Mutational signatures and increased retrotransposon insertions in xeroderma pigmentosum variant skin tumors.

Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.

Genetic and behavioral characterization of a Kmt2d mouse mutant, a new model for Kabuki Syndrome.

The recessive mutant mice bate palmas (bapa) - claps in Portuguese arose from N-ethyl-N-nitrosourea mutagenesis. A single nucleotide, T > C, change in exon 13, leading to a Thr1289 Ala substitution, was identified in the lysine (K)-specific methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a loss-of-function in the KMT2D gene on chromosome 12 in humans are responsible for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was performed using a behavioral test battery to evaluate the parameters related to general activity, the sensory nervous system, the psychomotor system, and the autonomous nervous system, as well as to measure motor function and spatial memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psychomotor impairments, such as hypotonia denoted by a surface righting reflex impairment and hindquarter fall, and a reduction in the auricular reflex, suggesting hearing impairment. Additionally, the enhanced general activity showed by the increased rearing and grooming frequency, distance traveled and average speed possibly presupposes the presence of hyperactivity of bapa mice compared with the control group. A slight motor coordination dysfunction was showed in bapa mice, which had a longer crossing time on the balance beam compared with BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such as a shorter stride length and shorter step length. In conclusion, the bapa mouse may be a valuable animal model to study the mechanisms involved in psychomotor and behavior impairments, such as hypotonia, fine motor coordination and hyperactivity linked to the Kmt2d mutation.

Draft genome sequences of KPC-2- and CTX-M-15-producing Klebsiella pneumoniae ST437 isolated from a clinical sample and urban rivers in Sao Paulo, Brazil.

OBJECTIVES: KPC-producing Klebsiella pneumoniae is considered one of the most worrisome multidrug-resistant micro-organisms in nosocomial infections. It has also been reported in wastewater and urban rivers in the city of Sao Paulo, Brazil. Here we report the draft genome sequences of three KPC-2- and CTX-M-15-producing K. pneumoniae sequence type 437 (ST437) isolates obtained from two urban rivers and from a clinical sample of a patient in Sao Paulo. METHODS: A genomic library was constructed using a Nextera XT Kit. An Illumina platform was used to perform whole-genome sequencing (WGS). RESULTS: WGS of environmental isolates Kp148/PINH-4900 and Kp196/TIET-4200 and clinical isolate Kp314/11 resulted in estimated genome sizes of 5464058, 5437723 and 5319218bp, respectively. Resistome analysis of the environmental and clinical strains revealed the presence of resistance genes to the following antimicrobials in all strains: aminoglycosides [aac(6')-Ib-cr]; ß-lactams (blaOXA-1, blaSHV-11, blaCTX-M-15 and blaKPC-2); fluoroquinolones [aac(6')-Ib-cr, oqxA and oqxB]; fosfomycin (fosAKP); macrolides [mph(A)]; phenicols (catB4); sulfonamides (sul1); and trimethoprim (dfrA30). The tetracycline resistance gene tetA was identified in Kp148/PINH-4900 and Kp314/11 only; the aminoglycoside resistance gene aph(3')-Ia was found only in environmental isolates, and aadA2 only in Kp314/11; and the phenicol resistance gene catA1 was identified only in Kp148/PINH-4900. CONCLUSIONS: The draft genome sequences of these strains help us to elucidate the dissemination of resistance genes in micro-organisms inside and outside the hospital and are useful for further comparisons between clinical and environmental strains.

Draft genome sequence of a blaCMY-2/IncI1-harbouring Escherichia coli D:ST457 isolated from coastal benthic organisms.

OBJECTIVES: Marine bivalves can act as bioindicators of marine environment pollution by multidrug-resistant (MDR) enteric bacteria of medical interest. The aim of this study was to report the draft genome sequence of a plasmid-encoded AmpC (pAmpC) (CMY-2)-carrying Escherichia coli isolate recovered from a marine bivalve sample in the coastal shore of Southeast Brazil. METHODS: The whole genome was sequenced on an Illumina NextSeq platform and was assembled using Velvet v.1.2.10. Data analysis was carried out using tools available from the Center of Genomic Epidemiology and Geneious R10 software. RESULTS: The genome size was calculated at 5198055bp, comprising a total of 5316 protein-coding sequences. The strain was assigned to ST457 and presented the blaCMY-2 pAmpC gene. In addition, the strain was clustered into the pathogenic phylogenetic group D. CONCLUSION: The release of this draft genome sequence can provide valuable information to better understand the dissemination of MDR enteric bacteria in marine environments.

Draft genome sequence of a CTX-M-8, CTX-M-55 and FosA3 co-producing Escherichia coli ST117/B2 isolated from an asymptomatic carrier.

