SNaPBceBcon: a Practical Tool for Identification and Genotyping of Burkholderia cepacia and Burkholderia contaminans.

We propose an optimized protocol for an extensive population analysis of Burkholderia cepacia and Burkholderia contaminans. Seven new polymorphisms were added to the recently proposed SNaPBcen assay, and a total of 18 markers ensured the clear identification and distinction of B. cepacia and B. contaminans isolates and high genotypic discrimination (Simpson index of 0.94) compared to those for multilocus sequence typing.

Development of a multiple-locus variable-number tandem-repeat typing scheme for genetic fingerprinting of Burkholderia cenocepacia and application to nationwide epidemiological analysis.

Organisms of the Burkholderia cepacia complex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization. B. cenocepacia and Burkholderia multivorans account for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for B. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences of B. cenocepacia strains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100 B. cenocepacia strains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of the B. cenocepacia IIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool in B. cenocepacia surveillance.

[An outbreak of Burkholderia cepacia bacteremia in a hemodialysis unit, Cadiz, 2014]. / Brote de bacteriemia por Burkholderia cepacia en una unidad de hemodiálisis de Cádiz, 2014.

INTRODUCTION: In January 2014 a possible outbreak of Burkholderia cepacia bacteremia occurred in a hemodialysis center situated in La Linea de la Concepción (Cadiz). An investigation was begun to confirm the outbreak, identify the source, and implement control measures. METHODS: A descriptive analysis was performed to describe the characteristics of the patients affected with Burkholderia cepacia bacteremia from November 2013 to February 2014. Environmental samples were taken. A molecular typing study was performed using pulsed field gel electrophoresis (SpeI PFGE) and MLST analysis in order to determine the genetic similarity between the isolates. RESULTS: The bacterium was isolated from blood cultures of 7 patients during the study period. Three of the samples (2 of which were also cases) were endoluminal fluid from catheter locks, and 4 chlorhexidine bottle samples. The patients were coincident in 2 of the 6 work shifts. The mean age of the cases was 67 years of whom 57% were women. Human samples and an environmental sample was analyzed and found to be genetically identical (ST653 clone). CONCLUSIONS: The analysis confirmed the outbreak of Burkholderia cepacia, with 7 cases among the patients of the hemodialysis center. The outbreak was due to the same strain, probably a common source and secondary transmission from person to person.

[Screening and optimization of cholesterol conversion strain].

OBJECTIVE: Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized. The transformation products were identified. Fermentation conditions were optimized for conversion. METHODS: Cholesterol was used as sole carbon source to isolate strain SE-1. Morphology, physiological and biochemical characteristics of strain SE-1 were studied. 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fermentation supernatants were extracted with chloroform, the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20. Their structures were identified by 1H-NMR and 13C-NMR. Fermentation medium including carbon and nitrogen, methods of adding substrates and fermentation conditions for Strain SE-1 were optimized. RESULTS: Strain SE-1 was a Gram-negative bacterium, exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis. The sequence analysis of 16S rRNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B. cepacia (Genbank No. U96927). The similarity was 99%. The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products, 7beta-hydroxycholesterol and the minor product was 7-oxocholesterol. The optimum culture conditions were: molasses 5%, (NH4 )2SO4 0.3%, 4% of inoculation, pH 7.5 and 36 degrees C. Under the optimum culture condition, the conversion rate reached 34.4% when concentration of cholesterol-Tween 80 was 1 g/L. Cholesterol 7beta-hydroxylation conversion rate under optimal conditions was improved by 20.8%. CONCLUSION: Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale.

Molecular analysis of bacterial microbiota associated with oysters (Crassostrea gigas and Crassostrea corteziensis) in different growth phases at two cultivation sites.

Microbiota presumably plays an essential role in inhibiting pathogen colonization and in the maintenance of health in oysters, but limited data exist concerning their different growth phases and conditions. We analyzed the bacterial microbiota composition of two commercial oysters: Crassostrea gigas and Crassostrea corteziensis. Differences in microbiota were assayed in three growth phases: post-larvae at the hatchery, juvenile, and adult at two grow-out cultivation sites. Variations in the microbiota were assessed by PCR analysis of the 16S rRNA gene in DNA extracted from depurated oysters. Restriction fragment length polymorphism (RFLP) profiles were studied using Dice's similarity coefficient (Cs) and statistical principal component analysis (PCA). The microbiota composition was determined by sequencing temperature gradient gel electrophoresis (TGGE) bands. The RFLP analysis of post-larvae revealed homology in the microbiota of both oyster species (Cs > 88 %). Dice and PCA analyses of C. corteziensis but not C. gigas showed differences in the microbiota according to the cultivation sites. The sequencing analysis revealed low bacterial diversity (primarily ß-Proteobacteria, Firmicutes, and Spirochaetes), with Burkholderia cepacia being the most abundant bacteria in both oyster species. This study provides the first description of the microbiota in C. corteziensis, which was shown to be influenced by cultivation site conditions. During early growth, we observed that B. cepacia colonized and remained strongly associated with the two oysters, probably in a symbiotic host-bacteria relationship. This association was maintained in the three growth phases and was not altered by environmental conditions or the management of the oysters at the grow-out site.

