper, mercury, and antimicrobial resistance appear to be derived from IS26 mediated acquisition of these genes from genomes carrying SGI-4 and the MREL. Animal isolates with the mercury and As/Cu/Ag resistance elements are strongly associated with porcine sources in Canada as has been shown previously for other jurisdictions. The data acquired in these investigations, as well as from the extensive literature on the subject, may aid source attribution in outbreaks of the organism and interventions to decrease the prevalence of this clone and reduce its impact on human disease.

A novel community-acquired MRSA clone, USA300-LV/J, uniquely evolved in Japan.

USA300 [ST8-staphylococcal cassette chromosome mec type IVa (ST8-IVa)/arginine catabolic mobile element (ACME) positive] is a major Panton-Valentine leucocidin (PVL)-positive community-acquired MRSA (CA-MRSA) clone. In Japan, we identified USA300-like strains with characteristics (ST8-IVc/ACME negative) similar to those of USA300. To reveal the evolution of the USA300-like strains. The whole-genome sequence of a USA300-like strain was determined and genome analysis was performed using Type Strain Genome Server, MUSCLE and progressiveMauve. Genome-based phylogenetic analysis showed that the USA300-like strain is more similar to the USA300-Latin American variant (USA300-LV), which is a PVL-positive CA-MRSA clone identified in South America, than to USA300. Instead of the ACME, copper and mercury resistance mobile elements were located on the genome of the USA300-like strain. In addition, the USA300-like strain possessed a unique mobile genetic element, ICE6013. Therefore, we named this novel USA300-LV variant identified in Japan as USA300-LV/J. Our findings strongly suggest that a PVL-positive CA-MRSA USA300-LV/J clone originating from abroad has uniquely evolved and disseminated in Japan.

Effects of Mercury II on Cupriavidus metallidurans Strain MSR33 during Mercury Bioremediation under Aerobic and Anaerobic Conditions

Mercury is a toxic element that harms organisms and disturbs biogeochemical cycles. Mercury bioremediation is based on the reduction of Hg (II) to Hg (0) by mercury-resistant bacteria. Cupriavidus metallidurans MSR33 possesses a broad-spectrum mercury resistance. This study aims to establish the effects of mercury on growth, oxygen uptake, and mercury removal parameters by C. metallidurans MSR33 in aqueous solution during aerobic and anaerobic mercury bioremediation. A new culture medium (GBC) was designed. The effects of mercury (II) (20 ppm) on growth parameters, oxygen uptake, and mercury removal were evaluated in GBC medium in a bioreactor (3 L) under aerobiosis. The anaerobic kinetics of mercury removal was evaluated by nitrogen replacement during mercury bioremediation in a bioreactor. Strain MSR33 reached a growth rate of µ = 0.43 h−1 in the bioreactor. Mercury inhibited oxygen uptake and bacterial growth; however, this inhibition was reversed after 5 h. Strain MSR33 was able to reduce Hg (II) under aerobic and anaerobic conditions, reaching, at 24 h, a metal removal of 97% and 71%, respectively. Therefore, oxygen was crucial for efficient mercury removal by this bacterium. Strain MSR33 was capable of tolerating the toxic effects of mercury (II) during aerobic bioremediation and recovered its metabolic activity.

Emergence of Multidrug-Resistant Salmonella enterica Subspecies enterica Serovar Infantis of Multilocus Sequence Type 2283 in German Broiler Farms.

