Search

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the ‘A-rule’. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.

Gemcitabine (GEM) is the drug used as first line to treat pancreatic cancer, one of the most devastating human tumors. This peculiar type of tumor develops resistance to several drugs, including GEM, due to its desmoplastic reaction and other features. The GEM chemoresistance has been investigated at molecular level aiming to find a pathway whose inhibition or activation should overcome it. Through next-generation sequencing was performed a chemogenomic assay of GEM using Saccharomyces cerevisiae as model cell and the results showed that more than 40% of genes related to GEM response in yeast possess unknown or dubious function. We choose two yeast mutants to individually validate the fitness defect results observed by chemogenomic assay, Δhmt1 and Δcsi1, and it was found that in addition to some already described pathways involved in GEM resistance, cells deficient in deneddylation enzyme Cop9 Signalosome Interactor 1 (Csi1p) presented a high sensitivity to GEM. This was confirmed by individual growth analyses of Δcsi1 cells exposed to GEM, and this phenotype was reverted with CSI1 complementation gene. Csi1p is a well-characterized homolog equivalent to human Csn6 subunit of COP9 signalosome (CSN) involved in deneddylation process. We highlighted too that epigenetic alterations, such as methylation mediated by protein arginine methyltransferase 1, play an important role in regulating gemcitabine treatment resistance. Our results point out new unexplored molecular pathways that can be used to overcome GEM resistance: the inhibition of CSN and the arginine methyltransferase activities. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00154-7) contains supplementary material, which is available to authorized users.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway as it removes different kinds of bulky lesions. Due to its essential role for genome integrity, it has appeared early in the evolution of species. However, most published studies are focused on humans, mice, yeast or bacteria. Considering the large amount of information on genome databases, it is currently possible to retrieve sequences from NER components in many organisms. Therefore, we have characterized the potential orthologs of 10 critical components of the human NER pathway in 12 eukaryotic species by using similarity and structural criteria through the use of bioinformatics tools. This approach has allowed us to characterize gene and protein structures comparatively, taking a glance at some evolutionary aspects of the NER pathway. We have obtained significant search results for the majority of the proteins in most of the organisms studied, mainly for factors that play a pivotal role in the pathway. However, we have revisited significant differences and found new aspects that may imply a distinct functioning of this pathway in different organisms. Through the demonstration of the heterogeneity of the gene structures and a variety in the protein architecture of the NER components evaluated, our results show important differences between human NER and evolutionarily distant eukaryotes. We highlight the lack of a canonical XPD in chicken, the divergence of XPA in plants and protozoans and the absence of XPE in the invertebrate species analyzed. In spite of this, it is remarkable the presence of this excision repair mechanism in a high number of evolutionary distant organisms, being present since the origin of eukaryotes.

The tremor mutant phenotype results from an autosomal recessive spontaneous mutation arisen in a Swiss–Webster mouse colony. The mutant mice displayed normal development until three weeks of age when they began to present motor impairment comprised by whole body tremor, ataxia, and decreased exploratory behavior. These features increased in severity with aging suggesting a neurodegenerative profile. In parallel, they showed audiogenic generalized clonic seizures. Results from genetic mapping identified the mutation tremor on chromosome 14, in an interval of 5 cM between D14Mit37 (33.21 cM) and D14Mit115 (38.21 cM), making Early Growth Response 3 (Egr3) the main candidate gene. Comparing with wild type (WT) mice, the tremor mice showed higher hippocampal gene expression of Egr3 and Gabra1 and increased concentrations of noradrenalin (NOR; p = .0012), serotonin (5HT; p = .0083), 5-hydroxyindoleacetic acid (5-HIAA; p = .0032), γ-amino butyric acid (GABA; p = .0123), glutamate (p = .0217) and aspartate (p = .0124). In opposition, the content of glycine (p = .0168) and the vanillylmandelic acid (VMA)/NOR ratio (p = .032) were decreased. Regarding to dopaminergic system, neither dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents nor the turnover rate of DA showed statistically significant differences between WT and mutant mice. Data demonstrated that audiogenic seizures of tremor mice are associated with progressive motor impairment as well as to hippocampal alterations of the Egr3 and Gabra1 gene expression and amino acid and monoamine content. In addition, the tremor mice could be useful for study of neurotransmission pathways as modulators of epilepsy and the pathogenesis of epilepsies occurring with generalized clonic seizures.

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.

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 5 464 058, 5 437 723 and 5 319 218 bp, 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]; β-lactams (blaOXA-1, blaSHV-11, blaCTX-M-15 and blaKPC-2); fluoroquinolones [aac(6ʹ)-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(3ʹ)-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.

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.

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 5 178 340 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.

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 6 436 450 bp, with 6357 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, β-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.

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 β-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.

Escherichia coli, the main host of the CTX-M-15 extended-spectrum β-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.

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 5 198 055 bp, 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.

Objectives Farm animals have been recognised as important carriers and reservoirs of extended-spectrum β-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 3 238 770 and E. coli 13B a total of 1 422 808 paired-end reads of ca. 190× and ca. 80×, respectively. The resistome revealed that both isolates carried resistance genes to aminoglycosides, β-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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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 5 028 498 bp and revealed the presence of 16S rRNA methylase rmtD and bla(CTX-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. (C) 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved. Univ São Paulo, Inst Biomed Sci, Dept Microbiol, São Paulo, Brazil Univ São Paulo, Sch Pharm, Dept Clin Anal, São Paulo, Brazil Univ Fed São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Univ São Paulo, Inst Biomed Sci, Genome Invest & Anal Lab GENIAL, São Paulo, Brazil Univ Fed São Paulo UNIFESP, Dept Med, Disciplina Infectol, Lab Alerta, São Paulo, Brazil Univ Fed São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Univ Fed São Paulo UNIFESP, Dept Med, Disciplina Infectol, Lab Alerta, São Paulo, Brazil FAPESP: 2016/08593-9 CNPq: 462042/2046-6 Web of Science

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.

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.

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 β-lactamases KPC-2 and CTX-M-15.

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.

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 5 228 857 bp with 5331 protein-coding sequences, revealing the presence of bla KPC-2 , bla CTX-M-15 and bla OXA-17 genes, responsible for resistance to all β-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.

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.