Bruna Fuga Araújo

Clinical and Molecular Epidemiology of Multidrug-Resistant P. aeruginosa Carrying aac(6')-Ib-cr, qnrS1 and blaSPM Genes in Brazil.

We described a comprehensive analysis of the molecular epidemiology of multidrug-resistant (MDR) P. aeruginosa. Molecular analysis included typing by Pulsed Field Gel Electrophoresis, identification of genes of interest through PCR-based assays and sequencing of target genes. Case-control study was conducted to better understand the prognostic of patients and the impact of inappropriate therapy in patients with bacteremia, as well as the risk factors of MDR infections. We observed a high rate of MDR isolates (40.7%), and 51.0% of them was independently associated with inappropriate antibiotic therapy. Bacteremia was detected in 66.9% of patients, and prolonged hospital stay was expressive in those resistant to fluoroquinolone. Plasmid-mediated quinolone resistance genes (PMQR), qnrS1 and aac(6’)Ib-cr, were detected in two different nosocomial isolates (5.3%), and the aac(6’)-Ib7 variant was detected at a high frequency (87.5%) in those negative to PMQR. The presence of mutations in gyrA and parC genes was observed in 100% and 85% of selected isolates, respectively. Isolates harboring PMQR genes or mutations in gyrA and parC were not closely related, except in those containing SPM (São Paulo metallo-β-lactamase) clone. In addition, there is no study published in Brazil to date reporting the presence of Pseudomonas aeruginosa isolates harboring both qnrS1 and aac(6’)Ib-cr genes, with alarming frequency of patients with inappropriate therapy.

Multidrug Resistance Related to Biofilm Formation in Acinetobacter baumannii and Klebsiella pneumoniae Clinical Strains from Different Pulsotypes.

The emergence of Acinetobacter baumannii and Klebsiella pneumoniae strains in the hospital environment has been associated with the presence of multiple genetic elements, virulence factors and the ability to form biofilms. This study evaluated the biofilm formation ability of clinical and environmental A. baumannii and K. pneumoniae strains, isolated from various sources and presenting different molecular characteristics, resistance profiles and pulsed-field gel electrophoresis patterns. Fifty-three isolates were recovered from 2009 to 2014 in a Brazilian university hospital. Investigation of biofilm formation was performed for 10 strains of each species assessed by an initial adhesion assay, biofilm cell concentration and biofilm biomass, evaluated by quantitative assays in replicates, in three independent experiments. All strains of A. baumannii were able to attach to polystyrene plates, although two strains adhered to a lesser degree than the control. K. pneumoniae strains showed opposite behaviour, where only three strains adhered significantly when compared to the control. Quantitative evaluation revealed that in five A. baumannii and four K. pneumoniae isolates the biomass production could be characterised as moderate. None of the isolates were strong biofilm producers. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst A. baumannii and K. pneumoniae clinical strains and it was not associated with certain clonal types; (2) no relationship between multidrug resistance and biofilm production was observed; (3) more virulent K. pneumoniae strains tended to present higher production of biofilm.

Molecular epidemiological survey of bacteremia by multidrug resistant Pseudomonas aeruginosa: the relevance of intrinsic resistance mechanisms

The bacterial factors associated with bacteremia by multidrug-resistant and extensively drug-resistant P. aeruginosa, including overexpression of efflux pumps, AmpC overproduction, and loss/alteration of the OprD porin in isolates that are non-Metallo-β-Lactamase producing were analyzed in a retrospective study. Molecular analyses included strain typing by Pulsed Field Gel Electrophoresis and identification of key genes via qualitative and quantitative PCR-based assays. Previous use of carbapenems and tracheostomy was independently associated with the development of bacteremia by extensively drug-resistant and multidrug-resistant strains of P. aeruginosa. A high consumption of antimicrobials was observed, and 75.0% of the isolates contained amplicons with the blaSPM-1 and blaVIM genes. Of the 47 non-Metallo-β-Lactamase isolates, none had another type of carbapenemase. However, the isolates exhibited high rates of hyperproduction of AmpC, loss of the OprD porin (71.4%) and the presence of MexABOprM (57.1%) and MexXY (64.3%). This study suggests that in non-Metallo-β-Lactamase isolates, the association of intrinsic resistance mechanisms could contributes to the expression of multidrug-resistant/extensively drug-resistant phenotypes.

Biofilm formation of Brazilian meticillin-resistant Staphylococcus aureus strains: prevalence of biofilm determinants and clonal profiles

Biofilms plays an important role in medical-device-related infections. This study aimed to determine the factors that influence adherence and biofilm production, as well as the relationship between strong biofilm production and genetic determinants in clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Fifteen strains carrying different chromosomal cassettes recovered from hospitalized patients were selected; five SCCmecII, five SCCmecIII and five SCCmecIV. The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. PFGE and multilocus sequence typing (MLST) techniques were also performed. The initial adhesion and biofilm formation were examined by quantitative assays. The surface tension and hydrophobicity of the strains were measured by the contact angle technique to evaluate the association between these parameters and adhesion ability. SCCmecIII and IV strains were less hydrophilic, with a high value for the electron acceptor parameter and higher adhesion in comparison with SCCmecII strains. Only SCCmecIII strains could be characterized as strong biofilm producers. The PFGE showed five major pulsotypes (A-E); however, biofilm production was related to the dissemination of one specific PFGE clone (C) belonging to MLST ST239 (Brazilian epidemic clonal complex). The genes agrI, fnbB and IS256 in SCCmecIII strains were considered as genetic determinants associated with strong biofilm-formation by an ica-independent biofilm pathway. This study contributes to the understanding of biofilm production as an aggravating factor potentially involved in the persistence and severity of infections caused by multidrug-resistant MRSA belonging to this genotype.