During the last decade, Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) has become more prevalent across Europe with an increased capability to persist in broiler farms. In this study, we aimed to identify potential genetic causes for the increased emergence and longer persistence of S. Infantis in German poultry farms by high-throughput-sequencing. Broiler derived S. Infantis strains from two decades, the 1990s (n = 12) and the 2010s (n = 18), were examined phenotypically and genotypically to detect potential differences responsible for increased prevalence and persistence. S. Infantis organisms were characterized by serotyping and determining antimicrobial susceptibility using the microdilution method. Genotypic characteristics were analyzed by whole genome sequencing (WGS) to detect antimicrobial resistance and virulence genes as well as plasmids. To detect possible clonal relatedness within S. Infantis organisms, 17 accessible genomes from previous studies about emergent S. Infantis were downloaded and analyzed using complete genome sequence of SI119944 from Israel as reference. In contrast to the broiler derived antibiotic-sensitive S. Infantis strains from the 1990s, the majority of strains from the 2010s (15 out of 18) revealed a multidrug-resistance (MDR) phenotype that encodes for at least three antimicrobials families: aminoglycosides [ant(3“)-Ia], sulfonamides (sul1), and tetracyclines [tet(A)]. Moreover, these MDR strains carry a virulence gene pattern missing in strains from the 1990s. It includes genes encoding for fimbriae clusters, the yersiniabactin siderophore, mercury and disinfectants resistance and toxin/antitoxin complexes. In depth genomic analysis confirmed that the 15 MDR strains from the 2010s carry a pESI-like megaplasmid with resistance and virulence gene patterns detected in the emerged S. Infantis strain SI119944 from Israel and clones inside and outside Europe. Genotyping analysis revealed two sequence types (STs) among the resistant strains from the 2010s, ST2283 (n = 13) and ST32 (n = 2). The sensitive strains from the 1990s, belong to sequence type ST32 (n = 10) and ST1032 (n = 2). Therefore, this study confirms the emergence of a MDR S. Infantis pESI-like clone of ST2283 in German broiler farms with presumably high tendency of dissemination. Further studies on the epidemiology and control of S. Infantis in broilers are needed to prevent the transfer from poultry into the human food chain.

Transcriptional regulation of drug resistance mechanisms in Salmonella: where we stand and what we need to know.

Salmonellae have evolved a wide range of molecular mechanisms to neutralize the effect of antibiotics and evade the host immune system response. These mechanisms are exquisitely controlled by global and local regulators and enable the pathogens to use its energy as per need and hence allow the pathogen to economize the consumption of energy by its cellular machinery. Several families that regulate the expression of different drug resistance genes are known; some of these are: the TetR family (which affects tetracycline resistance genes), the AraC/XylS family (regulators that can act as both transcriptional activators and repressors), two-component signal transduction systems (e.g. PhoPQ, a key regulator for virulence), mercury resistance Mer-R and multiple antibiotic resistance Mar-R regulators, LysR-type global regulators (e.g. LeuO) and histone-like protein regulators (involved in the repression of newly transferred resistance genes). This minireview focuses on the role of different regulators harbored by the Salmonella genome and characterized for mediating the drug resistance mechanisms particularly via efflux and influx systems. Understanding of such transcriptional regulation mechanisms is imperative to address drug resistance issues in Salmonella and other bacterial pathogens.

Molecular genetic analysis of an XDR Pseudomonas aeruginosa ST664 clone carrying multiple conjugal plasmids

A group of ST664 XDR Pseudomonas aeruginosa strains have been isolated from a burn clinic. Here we decipher their resistomes and likely mechanisms of resistance acquisition. The complete nucleotide sequences of representative isolates were determined, by PacBio and Illumina MiSeq sequencing, and analysed for antimicrobial resistance (AMR) genes as well as sequence variations. S1-PFGE was used to determine the sizes and numbers of plasmids harboured by the isolates. Purified plasmid DNA was further sequenced by PacBio technology, closed manually and annotated by RAST. The mobility of plasmids was determined by conjugation assays. The XDR P. aeruginosa ST664 clone carries 11 AMR genes, including a blaKPC-2 gene that confers resistance to carbapenems. Most of the ST664 isolates carry three coexisting plasmids. blaKPC-2 and a cluster of three AMR genes (aadB-cmlA1-sul1) are encoded on a 475 kb megaplasmid pNK546a, which codes for an IncP-3-like replication and partitioning mechanism, but has lost the conjugative transfer system. Interestingly, however, pNK546a is mobilizable and can be transferred to P. aeruginosa PAO1 with the help of a co-residing IncP-7 conjugative plasmid. The blaKPC-2 gene is carried by an IS6100-ISKpn27-blaKPC-2-ΔISKpn6-Tn1403 mobile element, which might be brought into the ST664 clone by another co-resident IncP-1α plasmid, which is inclined to be lost. Moreover, pNK546a harbours multiple heavy metal (mercury, tellurite and silver) resistance modules. To the best of our knowledge, pNK546a is the first fully sequenced blaKPC-2-carrying megaplasmid from P. aeruginosa. These results give new insights into bacterial adaptation and evolution during nosocomial infections.

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision.

