Decreasing the Cut-off Score Value of MALDI-ToF MS Increase the Identities of Burkholderia cepacia Complex Species.

Burkholderia cepacia complex (Bcc) comprises 24 related species genetically distinct, associated with high mortality in cystic fibrosis (CF) patients. Due to a high level of similarity among Bcc species, accurate identification has been problematic, and most conventional and automated phenotypic tests have shown low accuracy. We evaluated accuracy of MALDI-ToF MS decreasing the cut-off score value to distinguish Bcc species compared to recA gene sequencing. A total of 145 Bcc isolates were analyzed. B. vietnamiensis (41.37%), B. cenocepacia IIIA (23.44%), B. multivorans (20%), B. cenocepacia IIIB (11.03%), and B. contaminans (2.75%) among other species were identified by recA sequencing. MALDI-ToF MS identified 100% of Bcc isolates at the genus level and 53.1% at the species level. By decreasing cut-off values for ≥1.70, the correct identification at the species level increased to 74.5%. MALDI-ToF MS proved to be useful at the genus level identification, but it still requires improvements that allow more precise identification, requiring continuous updates and addition of new spectra to its database. A review of interpretative criteria is a field to be explored with a large collection of Bcc species.

Exploring the success of Brazilian endemic clone Pseudomonas aeruginosa ST277 and its association with the CRISPR-Cas system type I-C.

BACKGROUND: The Brazilian endemic clone Pseudomonas aeruginosa ST277 carries important antibiotic resistance determinants, highlighting the gene coding for SPM-1 carbapenemase. However, the resistance and persistence of this clone is apparently restricted to the Brazilian territory. To understand the differences between Brazilian strains from those isolated in other countries, we performed a phylogenetic analysis of 47 P. aeruginosa ST277 genomes as well as analyzed the virulence and resistance gene profiles. Furthermore, we evaluated the distribution of genomic islands and assessed in detail the characteristics of the CRISPR-Cas immunity system in these isolates. RESULTS: The Brazilian genomes presented a typical set of resistance and virulence determinants, genomic islands and a high frequency of the CRISPR-Cas system type I-C. Even though the ST277 genomes are closely related, the phylogenetic analysis showed that the Brazilian strains share a great number of exclusively SNPs when compared to other ST277 genomes. We also observed a standard CRISPR spacers content for P. aeruginosa ST277, confirming a strong link between sequence type and spacer acquisition. Most CRISPR spacer targets were phage sequences. CONCLUSIONS: Based on our findings, P. aeruginosa ST277 strains circulating in Brazil characteristically acquired In163 and PAGI-25, which can distinguish them from strains that do not accumulate resistance mechanisms and can be found on the Asian, European and North American continents. The distinctive genetic elements accumulated in Brazilian samples can contribute to the resistance, pathogenicity and transmission success that characterize the ST277 in this country.

Whole genome sequencing of a ST2594 MRSA strain causing non-mucosal preoperative colonization and low-grade postoperative infection.

We present a post-operative infection caused by a methicillin-resistant Staphylococcus aureus strain, previously isolated in the preoperative screening, in a patient submitted to femoral osteosynthesis, successfully treated with oral ciprofloxacin. The isolate exhibited in vitro resistance to ciprofloxacin, Staphylococcal Cassette Chromosome mec type IV, it was negative for the lukS-PV Panton-Valentine leucocidin gene and belonged to ST2594 in multilocus sequence typing analysis. Whole genome sequencing revealed a genome size of 2,818,289 base pairs. The annotated genomes of ST2594 and N315 strains were compared, looking for genes related to virulence and resistance. The lack of the tst, sec, sel genes, associated with a mutation in the clfA gene, may partially explain the low morbity in this case.

Multiclonal Expansion of Klebsiella pneumoniae Isolates Producing NDM-1 in Rio de Janeiro, Brazil.

We characterized NDM-1-producing Klebsiella isolates from Rio de Janeiro, Brazil. PCR was applied for resistance and virulence determinants. The genetic context of blaNDM was determined by S1 nuclease pulsed-field gel electrophoresis (PFGE) and hybridization. Genotyping was performed by PFGE and multilocus sequence typing (MLST). Most isolates carried multiple resistance genes and remained susceptible to amikacin, fosfomycin-trometamol, polymyxin B, and tigecycline. The spread of NDM-1-producing Klebsiella pneumoniae was not associated with clonal expansion and appears to be associated with Tn3000.

Monitoring clinical and microbiological evolution of a cystic fibrosis patient over 26 years: experience of a Brazilian CF Centre.