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya.

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79-82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53-83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.

[Burkholderia cepacia persistence in patients with mucoviscidosis].

AIM: Study features of persistence of Burkholderia cepacia in mucoviscidosis patients. MATERIALS AND METHODS: In the period from 2008 to 2009, 56 B. cepacia strains isolated from children with mucoviscidosis were obtained. 114 medical histories of children with mucoviscidosis from various age groups were analyzed. The developed algorithm for identification and typing including phenotype and molecular biology methods was used to identify B. cepacia bacteria. Strain genotyping was carried out by RAPD-PCR with random oligonucleotide primer as well as pulse-electrophoresis. RESULTS: Persistence of associations ofmicroogranisms in 59.4% of cases was established to be the feature of persistent infection in mucoviscidosis. The feature of persistence of B. cepacia strains in patients with diagnosis ofmuco-viscidosis mixed form, severe course is persistence in association with Pseudomonas aeruginosa. B. cepacia bacteria that can persist in mucoviscidosis patients are characterized by resistance to many antibiotics. A prolonged (up to 1 year and 5 months) persistence of B. cepacia strains isolated from 1 patient was proven by using microflora monitoring of lower respiratory tract. CONCLUSION: B. cepacia bacteria may colonize lower respiratory tract of mucoviscidosis patients, persist for a long time and be transmitted between patients.

A psychrotrophic Burkholderia cepacia strain isolated from refrigerated raw milk showing proteolytic activity and adhesion to stainless steel.

The proteolytic activity of a psychrotrophic strain of Burkholderia cepacia isolated from refrigerated raw milk was characterized. Bur. cepacia produced proteolytic activity during growth at refrigeration temperature, with maximum activity at pH 6-7. The enzyme showed relative thermal stability in the range 40-50°C during 25 min, and maintained 80% its initial activity at 76°C/30 s. Milk coagulation assay showed that the crude protease from Bur. cepacia caused coagulation from day 2 for skimmed milk, whereas coagulation was observed from day 5 for whole milk. The adherence of this strain to stainless steel was evaluated, and the substrata had around 107 CFU/cm2 after 15 to 60 min incubation. Results on biofilm development suggest that this bacterium could adhere and to form biofilms even at refrigeration temperatures. These results indicate that Bur. cepacia may represent a potential hazardous to milk and dairy products.

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.

Survival of phosphate-solubilizing bacteria against DNA damaging agents.

Phosphate-solubilizing bacteria (PSBs) were isolated from different plant rhizosphere soils of various agroecological regions of India. These isolates showed synthesis of pyrroloquinoline quinone (PQQ), production of gluconic acid, and release of phosphorus from insoluble tricalcium phosphate. The bacterial isolates synthesizing PQQ also showed higher tolerance to ultraviolet C radiation and mitomycin C as compared to Escherichia coli but were less tolerant than Deinococcus radiodurans. Unlike E. coli, PSB isolates showed higher tolerance to DNA damage when grown in the absence of inorganic phosphate. Higher tolerance to ultraviolet C radiation and oxidative stress in these PSBs grown under PQQ synthesis inducible conditions, namely phosphate starvation, might suggest the possible additional role of this redox cofactor in the survival of these isolates under extreme abiotic stress conditions.

[Isolation and identification of a coronatine producing strain].

OBJECTIVE: We screened and isolated coronatine-producing stains from various samples. METHODS: The strains were isolated and selected from samples by the methods of streak plate and serial dilution. The samples were sick leaves/branches and soil in which plants got sick according to the symptoms of leaf blight disease and tuber enlargement. Coronatine production was determined by high performance liquid chromatography (HPLC). The strain was characterized by the physiological and biochemical analysis, the determination of (G + C) mol% contents and 16S rDNA sequencing. The molecule structure of the fermentation product was identified based on the data of ultraviolet spectrum, infrared spectrum and mass spectrum. RESULTS: Strain BCC933 was gram-negative, polar flagella, short rod and non-spore-forming bacterium and accumulated poly-beta-hydroxybutyrate (PHB). It producted catalase, but not arginine dihydrolase nor oxidase,couldn't grow at temperature 41 degrees C. It hadn't the abilities to hydrolyze starch and gelatine. No nitrate reduction and denitrification activity was detected. The (G + C) mol% content was 67.2%. We analyzed 16S rDNA nucleotide sequence, and ascertained the phylogenetic position of the strain. CONCLUSION: Strain BCC933 with coronatine biosynthesis ability was identified as Burkholderia cepacia, which hasn't been reported up to date.

Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station.

The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.

Matrix-assisted laser desorption/ionization time-of-flight MS for the accurate identification of Burkholderia cepacia complex and Burkholderia gladioli in the clinical microbiology laboratory.

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.

Outbreak of Stenotrophomonas maltophilia and Burkholderia cepacia Bloodstream Infections at a Hemodialysis Center.