The epidemiological success of methicillin-resistant Staphylococcus aureus USA300 has been associated with the presence of two mobile elements, the arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER) element. These two mobile elements are associated with resistance to copper, which has been related to host fitness and survival within macrophages. Several studies found that ACME is more prevalent, and exhibits greater diversity, in Staphylococcus epidermidis while COMER has not been identified in S. epidermidis or any other staphylococcal species. We aimed in this study to evaluate the presence and diversity of ACME and COMER-like elements in our S. epidermidis clinical isolates. The genomes of 58 S. epidermidis clinical isolates, collected between 2009 and 2018 in a Scottish hospital, were sequenced. A core-genome phylogenetic tree and genome based MLST typing showed that more than half of the isolates belong to the clinically predominant sequence type2 (ST2) and these isolates have been found to split into two lineages within the phylogenetic tree. Analysis showed the presence of SCCmec in the majority of isolates. Comparative analysis identified a cluster of ACME-positive isolates with most of them belonging to ST48. ACME showed high variation even between isolates of the same ACME type and ST. COMER-like elements have been identified in one of the two major hospital adapted drug resistant ST2 lineages; and showed high stability. This difference in stability at the genomic level correlates well with the up to one hundred times higher excision frequency found for the SCCmec elements in ACME-containing isolates compared to COMER-like element containing isolates. ACME/COMER-like element positive isolates did not show a significant phenotype of decreased copper susceptibility, while resistance to mercury was over-represented in COMER-like element positive isolates. To the best of our knowledge, this is the first molecular characterization of COMER-like elements in S. epidermidis isolates. The presence of the COMER-like elements is the most prominent accessory genome feature of these successful lineages suggesting that this chromosomal island contributes to the success and wide clinical distribution of ST2 S. epidermidis.

MerP/MerT-mediated mechanism: A different approach to mercury resistance and bioaccumulation by marine bacteria

Currently, mechanism underlying mercury resistance and bioaccumulation of marine bacteria remains little understood. A marine bacterium Pseudomonas pseudoalcaligenes S1 is resistant to 120 mg/L Hg2+ with bioaccumulation capacity of 133.33 mg/g. Accordingly, Hg2+ resistance and bioaccumulation mechanism of S1 was investigated at molecular and cellular level. Annotation of S1 transcriptome reveals 772 differentially expressed genes, including Hg2+-relevant genes merT, merP and merA. Both merT and merP gene have three complete copies in S1 genome, while merA gene has only one. In order to evaluate the function of these Hg2+-relevant genes, three recombinant strains were constructed to express MerA (named as A), MerT/MerP (TP) and MerT/MerP/MerA (TPA), respectively. The results show that Hg2+ resistance of strain TP, TPA, and A are improved with minimum inhibition concentration (MIC) being 60 mg/L, 40 mg/L, and 20 mg/L, respectively compared to 2 mg/L of host strain. Strain TP and TPA exhibit enhanced Hg2+ bioaccumulation capacity, while strain A does not differ from the control. Their equilibrium Hg2+ bioaccumulation capacities are 110.48 mg/g, 94.49 mg/g, 83.76 mg/g and 82.29 mg/g, respectively. Summarily, different from most microorganisms that exhibit Hg2+ resistance by MerA-mediated mechanism, marine bacterium S1 achieves Hg2+ resistance and bioaccumulation capability via MerT/MerP-mediated strategy.

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element

BackgroundPseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories.ResultsHere we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, RifR, restriction-modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939 encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939. Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE) encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida cells, where it integrated in the genome and conferred the mercury resistance.ConclusionsThe high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research including e.g. investigation of horizontal gene transfer or comparative genomics.The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This indicates that the only known member of IncP-8 is in fact an ICE.

Study on the role of moonlighting proteins in resistance of cadmium and mercury of lactic acid bacteria

Study on the role of moonlighting proteins in resistance of cadmium and mercury of lactic acid bacteria

Native Plasmid-Encoded Mercury Resistance Genes Are Functional and Demonstrate Natural Transformation in Environmental Bacterial Isolates.

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.

Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24.

