Fourier Transform Infrared Spectroscopy for Typing Burkholderia cenocepacia ET12 Isolates.

The IR Biotyper and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using ClinProTools software (MALDI-TOF MS-ClinProTools) are two novel typing methods that rely on the analysis of carbohydrate and peptide residues in intact bacterial cells. These two methods have shown promising results in the rapid and accurate typing of bacteria. In this study, we evaluated these novel typing methods in comparison with genotypic typing for cluster analysis of Burkholderia cenocepacia epidemic strain ET12, isolated from adult cystic fibrosis patients. Sixty-six isolates of B. cenocepacia were used in this study, 35 of which were identified as the ET12 strain and 31 as non-ET12 strains by repetitive-element PCR (rep-PCR). Twelve isolates were used for the creation of typing models using IR Biotyper and MALDI-TOF MS-ClinProTools, and 54 isolates were used for external validation of the typing models. The IR Biotyper linear discriminant analysis (LDA) model had a diagnostic sensitivity of 84.6% for typing the epidemic strain, ET12. At a cutoff of 70%, MALDI-TOF MS-ClinProTools had 87.5% diagnostic sensitivity in detecting the ET12 strain (P = 1.00). Both methods had a diagnostic specificity of ≥80% for detecting the ET12 strain. In conclusion, IR Biotyper and MALDI-TOF MS-ClinProTools offer rapid typing using proteomics and analysis of small cellular molecules with a low running cost. Our pilot study showed suboptimal accuracy of both methods for typing outbreak strains of B. cenocepacia. Extending the spectral region analyzed by the IR Biotyper can improve the accuracy and has the potential of improving the generalizability of this technique for typing other organisms. IMPORTANCE Respiratory infections due to Burkholderia cenocepacia, particularly the ET12 epidemic strain, are considered sentinel events for persons with cystic fibrosis, as they are often associated with person-to-person transmission and accelerated decline in lung function and early mortality. Current typing methods are generally only available at reference centers, with long turn-around-times, which can affect the identification of outbreaks and critical patient triage. This pilot study aims to add to the growing literature illustrating the potential utility of Fourier transform infrared spectroscopy (FTIR), a novel rapid method, for the successful typing of clinically significant bacteria. In this study, we evaluated its utility to discriminate between the ET12 clone and non-ET12 isolates of B. cenocepacia and compared it to proteomics cluster analysis using MALDI-TOF MS and ClinProTools software. Both methods had encouraging but suboptimal accuracy (≥85% sensitivity and ≥83% specificity), which will likely be improved by extending the spectral region analyzed by the IR Biotyper with updated software.

Genomic analysis of Burkholderia cenocepacia isolated from a liver abscess in a patient with cystic fibrosis.

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B. cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B. cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

A case report of successful eradication of new isolates of Burkholderia cenocepacia in a child with cystic fibrosis.

Chronic respiratory infection with Burkholderia cenocepacia (Bc) in patients with cystic fibrosis (CF) is associated with accelerated decline in lung function and increased mortality. It is therefore important to attempt to eradicate new isolates, especially in children. However, there are no standardized guidelines to eradicate Bc. We report a case of successful eradication of new isolates of Bc in a 2-year-old child with CF using a combination of IV, nebulized antibiotics and sinus surgery.

Burkholderia cenocepacia ET12 transmission in adults with cystic fibrosis.

This report describes transmission of a Burkholderia cenocepacia ET12 strain (ET12-Bc) at the Toronto Adult Cystic Fibrosis (CF) Centre occurring from 2008 to 2017. Epidemiological and genomic data from 11 patients with CF were evaluated. Isolates were analysed using whole genome sequencing (WGS). Epidemiological investigation and WGS analysis suggested nosocomial transmission, despite enhanced infection control precautions. This was associated with subsequent deaths in 10 patients. ET12-Bc positive patients are no longer cared for on the same unit as ET12-Bc negative patients.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

Clinical characteristics of bacteraemia caused by Burkholderia cepacia complex species and antimicrobial susceptibility of the isolates in a medical centre in Taiwan.

