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.

Carbapenem-Resistant Pseudomonas aeruginosa in Chronic Lung Infection: Current Resistance Profile and Hypermutability in Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is associated with chronic and progressive lung disease and is closely related to increased morbidity and mortality in cystic fibrosis (CF) patients. Hypermutable (HPM) P. aeruginosa isolates have been described in these patients and are usually associated with antibiotic resistance. This study aimed to investigate the occurrence of carbapenem resistance and hypermutable phenotype in 179 P. aeruginosa isolates from 8 chronically CF patients assisted at two reference centers in Rio de Janeiro, Brazil. Using disk diffusion test, non-susceptible (NS) rates higher than 40% were observed for imipenem, amikacin, and gentamicin. A total of 79 isolates (44.1%), 71 (39.6%), and 8 (4.4%) were classified as carbapenem-resistant (CR resistance to at least one carbapenem), multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Minimal inhibitory concentration was determined for 79 CR P. aeruginosa and showed the following variations: 4 and 128 µg/mL to imipenem, 4 and 64 µg/mL to meropenem, and 4 and ≥ 32 µg/mL to doripenem. We have found only four (2.23%) HPM isolates from 4 patients. Analyzing the genetic relationship among the HPM isolates, 3 pulsed-field gel electrophoresis/pulsotypes (D, M, and J) were observed. Only M pulsotype was recovered from two patients in different years. Polymerase chain reaction screening for blaGES, blaIMP, blaKPC, blaNDM, blaOXA-48, blaSPM, and blaVIM genes was performed for all CR isolates and none of them were positive. Our results demonstrate a high occurrence of CR and MDR P. aeruginosa of CF patients follow-up in both centers studied, while the presence of HPM is still unusual.

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.

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.

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.

Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

Pre-operative Colonization by Staphylococcus aureus and Cephalosporin Non-susceptible Bacteria in Patients with Proximal Femoral Fractures.

Objective To estimate the frequency of Staphylococcus aureus and cephalosporin nonsusceptible bacteria colonization in patients with proximal femoral fracture during preoperative hospitalization. Methods Prevalence and incidence assessment in 63 hospitalized patients over 1 year. The median time of pretreatment hospitalization was 12 days. Samples were collected from the nostrils, groin skin and anal mucosa during the pretreatment hospitalization and were tested by the disc-diffusion technique. Results The hospital colonization incidence and the prevalence of positive results were 14.3 and 44.4% for S. aureus ; 3.2 and 6.4% for meticillin-resistant S. aureus ; 28.6 and 85.7% for meticillin-resistant coagulase-negative Staphylococcus ; 28.6 and 61.9% for cefazolin nonsusceptible Enterobacteriaceae (KFNSE); and 20.6 and 28.6% for cefuroxime nonsusceptible Enterobacteriaceae (CXNSE). In addition, factors such as to the duration of the pretreatment hospitalization period, being non-walker before fracture, antimicrobial use, American Society of Anesthesiologists (ASA) 4 surgical risk, and previous hospitalization, were related to an increase in the incidence of hospital acquisition and prevalence of colonization by the evaluated strains. The prevalence of colonization by KFNSE was three times higher than by CXNSE on admission, and twice as high at the time of fracture treatment. Conclusion There was a high incidence of hospital colonization and prevalence of colonization by all strains studied, which may guide the indication of prophylactic measures for infection.

Achromobacter xylosoxidans: characterization of strains in Brazilian cystic fibrosis patients.

We investigated the possibility of cross-infection among cystic fibrosis patients in two Brazilian reference centers. Achromobacter xylosoxidans isolates (n = 122) were recovered over a 5-year period from 39 patients. Isolates were genetically heterogeneous, but one genotype was present in 56% of the patients, suggesting that cross-infection may have occurred.

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection.

BACKGROUND: Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays. METHODS: B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase. RESULTS: ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs. CONCLUSION: B. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.

Genetic and biochemical characterization of GES-16, a new GES-type ß-lactamase with carbapenemase activity in Serratia marcescens.

We evaluated the genetic environment of blaGES-16 found in 2 carbapenem-resistant Serratia marcescens clinical isolates recovered from patients hospitalized at a tertiary hospital located in Rio de Janeiro, Brazil. We also compared the kinetics constants for GES-16 and GES-5 against several ß-lactams. Both S. marcescens isolates showed identical PFGE pattern and carried the carbapenemase-encoding gene blaGES-16 and the extended-spectrum ß-lactamase encoding gene blaOXA-10. The blaGES-16 was inserted at the first position of a defective class 1 integron, composed by a fragmented integrase gene that lacked its attI1 recombination site, followed by dfr22, aac(6')-IIc, and aadA1 genes. This integron was located on a 30-kb nonconjugative plasmid. The GES-16 showed 2 amino acid substitutions (Gln38Glu and Gly170Ser) compared to GES-1. Kinetic analysis showed that GES-16 presented hydrolytic activity against all ß-lactams tested, except for aztreonam. Imipenem was the carbapenem more efficiently hydrolyzed (highest kcat/Km) by GES-16. The kinetic parameters of GES-16 were similar to those of GES-5. In conclusion, we identified a new GES-type enzyme with carbapenemase activity in S. marcescens. The increasing diversity of such resistance determinants confirms the ongoing evolution of these ß-lactamases towards a broader spectrum of activity.

Pre-operative Colonization by Staphylococcus aureus and Cephalosporin Non-susceptible Bacteria in Patients with Proximal Femoral Fractures / Colonização pré-operatória por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur

Abstract Objective To estimate the frequency of Staphylococcus aureus and cephalosporin nonsusceptible bacteria colonization in patients with proximal femoral fracture during preoperative hospitalization. Methods Prevalence and incidence assessment in 63 hospitalized patients over 1 year. The median time of pretreatment hospitalization was 12 days. Samples were collected from the nostrils, groin skin and anal mucosa during the pretreatment hospitalization and were tested by the disc-diffusion technique. Results The hospital colonization incidence and the prevalence of positive results were 14.3 and 44.4% for S. aureus; 3.2 and 6.4% for meticillin-resistant S. aureus; 28.6 and 85.7% for meticillin-resistant coagulase-negative Staphylococcus; 28.6 and 61.9% for cefazolin nonsusceptible Enterobacteriaceae (KFNSE); and 20.6 and 28.6% for cefuroxime nonsusceptible Enterobacteriaceae (CXNSE). In addition, factors such as to the duration of the pretreatment hospitalization period, being non-walker before fracture, antimicrobial use, American Society of Anesthesiologists (ASA) 4 surgical risk, and previous hospitalization, were related to an increase in the incidence of hospital acquisition and prevalence of colonization by the evaluated strains. The prevalence of colonization by KFNSE was three times higher than by CXNSE on admission, and twice as high at the time of fracture treatment. Conclusion There was a high incidence of hospital colonization and prevalence of colonization by all strains studied, which may guide the indication of prophylactic measures for infection.

Resumo Objetivo Estimar a frequência da colonização por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur durante a internação pré-operatória. Métodos Avaliação da prevalência e incidência em 63 pacientes hospitalizados ao longo de um ano. O tempo médio de internação pré-tratamento foi de 12 dias. As amostras foram coletadas das narinas, pele da virilha e mucosa anal, durante a internação prévia ao tratamento e testadas pela técnica de disco-difusão. Resultados A incidência da colonização hospitalar e a prevalência de resultados positivos foram de 14,3% e 44,4% para Staphylococcus aureus; 3,2% e 6,4% para S. aureus resistente à meticilina; 28,6% e 85,7% para Staphylococcus coagulase-negativo resistente à meticilina; 28,6% e 61,9% para Enterobacteriaceae não suscetível à cefazolina (KFNSE); e 20,6% e 28,6% para Enterobacteriaceae não suscetível à cefuroxima (CXNSE). Além da duração do período de internação pré-tratamento, os pacientes não deambularam previamente à ocorrência da fratura e nem fizeram uso de antimicrobiano. Além disso, a duração do período de internação pré-tratamento cirúrgico, ser não-deambulador antes da fratura, uso de antimicrobianos, risco cirúrgico IV pela American Society of Anesthesiologists (ASA) e internação anterior, estiveram relacionados a um aumento na incidência de aquisição hospitalar e prevalência de colonização pelas cepas avaliadas. A prevalência de colonização pela KFNSE foi três vezes maior do que pela CXNSE na admissão e duas vezes maior no momento do tratamento da fratura. Conclusão Observou-se uma alta incidência da colonização hospitalar e prevalência da colonização por todas as cepas estudadas, o que pode orientar a indicação de medidas profiláticas contra a infecção.