BACKGROUND: Burkholderia cepacia complex is a group of opportunistic pathogens in cystic fibrosis (CF) patients believed to be associated with poor prognosis and patient-to-patient transmissibility. Little is known about clinical outcomes after B. vietnamiensis chronic colonization/infection. CASE PRESENTATION: A 33 yo male patient had diagnosis of CF by 7 yo, after recurrent pneumonia during infancy and lobectomy (left upper lobe) at 6 yo. Burkholderia cepacia complex (Bcc) was first isolated by 13 yo, and the patient fulfilled the criteria for chronic colonization by 15 yo. In the following 16 years (1997-2013), there was intermittent isolation of P. aeruginosa and continuous isolation of Bcc, identified as B. vietnamiensis. There was clinical and laboratorial stability for 16 years with annual rate of decline in forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) of 1.61 and 1.35%, respectively. From 2013 to 2015, there was significant clinical and lung function deterioration: annual rate of decline in FEV1 and FVC was 3 and 4.1%, respectively while body mass index decreased from 18.1 to 17.1. Episodes of hemoptysis and respiratory exacerbations (with hospital admissions) became more frequent. CF related diabetes was diagnosed (fasting glycemia: 116 mg/dL, oral glucose tolerance test: 305 mg/dL). Because of the severity of the disease in the last years, in addition to traditional microbiological surveillance, microbiome analysis by next generation sequencing (NGS) was performed on respiratory secretions. The NGS showed that 97% of the sequencing data were attributed to genus Burkholderia. CONCLUSIONS: We report the case of a 33-year-old male CF patient known to have chronic infection with B. vietnamiensis who remained clinically stable for 16 years and presented recent clinical and laboratorial deterioration. Microbiome analysis of respiratory secretions was performed in 3 samples collected in 2014-2015. Clinical deterioration overlapped with cystic fibrosis-related diabetes and microbiome composition revealed no significant differences when compared microbiome results to culture dependent methods.

First report of the emerging zoonotic agent Wohlfahrtiimonas chitiniclastica isolated from a retail frozen chicken in Rio de Janeiro, Brazil.

Wohlfahrtiimonas chitiniclastica is an emerging zoonotic bacterium commensally living in larvae of particular flies. It has been associated with human and animal infections but never isolated from food. In the present study, a whole chicken carcass was rinsed in buffered peptone water which was then inoculated into BHI and the growth plated onto selective medium. Species identification was performed by MALDI-TOF MS. Those bacteria identified as W. chitiniclastica were subjected to 16S rRNA sequencing for confirmation and MEGA software was used to obtain their phylogenetic position. The findings of this study raise concerns regarding the abattoir, transport and stock practices of frozen meat carcasses and should be of interest with regard to microbiology, entomology and food production.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Central role of PAFR signalling in ExoU-induced NF-κB activation.

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR-/- or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.

Fecal pollution source tracking in waters intended for human supply based on archaeal and bacterial genetic markers.

The determination of fecal pollution sources in aquatic ecosystems is essential to estimate associated health risks. In this study, we evaluate eight microbial source tracking (MST) markers including host-specific Bacteroidales and Methanobrevibacter spp. for discrimination between human, bovine, equine, and swine fecal contamination in waters intended for human supply. Overall, the novel host-specific archaeal and bacterial primers proposed in this study demonstrated high sensitivity and specificity. Markers for the Archaea domain were more prevalent in the fecal and water samples studied. We conclude that the investigations regarding the sources of fecal pollution in public water supplies can contribute to improve the quality of human health. To our knowledge, this is the first analysis using both archaeal and bacterial fecal MST markers on tropical water bodies of Rio de Janeiro city, Brazil.

Burkholderia cepacia complex: clinical course in cystic fibrosis patients.

BACKGROUND: Pulmonary deterioration after B.cepacia complex (BCC) colonization has a heterogeneous pattern. The aim was to investigate the clinical outcome of BCC colonization in CF patients chronically colonized with P. aeruginosa. METHODS: CF patients chronically colonized with P. aeruginosa were divided into three groups: intermittent (I), chronic (II) and no colonization (III) with BCC. Body mass index (BMI) percentile and spirometric parameters were analyzed at three different times in each group. RESULTS: Fifty-six patients chronically colonized with P. aeruginosa were included. Of these, 27 also had evidence of BCC colonization (13 intermittent and 14 chronic). BMI percentile was significantly lower among patients chronically colonized by both P. aeruginosa and BCC. Mean values of FEV1 and FVC % were also significantly lower in these patients, both at the time of chronic BCC colonization and 24 months forward. CONCLUSIONS: Chronic BCC colonization is associated with significant loss of lung function. Lower BMI might be a risk factor for chronic BCC colonization, preceding these events.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil.

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri (CCBH11880), Enterobacter hormaechei subsp. oharae (CCBH10892) and Klebsiella pneumoniae (CCBH13327), isolated in Brazil. Besides blaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2, aac(6')-Ib-cr] and quinolones (qnrA1, qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to the blaNDM-1 gene in all strains.