This study investigated the clinical characteristics and outcomes of bacteraemia due to Burkholderia cepacia complex (BCC) species among 54 patients without cystic fibrosis from January 2013 to February 2015. BCC isolates were identified to the species level by the Bruker Biotyper MALDI-TOF MS system and by sequencing analysis of the 16S rRNA and recA genes. Antimicrobial susceptibilities of the isolates were determined by the agar dilution method. Sequencing of the recA gene in the 54 blood isolates revealed 37 (68.5%) isolates of B. cenocepacia, 9 (16.7%) of B. cepacia, 4 (7.4%) of B. multivorans and one isolate each of B. arboris, B. pseudomultivorans, B. seminalis, and B. vietnamiensis. The overall performance of the Bruker Biotyper MALDI-TOF MS system for correctly identifying the 54 BCC isolates to the species level was 79.6%, which was better than that (16.7%) by 16S RNA sequencing analysis. Bacteraemic pneumonia (n = 23, 42.6%) and catheter-related bacteraemia (n = 21, 38.9%) were the most common types of infection. Higher rates of ceftazidime and meropenem resistance were found in B. cepacia isolates (33.3% and 22.2%, respectively) than in isolates of B. cenocepacia (21.6% and 10.8%, respectively) and other species (12.5% and 12.5%, respectively). Overall, the 30-day mortality rate was 38.9% (21/54). Bacteraemia caused by BCC species other than B. cenocepacia and B. cepacia (adjusted odds ratio [aOR] 20.005, P = 0.024) and high SOFA score (aOR 1.412, P = 0.003) were predictive of higher 30-day mortality. Different BCC species are associated with different outcomes of bacteraemia and exhibit different susceptibility patterns.

Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

Bacteria of the Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis (CF) patients, yet the precise mechanisms underlying inflammation, recurrent exacerbations and transition from chronic stages to acute infection and septicemia are not known. Bcc bacteria are generally believed to have a predominant extracellular biofilm life style in infected CF lungs, similar to Pseudomonas aeruginosa, but this has been challenged by clinical observations which show Bcc bacteria predominantly in macrophages. More recently, Bcc bacteria have emerged in nosocomial infections of patients hospitalized for reasons unrelated to CF. Research has abundantly shown that Bcc bacteria can survive and replicate in mammalian cells in vitro, yet the importance of an intracellular life style during infection in humans is unknown. Here we studied the contribution of innate immune cell types to fatal pro-inflammatory infection caused by B. cenocepacia using zebrafish larvae. In strong contrast to the usual protective role for macrophages against microbes, our results show that these phagocytes significantly worsen disease outcome. We provide new insight that macrophages are critical for multiplication of B. cenocepacia in the host and for development of a fatal, pro-inflammatory response that partially depends on Il1-signalling. In contrast, neutrophils did not significantly contribute to disease outcome. In subcutaneous infections that are dominated by neutrophil-driven phagocytosis, the absence of a functional NADPH oxidase complex resulted in a small but measurably higher increase in bacterial growth suggesting the oxidative burst helps limit bacterial multiplication; however, neutrophils were unable to clear the bacteria. We suggest that paradigm-changing approaches are needed for development of novel antimicrobials to efficiently disarm intracellular bacteria of this group of highly persistent, opportunistic pathogens.

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.

Using dendritic cells to evaluate how Burkholderia cenocepacia clonal isolates from a chronically infected cystic fibrosis patient subvert immune functions.

Infection with Burkholderia cepacia complex (Bcc) bacteria is a threat to cystic fibrosis (CF) patients, commonly leading to a fatal pneumonia, the cepacia syndrome. It causes a massive production of pro-inflammatory cytokines and leucocyte recruitment to airway epithelium without resolving infection and contributing to tissue lesion. To dissect how Bcc bacteria subvert the immune response, we developed a co-culture model with human dendritic cells (DCs) and B. cenocepacia clonal variants isolated from a chronically infected CF patient, who died with cepacia syndrome. We demonstrated that the two late variants were sevenfold and 17-fold (respectively) more internalized by DCs than the variant that initiated infection. The late variants showed improved survival within DCs (60.29 and 52.82 CFU/DC) compared to the initial variant (0.38 CFU/DC). All clonal isolates induced high expression of inflammatory cytokines IL-8, IL-6, IL-1ß, IL-12, IL-23, TNF-α and IL-1ß. This pro-inflammatory trait was significantly more pronounced in DCs infected with the late variants than in DCs infected with the variant that initiated patient's infection. All infected DCs failed to upregulate maturation markers, HLA-DR, CD80, CD86 and CD83. Nevertheless, these infected DCs activated approximately twice more T cells than non-infected DCs. Similar T cell activation was observable with respective conditioned media, suggesting a non-antigen-specific activation. Our data indicate that during prolonged infection, B. cenocepacia acquires ability to survive intracellularly, inducing inflammation, while refraining DC's maturation and stimulating non-antigen-specific T cell responses. The co-culture model here developed may be broadly applied to study B. cenocepacia-induced immunomodulation.

Biochemical Characterization of Glutamate Racemase-A New Candidate Drug Target against Burkholderia cenocepacia Infections.

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 µM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.

Multilocus sequence analysis reveals high genetic diversity in clinical isolates of Burkholderia cepacia complex from India.