OBJECTIVES: Asymptomatic carriers can act as reservoirs of multidrug-resistant (MDR) bacteria. The aim of this study was to describe the draft genome sequence of a MDR Escherichia coli lineage recovered from a faecal sample of a healthy carrier. METHODS: Genomic DNA was sequenced on an Illumina NextSeq platform. Sequence reads were de novo assembled using CLC Genomics Workbench and the whole genome sequence was evaluated through bioinformatics tools available from the Center of Genomic Epidemiology as well as additional in silico analysis. RESULTS: The genome size was calculated as 5178340 bp, with 5442 protein-coding sequences and 5492 total genes. Presence of the blaCTX-M-8, blaCTX-M-55 and fosA3 genes was detected in addition to other antimicrobial resistance genes. Interestingly, the strain was assigned to serotype O8:H4-fimH97 and was classified within the highly virulent phylogroup B2. CONCLUSION: This draft genome can provide helpful information to elucidate genetic features that contribute to colonisation and adaptation of MDR and virulent pathogens in asymptomatic carriers.

Draft genome sequence of an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST644 isolated from a footpad infection in a Magellanic penguin (Spheniscus magellanicus).

OBJECTIVES: The incidence of multidrug-resistant bacteria in wildlife animals has been investigated to improve our knowledge of the spread of clinically relevant antimicrobial resistance genes. The aim of this study was to report the first draft genome sequence of an extensively drug-resistant (XDR) Pseudomonas aeruginosa ST644 isolate recovered from a Magellanic penguin with a footpad infection (bumblefoot) undergoing rehabilitation process. METHODS: The genome was sequenced on an Illumina NextSeq® platform using 150-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.10, and the whole genome sequence was evaluated using bioinformatics approaches from the Center of Genomic Epidemiology, whereas an in-house method (mapping of raw whole genome sequence reads) was used to identify chromosomal point mutations. RESULTS: The genome size was calculated at 6436450bp, with 6357 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, ß-lactams, phenicols, sulphonamides, tetracyclines, quinolones and fosfomycin; in addition, mutations in the genes gyrA (Thr83Ile), parC (Ser87Leu), phoQ (Arg61His) and pmrB (Tyr345His), conferring resistance to quinolones and polymyxins, respectively, were confirmed. CONCLUSION: This draft genome sequence can provide useful information for comparative genomic analysis regarding the dissemination of clinically significant antibiotic resistance genes and XDR bacterial species at the human-animal interface.

Genome sequence analysis of a hypermucoviscous/hypervirulent and MDR CTX-M-15/K19/ST29 Klebsiella pneumoniae isolated from human infection.

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) with multidrug resistance (MDR) profile is a worrisome public health issue. We report the first draft genome sequence of a hypermucoviscous (positive string test) and MDR K. pneumoniae serotype K19, belonging to ST29, isolated from human infection. This strain harboured multiple antimicrobial resistance genes, including blaCTX-M-15, besides yersiniabactin and type 3 fimbriae virulence genes. In vivo experiments carried out with the Galleria mellonella infection model revealed that K. pneumoniae K19/ST29 killed 100% of the larvae at 24 h post-infection, in a similar way to the known hypermucoviscous hvKP K1/ST23 lineage.

Draft genome sequences of two fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST90 (ST23 complex) isolated from a calf and a dairy cow in South America.

OBJECTIVES: Farm animals have been recognised as important carriers and reservoirs of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. The aim of this study was to report the draft genome sequences of two multidrug-resistant (MDR) CTX-M-15-producing E. coli strains (47VL and 13B) isolated from different bovine hosts (a calf and a dairy cow), housed separately in a commercial dairy farm in Brazil. METHODS: Total genomic DNA of the E. coli isolates was sequenced using an Illumina MiSeq paired-end 300-bp sequencing platform. Sequence reads were de novo assembled using the A5-miseq pipeline and polishing assembly in Geneious v.R9. The NCBI Prokaryotic Genome Annotation Pipeline v.3.2 was used for genome annotation, whereas whole-genome sequences were analysed using bioinformatic tools from the Center of Genomic Epidemiology and EnteroBase. RESULTS: E. coli 47VL generated a total of 3238770 and E. coli 13B a total of 1422808 paired-end reads of ca. 190× and ca. 80×, respectively. The resistome revealed that both isolates carried resistance genes to aminoglycosides, ß-lactams, macrolides, sulphonamides, trimethoprim and tetracycline. Comparative analyses revealed clonal relatedness. In fact, both isolates belonged to sequence type ST90 (clonal complex CC23) and phylogroup AxB1. CONCLUSION: To our knowledge, these are the first draft genome sequences of CTX-M-15-producing E. coli ST90 isolated from bovines in South America. These data can be used to elucidate genetic features that contribute to colonisation and adaptation of CTX-M-15-producing E. coli in dairy cattle.