A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 5′-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

557. Impact of Heavy Metal Exposure in the Transcriptional Response of Methicillin--Resistant Staphylococcus aureus (MRSA)-USA300 Latin-American Variant (USA300-LV)

BackgroundUSA 300-LV is the predominant MRSA clone in Colombia and contains a genomic island designated “COMER” with genes for copper (Cu) and mercury (Hg) resistance. HM environmental contamination is a serious threat to public health in Colombia and could also influence the selection and evolution of HM resistance genes in MRSA. Here, we investigate the global transcriptomic responses of USA300-LV after exposure to HM under the hypothesis that USA300-LV strains are highly capable of sustaining higher HM concentrationsMethodsWe performed comparative RNAseq experiments in USA300-LV clinical strain (CA-MRSA12). Total RNA was isolated in exponential phase in the absence and presence of sub-inhibitory concentrations of Cu and Hg (3 replicates). cDNA libraries were prepared and sequenced on an Illumina platform. Differentially expressed genes (DEG) were calculated by DeSeq2 (p-adjusted value ˂ 0.01) and results on 19 selected genes were confirmed by qRT-PCR.ResultsUS300-LV exhibited a larger number of differentially expressed genes when exposed to Hg (n = 114) compared with Cu treatment (n = 16). The most common functional groups of genes upregulated after Hg exposure included those involved in amino acid metabolism (n = 18). In contrast, 45 genes were downregulated after Hg exposure, mostly associated to host immune system defense (n = 11). qRT-PCR confirmed that the most upregulated genes were those involved in murein hydrolase activity, Hg resistance and the transcriptional regulator Cro/CI. Of 9 genes that were downregulated, functional groups included ype VII secretion system, immune modulators and leucocidins. Copper treatment resulted in only 12 genes that were upregulated including those in the COMER element (n = 6), aminoacid metabolism (n = 3), ROS response (n = 1), host immune system defense (n = 1) and unknown function (n = 1). Downregulated genes were those associated to host immune system defense (n = 2), energy generation (n = 1) and unknown function (n = 1).ConclusionDifferential adaptive responses after exposure to HM in USA300-LV suggest a role in the evolution of antimicrobial resistance and successful spread in the region. Metabolic adaptations involving amino acid metabolism seem to play a role in the evolution of HM resistance in MRSA.Disclosures All authors: No reported disclosures.

556. Phylogenomic Epidemiology of Methicillin-Resistant Staphylococcus aureus (MRSA) Chilean-Cordobes Clone in Latin America

BackgroundThe MRSA Chilean-Cordobes (ChC) clone belongs to the clonal complex 5 (CC5) and typically carries SCCmec I. The ChC clone predominated widely throughout several countries of Latin America (LA), but during the mid-2000s a CA-MRSA CC8 LA variant (USA300-LV) quickly replaced the ChC in Colombia and Ecuador. Most notably, this replacement was not observed in Peru or Chile. Here, we aimed to understand the phylogenomic relatedness of the CC5 ChC clone obtained from different countries of LA.MethodsWe sequenced and analyzed the genomes of 115 MRSA isolates obtained between 2011–2014 from bloodstream infections in 6 LA countries (Argentina, Brazil, Colombia, Chile, Peru, and Venezuela). All isolates were confirmed as ChC clone by pulsed-field gel electrophoresis (PFGE). We used core genome-based phylogenomic reconstructions and molecular clock analysis to infer the relationships and time of divergence between clades.ResultsWhole-genome-based multilocus sequence typing determined that 110/115 isolates belonged to ST5 and carried SCCmec I. The phylogenomic reconstruction showed ChC isolates clustered into 4 major clades distinctly segregated by country of origin (Figure 1). Interestingly, isolates recovered from Chile divided into 2 different clades that segregate according to the city of origin (Santiago [SCL] or Concepción [CON]), suggesting these clades evolved independently. Molecular clock analyses suggested all clades share a common ancestor with the divergence of the Chilean clades occurring earlier (Figure 2). Of note, analysis of heavy metal genes suggested the divergence between Chilean isolates was characterized by the loss of a mercury resistance gene cluster, which is present in an 88% of CON isolates, but only in 28% of SCL (Figure 2).ConclusionMRSA isolates belonging to the ChC clone from 6 LA countries clustered in 4 clades according to the geographical region of isolation. This segregation suggests divergent adaptations that may respond to different selective pressures. Heavy metal resistance could play a role in the ability of the MRSA ChC to disseminate in specific geographical locations. Disclosures All authors: No reported disclosures.