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil.

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii(ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.

Comparative analysis of the antimicrobial resistance and virulence traits in ESBL-producing-Klebsiella pneumoniae ST307 strains colonizing the gastrointestinal tract and causing a fatal bloodstream infection in a leukemia patient.

Klebsiella pneumoniae is an opportunistic pathogen that can colonize the gastrointestinal tract (GIT) of humans. The mechanisms underlying the successful translocation of this pathogen to cause extra-intestinal infections remain unknown, although virulence and antimicrobial resistance traits likely play significant roles in the establishment of infections. We investigated K. pneumoniae strains isolated from GIT colonization (strains Kp_FZcol-1, Kp_FZcol-2 and Kp_FZcro-1) and from a fatal bloodstream infection (strain Kp_HM-1) in a leukemia patient. All strains belonged to ST307, carried a transferable IncF plasmid containing the blaCTX-M-15 gene (pKPN3-307 TypeA-like plasmid) and showed a multidrug-resistance phenotype. Phylogenetic analysis demonstrated that Kp_HM-1 was more closely related to Kp_FZcro-1 than to the other colonizing strains. The Kp_FZcol-2 genome showed 81 % coverage with the Kp_HM-1 246,730 bp plasmid (pKp_HM-1), lacking most of its putative virulence genes. Searching public genomes with similar coverage, we observed the occurrence of this deletion in K. pneumoniae ST307 strains recovered from human colonization and infection in different countries. Our findings suggest that strains lacking the putative virulence genes found in the pKPN3-307 TypeA plasmid are still able to colonize and infect humans, highlighting the need to further investigate the role of these genes for the adaptation of K. pneumoniae ST307 in distinct human body sites.

Gene expression analysis by real-time PCR: experimental demonstration of PCR detection limits.

Reverse transcription followed by real-time PCR (RT-qPCR) is the gold standard for quantifying gene expression. However, because of PCR detection limits, theorized to be three template copies, the quantification of genes exhibiting great expression variability is challenging. Using genes with high to low expression in rat tissues we experimentally demonstrated this limit and found it to be applicable only for describing reactions in which stochastic events and the Monte Carlo effect are present. We also determined the lower limits of RNA input that should be used to prevent artifactual template quantification and we propose a methodology to assess RT-qPCR detection limits in any qPCR platform.

Outbreak report of polymyxin-carbapenem-resistant Klebsiella pneumoniae causing untreatable infections evidenced by synergy tests and bacterial genomes.

Polymyxin-carbapenem-resistant Klebsiella pneumoniae (PCR-Kp) with pan (PDR)- or extensively drug-resistant phenotypes has been increasingly described worldwide. Here, we report a PCR-Kp outbreak causing untreatable infections descriptively correlated with bacterial genomes. Hospital-wide surveillance of PCR-Kp was initiated in December-2014, after the first detection of a K. pneumoniae phenotype initially classified as PDR, recovered from close spatiotemporal cases of a sentinel hospital in Rio de Janeiro. Whole-genome sequencing of clinical PCR-Kp was performed to investigate similarities and dissimilarities in phylogeny, resistance and virulence genes, plasmid structures and genetic polymorphisms. A target phenotypic profile was detected in 10% (12/117) of the tested K. pneumoniae complex bacteria recovered from patients (8.5%, 8/94) who had epidemiological links and were involved in intractable infections and death, with combined therapeutic drugs failing to meet synergy. Two resistant bacterial clades belong to the same transmission cluster (ST437) or might have different sources (ST11). The severity of infection was likely related to patients' comorbidities, lack of antimicrobial therapy and predicted bacterial genes related to high resistance, survival, and proliferation. This report contributes to the actual knowledge about the natural history of PCR-Kp infection, while reporting from a time when there were no licensed drugs in the world to treat some of these infections. More studies comparing clinical findings with bacterial genetic markers during clonal spread are needed.

Terpenic fraction of Pterodon pubescens inhibits nuclear factor kappa B and extracellular signal-regulated protein kinase 1/2 activation and deregulates gene expression in leukemia cells.