Burkholderia cepacia complex (Bcc) is a complex group of bacteria causing opportunistic infections in immunocompromised and cystic fibrosis (CF) patients. Herein, we report multilocus sequence typing and analysis of the 57 clinical isolates of Bcc collected over the period of seven years (2005-2012) from several hospitals across India. A total of 21 sequence types (ST) including two STs from cystic fibrosis patient's isolates and twelve novel STs were identified in the population reflecting the extent of genetic diversity. Multilocus sequence analysis revealed two lineages in population, a major lineage belonging to B. cenocepacia and a minor lineage belonging to B. cepacia. Split-decomposition analysis suggests absence of interspecies recombination and intraspecies recombination contributed in generating genotypic diversity amongst isolates. Further linkage disequilibrium analysis indicates that recombination takes place at a low frequency, which is not sufficient to break down the clonal relationship. This knowledge of the genetic structure of Bcc population from a rapidly developing country will be invaluable in the epidemiology, surveillance and understanding global diversity of this group of a pathogen.

Whole-Genome Sequencing of Three Clonal Clinical Isolates of B. cenocepacia from a Patient with Cystic Fibrosis.

Burkholderia cepacia complex bacteria are amongst the most feared of pathogens in cystic fibrosis (CF). The BCC comprises at least 20 distinct species that can cause chronic and unpredictable lung infections in CF. Historically the species B. cenocepacia has been the most prevalent in CF infections and has been associated in some centers with high rates of mortality. Modeling chronic infection by B. cenocepacia in the laboratory is challenging and no models exist which effectively recapitulate CF disease caused by BCC bacteria. Therefore our understanding of factors that contribute towards the morbidity and mortality caused by this organism is limited. In this study we used whole-genome sequencing to examine the evolution of 3 clonal clinical isolates of B. cenocepacia from a patient with cystic fibrosis. The first isolate was from the beginning of infection, and the second two almost 10 years later during the final year of the patients' life. These isolates also demonstrated phenotypic heterogeneity, with the first isolate displaying the mucoid phenotype (conferred by the overproduction of exopolysaccharide), while one of the later two was nonmucoid. In addition we also sequenced a nonmucoid derivative of the initial mucoid isolate, acquired in the laboratory by antibiotic pressure. Examination of sequence data revealed that the two late stage isolates shared 20 variant nucleotides in common compared to the early isolate. However, despite their isolation within 10 months of one another, there was also considerable variation between the late stage isolates, including 42 single nucleotide variants and three deletions. Additionally, no sequence differences were identified between the initial mucoid isolate and its laboratory acquired nonmucoid derivative, however transcript analysis indicated at least partial down regulation of genes involved in exopolysaccharide production. Our study examines the progression of B. cenocepacia throughout chronic infection, including establishment of sub-populations likely evolved from the original isolate, suggestive of parallel evolution. Additionally, the lack of sequence differences between two of the isolates with differing mucoid phenotypes suggests that other factors, such as gene regulation, come into play in establishing the mucoid phenotype.

Pseudomonas aeruginosa displays an altered phenotype in vitro when grown in the presence of mannitol.

D-mannitol has been approved in dry powder formulation as an effective antimucolytic agent in patients with cystic fibrosis. What is not known is the effect of adding a metabolisable sugar on the biology of chronic bacterial pathogens in the CF lung. Therefore, a series of simple in vitro experiments were performed to examine the effect of adding D-mannitol on the phenotype of the CF respiratory pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia. Clinical isolates (n = 86) consisting of P. aeruginosa (n = 51), B. cenocepacia (n = 26), P. putida (n = 4), Stenotrophomonas maltophila (n = 3) and Pseudomonas spp. (n = 2) were examined by supplementing basal nutrient agar with varying concentrations of D-mannitol (0-20% [w/v]) and subsequently examining for any change in microbial phenotype. The effect of supplementation with mannitol was four-fold, namely i) To increase the proliferation and increase in cell density of all CF organisms examined, with an optimal concentration of 2-4% (w/v) D-mannitol. No such increase in cell proliferation was observed when mannitol was substituted with sodium chloride. ii) Enhanced pigment production was observed in 2/51 (3.9%) of the P. aeruginosa isolates examined, in one of the P. putida isolates, and in 3/26 (11.5%) of the B. cenocepacia isolates examined. iii). When examined at 4.0% (w/v) supplementation with mannitol, 11/51 (21.6%) P. aeruginosa isolates and 3/26 (11.5%) B. cenocepacia isolates were seen to exhibit the altered adhesion phenotype. iv). With respect to the altered mucoid phenotype, 5/51 (9.8%) P. aeruginosa produced this phenotype when grown at 4% mannitol. Mucoid production was greatest at 4%, was poor at 10% and absent at 20% (w/v) mannitol. The altered mucoid phenotype was not observed in the B. cenocepacia isolates or any of the other clinical taxa examined. Due consideration therefore needs to be given, where there is altered physiology within the small airways, leading to a potentially altered biological state of the colonising microorganisms in novel inhaled pharmaceutical interventions in CF, particularly those, which are not designated as antimicrobial agents.

