Carbapenems represent the mainstay therapy for the treatment of serious P. aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-β-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centers. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the blaSPM−1 gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together, these factors could contribute to the marked resistance and persistence of the SPM-1-producing P. aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa . To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

Pseudomonas aeruginosa is one of the most common pathogens related to healthcare-associated infections. The Brazilian isolate, named CCBH4851, is a multidrug-resistant clone belonging to the sequence type 277. The antimicrobial resistance mechanisms of the CCBH4851 strain are associated with the presence of the bla SPM-1 gene, encoding a metallo-beta-lactamase, in combination with other exogenously acquired genes. Whole-genome sequencing studies focusing on emerging pathogens are essential to identify key features of their physiology that may lead to the identification of new targets for therapy. Using both illumina and pacBio sequencing data, we obtained a single contig representing the CCBH4851 genome with annotated features that were consistent with data reported for the species. However, comparative analysis with other Pseudomonas aeruginosa strains revealed genomic differences regarding virulence factors and regulatory proteins. In addition, we performed phenotypic assays that revealed CCBH4851 is impaired in bacterial motilities and biofilm formation. On the other hand, CCBH4851 genome contained acquired genomic islands that carry transcriptional factors, virulence and antimicrobial resistance-related genes. presence of single nucleotide polymorphisms in the core genome, mainly those located in resistance-associated genes, suggests that these mutations may also influence the multidrug-resistant behavior of CCBH4851. Overall, characterization of Pseudomonas aeruginosa CCBH4851 complete genome revealed the presence of features that strongly relates to the virulence and antibiotic resistance profile of this important infectious agent. Pseudomonas aeruginosa is one of the most common pathogens related to healthcare-associated infections in hospitalized individuals worldwide. Multidrug-resistant (MDR) isolates, particularly those non-susceptible to carbapenems, have become a major concern of health institutions. In addition to mechanisms such as loss of porins or overexpression of efflux pumps, carbapenem resistance is produced by the acquisition of genes encoding carbapenem-hydrolyzing beta-lactamases 1. These enzymes are classified into classes A and D, the activesite serine beta-lactamases (SBLs), and class B, the zinc-dependent or metallo-beta-lactamases (MBLs). SBLs have a broad spectrum of activity against beta-lactams but are inhibited by common beta-lactamase inhibitors.

Background -Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide.Gene regulatory networks computationally represent interactions among regulatory genes and their targets, an important approach to understand bacterial behavior and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. Objectives -Our goal in this manuscript is to present a reconstruction of multidrug-resistant P. aeruginosa gene regulatory network and to analyze its topological properties. Methods -The methodology was based on gene orthology inference by the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This multidrug-resistant strain is representative of an endemic outbreak in Brazilian territory belonging to ST277. Findings -As the main finding, we obtained a network with a larger number of regulatory genes, target genes and interactions compared to previous work. Topological analysis results are accordant to the complex network representation of biological processes. Main conclusions -The network properties are consistent with P. aeruginosa biological features. To the best of our knowledge, the P. aeruginosa gene regulatory network presented here is the most complete version available to date. Pseudomonas aeruginosa | gene regulatory network | multidrug resistanceCorrespondence: ana.pbn@gmail.com

Agradeço a Deus a oportunidade da vida. Sem ti, querido Pai, não estaria aqui. Agradeço as agências financiadoras, CAPES e FAPESP, o suporte ao projeto. Agradeço a Universidade de São Paulo e o Instituto de Química a infraestrutura acadêmica. Agradeço a Doutora Regina Lúcia Baldini a confiança na minha capacidade de realizar este trabalho. Mais que isso, agradeço a orientação, a amizade, o carinho e os conselhos dados a respeito da caminhada científica e da vida. Compartilhar da sua experiência acrescentou apenas bons aspectos à minha formação como profissional e como pessoa. Agradeço a Doutora Suely Lopes Gomes a disposição do espaço físico, os recursos e o zelo pela organização sempre impecáveis do ambiente de trabalho. Agradeço aos membros que compuseram minha banca de qualificação, os Doutores Daniela Sanchez Basseres, Maria Terêsa Machini e Beny Spira, as críticas construtivas e sugestões. Agradeço o Doutor Bayardo Baptista Torres o ensino da Bioquímica de maneira tão fluída e prazerosa, sem assistir suas aulas jamais teria sido capaz de iniciar esta jornada. Agradeço a todos os professores que contribuíram para a realização deste trabalho e para a minha formação acadêmica através de aulas, discussões informais e acesso a equipamentos e reagentes, em especial a Doutora Marilis do Valle Marques. Agradeço aos amigos do grupo de pesquisa das professoras Regina e Suely. Desde 2005, quando iniciei meu estágio no laboratório, até o atual momento, muitas pessoas passaram por minha vida e deixaram sua contribuição.