Draft genome sequence of a multidrug-resistant KPC-2-producing Enterobacter aerogenes isolated from a hospitalised patient in Brazil.

OBJECTIVES: Multidrug-resistant (MDR) Enterobacter aerogenes strains are frequently associated with nosocomial infections and high mortality rates, representing a serious public health problem. The aim of this study was to present the draft genome sequence of a MDR KPC-2-producing E. aerogenes isolated from a perineal swab of a hospitalised patient in Brazil. METHODS: Genomic DNA was sequenced using an Illumina MiSeq platform. De novo genome assembly was carried out using the A5-Miseq pipeline, and whole-genome sequence analysis was performed using tools from the Center for Genomic Epidemiology. RESULTS: The strain harboured resistance genes to ß-lactams, aminoglycosides, sulphonamides and trimethoprim in addition to genes encoding multidrug efflux system proteins, a quaternary ammonium transporter and heavy metal efflux system proteins. In addition, the strain harboured genes encoding diverse virulence factors. CONCLUSION: These data might allow a better understanding of the genetic basis of antimicrobial resistance and virulence in E. aerogenes strains.

Draft genome sequence of Enterobacter cloacae ST520 harbouring blaKPC-2, blaCTX-M-15 and blaOXA-17 isolated from coastal waters of the South Atlantic Ocean.

OBJECTIVES: Aquatic environments have contributed to the dissemination of multidrug-resistant (MDR) bacteria, representing a risk for humans and animals. The aim of this study was to report the first draft genome sequence of a MDR Enterobacter cloacae strain recovered from seawater in a public beach in Brazil. METHODS: The genome was sequenced on an Illumina MiSeq platform. De novo genome assembly was performed using SPAdes 3.10.1 and the whole genome sequence was analysed using bioinformatics tools from the Center of Genomic Epidemiology. RESULTS: This draft genome resulted in 5 228 857bp with 5331 protein-coding sequences, revealing the presence of blaKPC-2, blaCTX-M-15 and blaOXA-17 genes, responsible for resistance to all ß-lactam antibiotics. In addition, the strain was assigned to sequenced type 520 (ST520). CONCLUSION: These data provide useful information for comparative genomic analysis regarding the dissemination of antibiotic resistance genes.

Draft genome sequence of a multidrug-resistant Aeromonas hydrophila ST508 strain carrying rmtD and blaCTX-M-131 isolated from a bloodstream infection.

Here we report the draft genome sequence of a multidrug-resistant (MDR) Aeromonas hydrophila strain belonging to sequence type 508 (ST508) isolated from a human bloodstream infection. Assembly and annotation of this draft genome resulted in 5028498bp and revealed the presence of 16S rRNA methylase rmtD and blaCTX-M-131 genes encoding high-level resistance to aminoglycosides and cephalosporins, respectively, as well as multiple virulence genes. This draft genome can provide significant information for understanding mechanisms on the establishment and treatment of infections caused by this pathogen.

Draft genome sequence of a CTX-M-15-producing Escherichia coli ST345 from commercial chicken meat in Brazil.

Escherichia coli, the main host of the CTX-M-15 extended-spectrum ß-lactamase (ESBL) enzyme, is widely distributed and exchanged between the environment, animals and humans. Therefore, identification of blaCTX-M-15-positive lineages in food has a significant impact on public health. In this regard, until the end of 1990s, ESBL-producing isolates were mainly associated with hospital-acquired infections, with a predominance of SHV- and TEM-type enzymes. In recent years, a new trend has been observed among ESBL-producers, where most isolates now harbour CTX-M-type, being further isolated from community-acquired infections. Nowadays, CTX-M-15 has been recognised as the most important ESBL variant, invading virtually all human and animal compartments, leading to a global pandemic. Thus, whilst the rapid emergence and dissemination of CTX-M-15 among E. coli isolates has generated a large genetic reservoir from which other members of the Enterobacteriaceae family can easily acquire this resistance gene, there are an increasing number of new reservoirs and transmission mechanisms that must be investigated. In this study, we present the draft genome sequence of a CTX-M-15-producing E. coli ST345 isolated from commercial chicken meat in Brazil. This draft genome can be used as a reference sequence for comparative analysis among CTX-M-15-producers.

Draft Genome Sequences of Colistin-Resistant MCR-1-Producing Escherichia coli ST1850 and ST74 Strains Isolated from Commercial Chicken Meat.

We present here the draft genome sequences of two colistin-resistant mcr-1-carrying Escherichia coli strains belonging to sequence type 74 (ST74) and ST1850, isolated from commercial chicken meat in Brazil. Assembly of this draft genome resulted in 5,022,083 and 4,950,681 bp, respectively, revealing the presence of the IncX4 plasmid-mediated mcr-1 gene responsible for resistance to colistin.