BACKGROUND: Plant derived compounds have been shown to be important sources of several anti-cancer agents. As cell cycle deregulation and tumor growth are intimately linked, the discovery of new substances targeting events in this biochemical pathway would be of great value. The anti-leukemic effect of an ethanolic extract of Pterodon pubescens seeds (EEPp) has been previously demonstrated and now we show that a terpenic subfraction (SF5) of EEPp containing farnesol, geranylgeraniol and vouacapan derivatives induces apoptosis in the human chronic myelogenous leukemia cell line K562. This work addresses SF5's antiproliferative mechanisms in these cells since they are still unclear. METHODS: DNA synthesis in K562 cells was assessed by [3H]-methyl-thymidine incorporation and cell cycle status by flow cytometry. The expression of cyclins D1 and E2, of the cell cycle inhibitor p21 and of the proto-oncogene c-myc was evaluated by semi-quantitative RT-PCR. Extracellular-signal-regulated kinases (ERK) 1/2 and nuclear factor kappa B (NF-κB) activation was evaluated by western blotting. RESULTS: In K562 cells, SF5 treatment induced a higher inhibition of DNA synthesis and cell growth than the original EEPp hexanic fraction from which SF5 originated, and also arrested the cell cycle in G1. Exposure of these cells to SF5 led to a decrease in cyclin E2 and c-myc expression while p21 mRNA levels were increased. Furthermore, SF5 inhibited the activation of mitogen-activated protein kinase (MAPK) ERK 1/2 and NF-κB. CONCLUSIONS: This work suggests that the anti-leukemic action of SF5 is linked to the inhibition of ERKs, NF-κB and c-myc signaling pathways resulting in reduced cyclin E2 mRNA expression and cell cycle arrest in the G1 phase.

CABGen: A Web Application for the Bioinformatic Analysis of Bacterial Genomes.

Due to recent developments in NGS technologies, genome sequencing is generating large volumes of new data containing a wealth of biological information. Understanding sequenced genomes in a biologically meaningful way and delineating their functional and metabolic landscapes is a first-level challenge. Considering the global antimicrobial resistance (AMR) problem, investments to expand surveillance and improve existing genome analysis technologies are pressing. In addition, the speed at which new genomic data is generated surpasses our capacity to analyze it with available bioinformatics methods, thus creating a need to develop new, user-friendly and comprehensive analytical tools. To this end, we propose a new web application, CABGen, developed with open-source software. CABGen allows storing, organizing, analyzing, and interpreting bioinformatics data in a friendly, scalable, easy-to-use environment and can process data from bacterial isolates of different species and origins. CABGen has three modules: Upload Sequences, Analyze Sequences, and Verify Results. Functionalities include coverage estimation, species identification, de novo genome assembly, and assembly quality, genome annotation, MLST mapping, searches for genes related to AMR, virulence, and plasmids, and detection of point mutations in specific AMR genes. Visualization tools are also available, greatly facilitating the handling of biological data. The reports include those results that are clinically relevant. To illustrate the use of CABGen, whole-genome shotgun data from 181 bacterial isolates of different species collected in 5 Brazilian regions between 2018 and 2020 were uploaded and submitted to the platform's modules.

Description of a novel IncP plasmid harboring blaKPC-2 recovered from a SPM-1-producing Pseudomonas aeruginosa from ST277.

The high rates of carbapenem resistance among Brazilian Pseudomonas aeruginosa isolates are mainly associated with the clone ST277 producing the carbapenemase SPM-1. Here, the complete genetic composition of a IncP plasmid harboring blaKPC-2 in isolates of this endemic clone carrying chromosomal blaSPM-1 was described using whole genome sequencing. These results confirm the association of these two carbapenemases in ST277 and also describe the genetic composition of a novel blaKPC-2-plasmid. Considering the fact that this association occurs in a high-risk clone, monitoring the dissemination of this plasmid should be a public health concern.

Mobile resistome of microbial communities and antimicrobial residues from drinking water supply systems in Rio de Janeiro, Brazil.

Antibiotic resistance genes (ARGs) are widespread in the environment due to the overuse of antibiotics and other pollutants, posing a threat to human and animal health. In this study, we evaluated antimicrobial residues, bacterial diversity and ARGs in two important watersheds, Guandu and São João, that supply drinking water to Rio de Janeiro city, Brazil. In addition, tap water samples were collected from three different cities in Rio de Janeiro State, including the metropolitan area of Rio de Janeiro city. Clarithromycin, sulfamethoxazole and azithromycin were found in untreated water and drinking water in all samples. A greater abundance of Proteobacteria was observed in Guandu and São João watersheds, with most of the sequences belonging to the Gammaproteobacteria class. A plasmidome-focused metagenomics approach revealed 4881 (Guandu), 3705 (São João) and 3385 (drinking water) ARGs mainly associated with efflux systems. The genes encoding metallo-ß-lactamase enzymes (blaAIM, blaGIM, blaIMP, and blaVIM) were detected in the two watersheds and in drinking water samples. Moreover, we demonstrated the presence of the colistin resistance genes mcr-3 and mcr-4 (both watersheds) and mcr-9 (drinking water and Guandu) for the first time in Brazil. Our data emphasize the importance of introducing measures to reduce the disposal of antibiotics and other pollutants capable of promoting the occurrence and spread of the microbial resistome on aquatic environments and predicting possible negative impacts on human health.