Genomic Comparative of Pseudomonas aeruginosa Small Colony Variant, Mucoid and Non-mucoid Phenotypes Obtained from a Patient with Cystic Fibrosis During Respiratory Exacerbations.

Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.

Bacterial communities in the rhizosphere of biofortified BRS pontal and conventional carioca bean (Phaseolus vulgaris) plants.

The current knowledge of bean plants microbiome is far from being complete. Unraveling the complexity of the bacterial communities within common bean cultivars that may significantly contribute to plant health, growth and protection against soil pathogens, represents a major challenge. In this study, the richness and composition of the bacterial communities from bean plant rhizospheres and control bulk soils were analyzed by high-throughput sequencing and comparative analyses. From the classified sequences, Proteobacteria represented the most abundant phylum (42-66%), followed by Acidobacteria (12-36%) and Actinobacteria (11-18%), Bacterial community structures were different between bulk soil and rhizosphere samples. Comparing the bean cultivars, the biofortified cultivar presented high number of sequences affiliated to the genera Burkholderia and Rhodanobacter. Interestingly, despite the presence of stable and persistent core bacterial taxa associated with the common bean varieties, our analysis suggested that the biofortified common bean cultivar could select and maintain differential abundances of microbial groups in the rhizosphere environment.

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.

XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements.

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5' cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.

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.

Autochthonous Transmission of East/Central/South African Genotype Chikungunya Virus, Brazil.

We isolated East/Central/South African genotype chikungunya virus during the 2016 epidemic in Rio de Janeiro, Brazil. Genome sequencing revealed unique mutations in the nonstructural protein 4 (NSP4-A481D) and envelope protein 1 (E1-K211T). Moreover, all Brazil East/Central/South isolates shared the exclusive mutations E1-M407L and E2-A103T.

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.

Comprehensive analysis of the cellulolytic system reveals its potential for deconstruction of lignocellulosic biomass in a novel Streptomyces sp.

The giant snail Achatina fulica is considered an invasive species in most territories in which it was introduced, due to its ability to process a large amount of lignocellulose as a consequence of the presence of a cellulolytic-associated microflora. Streptomyces are well known as crucial agents in the decomposition of complex polymers in soil environments and also as cellulolytic symbionts commonly associated with herbivore insects. Here, we employed a combination of genomic and biochemical tools for a detailed evaluation of the cellulolytic potential of Streptomyces sp. I1.2, an aerobic bacterium isolated from the intestinal lumen of A. fulica in a screening for cellulolytic bacteria. Genomic analysis revealed that the ratio and diversity of CAZy domains and GH families coded by Streptomyces sp. I1.2 are comparable to those present in other highly cellulolytic bacteria. After growth on crystalline cellulose or sugarcane bagasse as sole carbon sources, the functionality of several genes encoding endoglucanases, cellobiohydrolases, xylanases, CBMs, and one ß-glucosidase were confirmed by the combination of enzymatic activity measurements, zymography, TLC, and cellulose-binding assays. The endoglucanases secreted by this isolate were stable at 50 °C and exhibited activity over a broad pH range between 4.0 and 8.0. The endoglucanases and cellobiohydrolases secreted by Streptomyces sp. I1.2 exhibited specific activities that were similar to the levels present in a commercial cellulase preparation from Trichoderma reesei, while I1.2 xylanase levels were even 350 % higher. The results presented here show that Streptomyces sp. I1.2 is promising for future biotechnological applications, since it is able to produce endoglucanases, cellobiohydrolases, and xylanases in appreciable amounts when grown on a low-cost residue such as sugarcane bagasse.

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.

Genomic information on multidrug-resistant livestock-associated methicillin-resistant Staphylococcus aureus ST398 isolated from a Brazilian patient with cystic fibrosis.

Alarmingly, the isolation of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing among patients with cystic fibrosis (CF). During a previous molecular characterisation of MRSA isolates obtained from patients with CF from Rio de Janeiro, Brazil, one isolate was identified as the ST398 clone, a livestock-associated (LA) MRSA. In this study, we report the draft genome sequence of an LA-MRSA ST398 clone isolated from a patient with CF.

Alterations in glucose metabolism proteins responsible for the Warburg effect in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal tumor in the world. ESCC presents late diagnosis, highly aggressive behavior and poor survival. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1 (GLUT-1), and transcription factor induced by hypoxia (HIF1α), responsible for the transcription of proteins cited. The objective of this study is to evaluate the alterations of these proteins and their association with clinicopathological data in ESCC. We performed immunohistochemistry to determine HIF-1α, GLUT-1, PKM1, PKM2, HK2 and Ki67-expression in ESCC patients and controls. Also, we used RT-qPCR to evaluated mRNA expression of GLUT-1 in esophageal mucosa of individuals without cancer, but are alcohol drinkers and tobacco smokers. Our results showed the exclusively expression of GLUT-1 in tumors cells and dysplastic samples. We also observed a compartmentalization of the expression of PKM1 and PKM2 in relation to tumor cells and stroma associated to tumor areas. All of the proteins evaluated, excepted GLUT-1, were frequently detected in normal mucosa. No correlations between clinicopathological features and protein expressions were observed. GLUT-1 expression appears in initial tumor lesions and is maintained through ESCC evolution. We reported for the first time PKM1 staining in normal esophagus and ESCC, being mostly present in more differentiated cells.

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.

Draft genome sequences of four Achromobacter ruhlandii strains isolated from cystic fibrosis patients.

Achromobacter species are being increasingly isolated from the respiratory tract of cystic fibrosis patients. Recent reports indicate that Achromobacter ruhlandii is a potential human pathogen in cystic fibrosis-related infections. Here we report the draft genome of four A. ruhlandii strains isolated from cystic fibrosis patients in Brazil. This report describes A. ruhlandii as a potential opportunistic pathogen in cystic fibrosis and provides a framework to for additional enquires into potential virulence factors and resistance mechanisms within this species.

Draft genome sequence of Acinetobacter pittii ST643 shared by cystic fibrosis patients.

Acinetobacter pittii has emerged as an important hospital pathogen that is associated with outbreaks and drug resistance. In cystic fibrosis (CF) patients, the detection of Acinetobacter spp. is rare; however, we isolated the A. pittii sequence type ST643 in several Brazilian CF patients treated in the same centre. The current study describes the draft genome of A. pittii ST643.

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.

Characterization of Achromobacter Species in Cystic Fibrosis Patients: Comparison of bla(OXA-114) PCR Amplification, Multilocus Sequence Typing, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Molecular methodologies were used to identify 28 Achromobacter spp. from patients with cystic fibrosis (CF). Multilocus sequence typing (MLST) identified 17 Achromobacter xylosoxidans isolates (all bla(OXA-114) positive), nine Achromobacter ruhlandii isolates (all bla(OXA-114) positive), one Achromobacter dolens isolate, and one Achromobacter insuavis isolate. All less common species were misidentified as A. xylosoxidans by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Chronic colonization by clonally related A. ruhlandii isolates was demonstrated.

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.