Hypervirulence and biofilm production in KPC-2-producing Klebsiella pneumoniae CG258 isolated in Brazil

In this study, we describe the frequency of virulence genes in Klebsiella pneumoniae carbapenemase-2-producing Klebsiella pneumoniae (KPC-KP), including hypervirulent (hv) and hypermucoviscous (hm) strains by whole-genome sequencing. We also evaluate the capacity for biofilm formation by using phenotypic techniques. The occurrence of several virulence genes (fimABCDEFGHIK, mrkABCDFHJ, ecpA, wabG, entB, ugE, irp1, irp2, traT, iutA and ureADE) and a high frequency of hvhmKPC-KP isolates was found. Most hospital-associated lineages of KPC-KP belong to the international clonal group 258 (CG258). Biofilm formation was a constant feature among 90.9 % of KPC-KP strains. This report suggests a close relationship between ST437 and weak biofilm production, given that all weakly biofilm-producing strains belonged to this sequence type. This also supports the dissemination of KPC-KP containing numerous virulence determinants belonging to the biofilm-producing CG258 type in Brazil, including hv and hm strains. These factors allow this pathogen to cause infections, leading to its rapid expansion and persistence in hospital settings.

IncX3 plasmid harboring a non-Tn4401 genetic element (NTEKPC) in a hospital-associated clone of KPC-2-producing Klebsiella pneumoniae ST340/CG258

IncX-type plasmids have achieved clinical significance for their contribution in the dissemination of genes confering resistance to carbapenems (most blaKPC- and blaNDM-type genes) and polymyxins (mcr-type genes), both antibiotics considered last resort for multidrug-resistant Gram-negative infections. In this study, we report the identification and complete sequence analysis of an IncX3 plasmid (designated pKP1194a) carrying a non-Tn4401 genetic element (NTEKPC) of tnpR-tnpA (partial)-blaKPC-2-ΔISKpn6/traN, originating from a hospital-associated lineage of K. pneumoniae belonging to the ST340/CG258, with epidemiological link to Brazil.

Molecular characterization and clonal dynamics of nosocomial blaOXA-23 producing XDR Acinetobacter baumannii

The emergence of infections associated to new antimicrobial resistance in Acinetobacter baumannii (Ab) genotypes represents a major challenge. In this context, this study aimed to determine the diversity of resistance mechanisms and investigate clonal dissemination and predominant sequence types (STs) in multidrug-resistant Ab strains of clinical (tracheal aspirate, n = 17) and environmental (surface, n = 6) origins. Additionally, the major clones found in clinical (A) and environmental (H) strains had their complete genomes sequenced. All strains were submitted to polymerase chain reactions (PCR) for the detection of the ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like genes, while the expression of genes encoding the carO porin, AdeABC (adeB), AdeFGH (adeG), and AdeIJK (adeJ) efflux pumps was determined by real time PCR (qPCR). Most of the strains were characterized as extensively drug-resistant (XDR) with high minimal inhibitory concentrations (MICs) detected for tigecycline and carbapenems. Associations between ISAba1/OXA-51 and ISAba1/OXA-23 were observed in 91.3% and 52.2% of the strains, respectively. Only the adeB gene was considered hyper-expressed. Furthermore, most of the strains analyzed by the MuLtilocus Sequence-Typing (MLST) were found to belong to the clonal complex 113 (CC113). In addition, a new ST, ST1399, belonging to CC229, was also discovered herein. Strains analyzed by whole genome sequencing presented resistance genes linked to multidrug-resistance phenotypes and confirmed the presence of Tn2008, which provides high levels carbapenem-resistance.

High frequency of the combined presence of QRDR mutations and PMQR determinants in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections

Plasmid-mediated quinolone resistance (PMQR) determinants combined with mutations in quinolone resistance-determining regions (QRDRs) and clonal dissemination were investigated in 40 fluoroquinolone-resistant Klebsiellapneumoniae and Escherichiacoli isolates from nosocomial and community-acquired infections. We observed nucleotide substitutions in gyrA (Ser83Ile, Val37Leu, Lys154Arg, Ser171Ala, Ser19Asn, Ile198Val, Ser83Tyr, Ser83Leu, Asp87Asn and Asp87Gly) and parC genes (Ser80Ile, Glu84Lys, Ala129Ser, Val141Ala and Glu84Gly). Two novel substitutions were detected in the gyrA gene (Val37Leu and Ile198Val). The presence of PMQR genes predominated in community isolates (55.5 %). In addition to the frequent presence of the class 1 integron in isolates from community-acquired infections, the genetic similarity results obtained by PFGE showed high genomic diversity. This study suggests that management of multidrug-resistant Enterobacteriaceae isolates from the community are a possible source of genetic mobile elements that carry genes that confer resistance to fluoroquinolones. More attention should be paid to the surveillance of community-acquired infections.