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.

Lung transplant for a patient with cystic fibrosis and active Burkholderia Cenocepacia pneumonia.

Lung transplant for cystic fibrosis has been considered contraindicated in patients who have Burkholderia Cenocepacia infection. A 24-year-old white woman who had cystic fibrosis presented with respiratory failure caused by B. Cenocepacia pneumonia. She was treated with broad-spectrum antibiotics and a double-lung transplant. The chest cavity and both bronchi were irrigated with 0.5% povidone-iodine solution. For immunosuppression, she received induction therapy with alemtuzumab (15 mg) and methylprednisolone and maintenance therapy with tacrolimus, mycophenolate mofetil, and prednisone (5 mg daily). Postoperative antibiotics included intravenous meropenem for 3 weeks; vancomycin for 10 days; and inhaled ceftazidime, oral trimethoprim-sulfamethoxazole, and doxycycline for several months. Follow-up at 25 months after transplant showed that chest radiographs were clear and lung function was normal. At 6 years after transplant, she was working full time and had no recurrence of infection from B. Cenocepacia. This case suggests that patients who have cystic fibrosis and active B. Cenocepacia pneumonia may be successfully treated with a lung transplant.

Contamination of hypertonic saline solutions in use by cystic fibrosis patients in Israel.

BACKGROUND: Treatment of cystic fibrosis (CF) patients with inhaled hypertonic saline (HS) solutions is safe, beneficial and reduces exacerbation rates. We studied contamination of solutions used by Israeli CF patients for prolonged periods. METHODS: The study addressed whether daily opening of previously unopened solutions caused contamination, survival of 6 CF-associated bacteria in artificially inoculated solutions, in-use contamination of solutions and patterns of their use by patients. RESULTS: Repeated opening did not contaminate solutions and survival of indicator bacteria was variable. Mycobacterium abscessus survived in 3% HS solution for 6 weeks and Burkholderia cenocepacia and Pseudomonas aeruginosa were longer. In 30/76 (39.5%) of used solutions 49 contaminants were found, none being common CF-associated pathogens. CONCLUSIONS: Most CF-related bacteria survived to some degree in HS. Approximately 40% of solutions used by patients were contaminated by organisms of uncertain significance. Our findings highlight the potential risk posed by contamination of HS solutions and support recommendations to use sterile unit-dose formulations.

The use of doripenem in pediatric cystic fibrosis patients in case of meropenem shortages.

Ceftazidime is the only anti-pseudomonal beta-lactam that has been reported to be administered by extended infusion in pediatric cystic fibrosis (CF) patients. A small pediatric pharmacokinetic/pharmacodynamic study has been published regarding the use of intermittent extended infusion doripenem in the treatment of an acute pulmonary exacerbation (APE) in pediatric CF patients; however, clinical use of intermittent extended infusion doripenem in pediatric CF patients has not been previously reported. We present three cases administering intermittent extended infusion doripenem in pediatric CF patients for the treatment of an APE in the case of replacing meropenem due to shortage. The delivery of beta-lactam antibiotics via intermittent extended infusion should be considered in order to optimize the pharmacodynamics of beta-lactams in the treatment of an APE.

Identification and molecular epidemiology of nosocomial outbreaks due to Burkholderia cepacia in cystic fibrosis patients of Masih Daneshvary Hospital, Iran.

INTRODUCTION: B. cepacia complex have emerged as an important opportunistic pathogen in hospitalized and immunocompromised patients. Small hospital outbreaks are frequent and are usually due to a single contaminated environmental source. In this study we were going to investigate the role of B.cepacia complex in those patients suspected to involve with cystic fibrosis and evaluate responsible types in Masih Daneshvary Hospital. METHODS: One hundred specimens were collected from all admitted patients who were suspected to cystic fibrosis to Masih Daneshvary hospital during one year April 2011 till end of March 2012. All were culture and identified standard procedure. All samples were checked by API system (API20NE) and by specific PCR method for genus Bulkhorderia and Bcc as well. Identified strains were finally tested by PFGE system to identifying specific involving pulse-types. RESULT: . Isolation and identification methods revealed 5 specimens were B.cepasia, The frequency of the cystic fibrosis detected at this study was lower than other similar study previously reported. All these isolates showed similar pattern by PFGE standard protocol that may have spread from a single source and could not be attributed to cross infections from patient to patients. DISCUSSION: Application of PFGE and identification of pulse-type is a potential tool to enhance the investigation of apparent nosocomial outbreaks of B.cepacia. However it needs to be adjusted with environmental findings. Implementation of educational programs and adherence to infection control policies are obviously the main element for complete elimination of an outbreak.

Genetic similarity of Burkholderia cenocepacia from cystic fibrosis patients.

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia.