Tânia Wrobel Folescu

Burkholderia cenocepacia, B. multivorans, B. ambifaria and B. vietnamiensis isolates from cystic fibrosis patients have different profiles of exoenzyme production

Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC‐infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

ABSTRACT Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

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.

Antibody response to Pseudomonas aeruginosa in children with cystic fibrosis

Cystic fibrosis (CF) is the most frequent life threatening autosomal recessive disease in white subjects. The primary cause of morbidity and mortality in children with CF is chronic pulmonary infection, mainly caused by Pseudomonas aeruginosa. The purpose of this study was to assess the value of the measurement of antibodies to P. aeruginosa in diagnosing lung infection by the bacteria in CF patients. We assessed P. aeruginosa antibody titers in CF patients from Rio de Janeiro, Brazil, using cell lysate antigens as well as recombinant PcrV, a Type III Secretion System protein. Sputum (more than 70% of the specimens) or oropharyngeal swabs were obtained whenever patients were regularly followed for their pulmonary disease. Blood samples were obtained with an average interval of 6 months for a period of 2 years. The ELISA cut‐offs were assigned as the positive 95% confidence interval of the mean antibody levels from non‐fibrocystic controls. Our data showed that most CF patients (81%) of whom were not chronically infected by P. aeruginosa (Groups I and II), had their first serology positive for rPcrV. Cell‐lysate ELISA was able to detect P. aeruginosa antibodies before positive culture in the first serum sample of 44% of the patients from Groups I and II. When serum reactivity to rPcrV and cell lysate were combined, 94% of CF patients from Groups I and II (n = 16) had the first serology positive for P. aeruginosa over a mean time of 20 months before the first isolation of P. aeruginosa. In conclusion, longitudinal P. aeruginosa serology should become part of respiratory care follow‐up, in conjunction with other lung parameter functions. Pediatr Pulmonol. 2009; 44:392–401.

Comparison of the worldwide transmissible Pseudomonas aeruginosa with isolates from brazilian cystic fibrosis patients

Cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients is a rare occurrence. However, the emergence of transmissible strains has been reported between unrelated individuals. We analyzed the genetic relationship among P. aeruginosa isolates from Brazilian CF patients and transmissible clones which are worldwide spread. The data does not indicate the presence of closely related variant clones.

Chronic infection of cystic fibrosis patient airways by a single clone of Burkholderia cepacia: replacement of non-mucoid to mucoid morphotype

Mucoid Burkholderia cepacia morphotype emerged within a nine year follow-up of a cystic fibrosis patient. Clinical data suggested a linkage between the mucoid phenotype isolation and the deterioration of the patients condition. Despite of the phenotypic variation, molecular typing showed that the patient was chronically infected with B. cepacia complex isolates belonging to a same genetic clone.

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.

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.

Enterobacter cloacae harbouring blaKPC-2 and qnrB-1 isolated from a cystic fibrosis patient: a case report

We describe the first detection of a KPC-2- and QnrB-producing Enterobacter cloacae from a patient with cystic fibrosis. The blaKPC-2 and qnrB-1 genes were located in a 79.8-kb plasmid. The presence of blaKPC-2 and qnrB-1 genes was determined by PCR and sequencing. Mobilization of plasmid containing blaKPC2 gene was assayed by conjugation.

Methicillin-resistant Staphylococcus aureus in cystic fibrosis patients: do we need to care? A cohort study

CONTEXT AND OBJECTIVE The prevalence of a variety of potentially pathogenic microorganisms in cystic fibrosis patients, such as methicillin-resistant Staphylococcus aureus (MRSA), has increased over the past decade. Given the increasing prevalence of MRSA and the few data available in the literature, better understanding of the clinical repercussions of colonization by this bacterium in cystic fibrosis patients becomes essential. This study aimed to evaluate the repercussions of chronic colonization by MRSA in cystic fibrosis patients. DESIGN AND SETTING Retrospective cohort study from January 2004 to December 2013 in a cystic fibrosis reference center. METHODS Each patient with cystic fibrosis was evaluated for nutritional status (body mass index, BMI, and BMI percentile), pulmonary function and tomographic abnormalities (modified Bhalla scores) at the time of chronic colonization by MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) and throughout the study period. RESULTS Twenty pairs of patients were included. There were no significant differences between the groups regarding nutritional characteristics. Spirometric data showed a trend towards greater obstruction of the airways in patients with MRSA. Patients with MRSA presented greater structural damage to their lungs, demonstrated not only by the total Bhalla score but also by its parameters individually. CONCLUSIONS Patients colonized by MRSA presented greater functional and structural respiratory impairment at the time of chronic colonization. Disease progression was also faster in patients chronically colonized by MRSA than in those with MSSA. This was shown through comparisons that avoided possible confounding variables.