Genome Sequence of Airborne Acinetobacter sp. Strain 5-2Ac02 in the Hospital Environment, Close to the Species of Acinetobacter towneri.

Acinetobacter spp. are found in 53% of air colonization samples from the hospital environment. In this work, we sequenced all the genome of airborne Acinetobacter sp. strain 5-2Ac02. We found important features at the genomic level in regards to the rhizome. By phylogenetic analysis, A. towneri was the species most closely related to Acinetobacter sp. 5-2Ac02.

Transient isolation of Burkholderia multivorans and Burkholderia cenocepacia from a Brazilian cystic fibrosis patient chronically colonized with Burkholderia vietnamiensis.

Fifteen serial Burkholderia cepacia complex isolates recovered over a period of 4 years from a single cystic fibrosis patient were analysed for genomovar status by means of recA sequence determination, and genetic relatedness by RAPD-PCR. Twelve isolates were assigned as Burkholderia vietnamiensis, two as Burkholderia cenocepacia and one as Burkholderia multivorans. B. vietnamiensis persisted in the airways during 4 years, except in three occasions when B. cenocepacia or B. multivorans were isolated. The patient was chronically colonized by B. vietnamiensis with the RAPD-profile 12 and transiently by the RAPD-profile 15.

Multidrug resistant Mycobacterium tuberculosis: a retrospective katG and rpoB mutation profile analysis in isolates from a reference center in Brazil.

BACKGROUND: Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in Mycobacterium tuberculosis isolates from a national reference center. METHODS AND FINDINGS: Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical IS6110-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 rpoB mutations while codon 531 mutations prevailed in the other isolates. CONCLUSIONS: Our data suggest that there were no major multidrug resistant M. tuberculosis strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.

Type III apparatus of Pseudomonas aeruginosa as a tool to diagnose pulmonary infection in cystic fibrosis patients.

Pseudomonas aeruginosa is associated with increased mortality in cystic fibrosis (CF) patients, and expresses type III secretion system proteins (TTSP), which is a common mechanism used by gram-negative pathogens for delivery of anti-host factors. Our aim was to investigate whether or not these antigens (TTSP) would be recognized by CF sera, by Western blot reaction. We have showed herein that all patients (n = 11) not chronically infected by P. aeruginosa had their first serum positive for TTSP (ExoS, ExoT, PopB, and/or PopD). All chronic patients had a strong positive serology to TTSP, although relatively weak reactions to TTSP were observed for some individuals in the negative control group. Therefore, TTSP that were early produced in P. aeruginosa infected CF patients, induced a detectable antibody response in those patients and were easily detected by Western-blot reaction.

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.

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.

KPC-2 carbapenemase-producing Klebsiella pneumoniae isolates from patients with Cystic Fibrosis.

Bacteria producing Klebsiella pneumoniae carbapenemases (KPCs) are rapidly emerging as a cause of multidrug-resistant infections worldwide. KPCs enzyme are plasmid-borne and can accumulate and transfer resistance determinants to other classes of antibiotics.We report two cases of KPC-2 carbapenemase-producing Klebsiella pneumoniae isolates from Cystic Fibrosis patients.