Draft genome sequence of an environmental multidrug-resistant Klebsiella pneumoniae ST340/CC258 harbouring blaCTX-M-15 and blaKPC-2 genes.

Anthropogenic activities, including the release of wastewater and sewage from hospitals, have contributed to the contamination of aquatic environments, raising a concern to public health. In this study, we present the first draft genome sequence of a Klebsiella pneumoniae strain (Kp171, TIET-4200) belonging to the high-risk hospital-associated clonal lineage ST340/CC258, which was recovered from a water sample collected in an urban river in Brazil.

Draft genome sequence of an aminoglycoside-resistant RmtG-producing Pseudomonas aeruginosa ST235 isolated from a cystic fibrosis patient.

Cystic fibrosis (CF) patients are often chronically colonised or infected by non-fermenting Gram-negative bacilli, with Pseudomonas aeruginosa being the most prevalent. In this study, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain belonging to sequence type ST235, isolated from the respiratory tract of a CF patient with chronic colonisation. Whole-genome sequencing analysis revealed a 6.7Mb genome size and the presence of 12 antibiotic resistance genes, including the rmtG gene conferring high-level aminoglycoside resistance, located on the chromosome.

Draft Genome Sequence of a Hospital-Associated Clone of Klebsiella pneumoniae ST340/CC258 Coproducing RmtG and KPC-2 Isolated from a Pediatric Patient.

We report here the draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to the hospital-associated sequence type 340 (ST340; clonal complex CC258), isolated from a catheter tip culture from a pediatric patient. The multidrug-resistant strain coproduced the 16S rRNA methyltransferase rRNA RmtG and ß-lactamases KPC-2 and CTX-M-15.

Draft genome sequence of a CTX-M-15-producing Klebsiella pneumoniae sequence type 340 (clonal complex 258) isolate from a food-producing animal.

Klebsiella pneumoniae carrying blaCTX-M-15 have been widely disseminated in hospital settings. In this regard, most clinically important strains belong to clonal complex 28 (CC258), which includes sequence type 340 (ST340). In this study, we present the draft genome sequence of a CTX-M-15-producing ST340 K. pneumoniae strain isolated from a food-producing animal in Brazil.

Cytotoxicity and genotoxicity of silver nanoparticles of different sizes in CHO-K1 and CHO-XRS5 cell lines.

Nanoparticles (NPs) have been used in a range of products due to their unique properties. Nevertheless, these NPs can cause adverse biological effects and because of that, there is a great concern about the health and environmental risks related to their use. Recently, silver nanoparticles (Ag NPs) have been used in a variety of cytotoxicity and genotoxicity studies, but there are still controversies regarding the association between the size and the toxicity of these particles. Therefore, in this study, we aimed to evaluate the cytotoxicity and genotoxicity of Ag NPs (10 and 100 nm) in two different cell lines, CHO-K1 and CHO-XRS5, by performing cell viability assay (XTT), clonogenic assay, micronucleus test, comet assay, as well as by investigating the cell cycle kinetics using the flow cytometry. Cell cultures were exposed to different concentrations of AgNPs (0.025-5.0 µg/ml) for 24 h. Our results indicated that cytotoxicity and genotoxicity induced by the 100 nm-Ag NPs were greater than those induced by the 10 nm-Ag NPs for both cell lines, which suggests that the exposure to greater size particles (100 nm) can cause more adverse biological effects than the exposure to the smaller ones (10 nm).

Cyto- and genotoxic effects of metallic nanoparticles in untransformed human fibroblast.

Metallic nanoparticles such as silver (Ag), cerium dioxide (CeO2) and titanium dioxide (TiO2) are produced at a large scale and included in many consumer products. It is well known that most metallic NPs are toxic to humans which raise concerns about these engineered particles. Various studies have already been published on the subject, however, almost all of these studies have been conducted in cancer or transformed cell lines. In this work we performed a comparative evaluation of these metallic NPs on normal untransformed human fibroblasts (GM07492) detecting cyto- and geno-toxic responses after exposure to these NPs. Our results showed that all three metallic NPs were able to cross the plasma membrane and were mainly found in endocytic vesicles. The Ag and TiO2 NPs affected mitochondrial enzymatic activity (XTT), increased DNA fragmentation, oxidative damage (Comet assay) and induced cell death mainly by the apoptotic pathway. Ag NPs increased GADD45α transcript levels and the phosphorylation of proteins γH2AX. Transient genotoxicity was also observed from exposure to CeO2 NPs while TiO2 NPs showed no increase in DNA damage at sub-cytotoxic concentrations. In comparison, Ag NPs were found to be the most cyto-genotoxic NPs to fibroblasts. Thus, these results support the use of normal fibroblast as a more informative tool to detect the mechanisms of action induced by metallic NPs.