Patients undergoing hemodialysis are at an increased risk for bloodstream infections (BSIs). Infection usually occurs because of contamination of water supply, water treatment, distribution systems, or reprocessing dialyzers. Here, we report an outbreak of BSIs caused by Stenotrophomonas maltophilia (n = 21) and Burkholderia cepacia (n = 22) among dialyzed patients at a large hemodialysis center in Brazil. Overall, three patients died (7%), two of which had bacteremia caused by S. maltophilia and the other had a B. cepacia infection. We collected water samples from different points of the hemodialysis system for culture and typing. Genetic patterns were identified through polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) and pulsed-field gel electrophoresis. The same genotypes of S. maltophilia and B. cepacia recovered from blood cultures were found in dialysis water. Also, multiple genetic profiles were identified among water isolates, suggesting heavy contamination. Bacteremia cases persisted even after implementing standard control measures, which led us to believe that the piping system was contaminated with microbial biofilms. Soon after we changed the entire plumbing system, reported cases dropped back to the number typically expected, and the outbreak came to an end.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Epidemiological investigation and successful management of a Burkholderia cepacia outbreak in a neurotrauma intensive care unit.

OBJECTIVE: The detailed epidemiological and molecular characterization of an outbreak of Burkholderia cepacia at a neurotrauma intensive care unit of a level 1 trauma centre is described. The stringent infection control interventions taken to successfully curb this outbreak are emphasized. METHODS: The clinical and microbiological data for those patients who had more than one blood culture that grew B. cepacia were reviewed. Bacterial identification and antimicrobial susceptibility testing was done using automated Vitek 2 systems. Prospective surveillance, environmental sampling, and multilocus sequence typing (MLST) were performed for extensive source tracking. Intensive infection control measures were taken to further control the hospital spread. RESULTS: Out of a total 48 patients with B. cepacia bacteraemia, 15 (31%) had central line-associated blood stream infections. Two hundred and thirty-one environmental samples were collected and screened, and only two water samples grew B. cepacia with similar phenotypic characteristics. The clinical strains characterized by MLST typing were clonal. However, isolates from the water represented a novel strain type (ST-1289). Intensive terminal cleaning, disinfection of the water supply, and the augmentation of infection control activities were done to curb the outbreak. A subsequent reduction in bacteraemia cases was observed. CONCLUSION: Early diagnosis and appropriate therapy, along with the rigorous implementation of essential hospital infection control practices is required for successful containment of this pathogen and to curb such an outbreak.

[Identification of clinical isolates of Burkholderia cepacia and assessment of their pathogenicity in animal experiments].

In experiments on animals study of pathogenicity of 9 clinical strains of Burkholderia cepacia isolated from patients with chronic lung diseases was performed. Preliminary identification of studied strains by means of biochemical and genetic methods allowed to establish their belonging to B. cepacia species. It was determined that 6 of 9 strains are epidemiologically significant. Experiments showed that bacteria of studied strains are not able to cause infectious process in white mice and hamadryas baboons. Conclusion about appropriateness of development and use of other biological models was made.

Outbreak of Burkholderia cepacia pseudobacteraemia caused by intrinsically contaminated commercial 0.5% chlorhexidine solution in neonatal intensive care units.

BACKGROUND: Burkholderia cepacia is intrinsically resistant to certain antiseptics. The authors noted a sudden increase in the frequency of isolation of B. cepacia from blood cultures in a neonatal intensive care unit (NICU) of a university-affiliated hospital. AIM: To identify the source and intervene in the ongoing infections. METHODS: The cases were defined as patients with positive blood cultures for B. cepacia in an NICU between November 2014 and January 2015. Medical records were reviewed and NICU healthcare workers were interviewed. Samples of suspected antiseptics, blood culture bottles, cotton balls, gauze and a needle used in the NICU were analysed microbiologically. FINDINGS: During the outbreak period, B. cepacia was identified in 25 blood cultures obtained from 21 patients. The clinical features of the patients were suggestive of pseudobacteraemia. Regarding environmental samples, B. cepacia was cultured from 0.5% chlorhexidine gluconate (CHG) solution products that had been used as a skin antiseptic during blood drawing in the NICU. The clinical B. cepacia isolate and two strains obtained from 0.5% CHG exhibited identical pulsed-field gel electrophoresis patterns. After the CHG products were withdrawn, the outbreak was resolved. CONCLUSIONS: The pseudobacteraemia cases were caused by contaminated 0.5% CHG produced by a single manufacturer. Stricter government regulation is needed to prevent contamination of disinfectants during manufacturing. In addition, microbial contamination of antiseptics and disinfectants should be suspected when a B. cepacia outbreak occurs in hospitalized patients.

Outbreak of Burkholderia cepacia bacteraemia in a tertiary care centre due to contaminated ultrasound probe gel.

BACKGROUND: Burkholderia cepacia is an important opportunistic organism in hospitalized and immunocompromised patients, particularly in cystic fibrosis. AIMS: To describe the epidemiological investigation of an outbreak of B. cepacia bacteraemia. METHODS: The study examined 14 patients during their admission to three intensive care units in a tertiary care hospital between January and June 2016. The outbreak involved nine (57%) female and six (43%) male patients. All patients were adults of ages ranging from 19 to 85 years with a median age of 52 years. Patients' medical charts, laboratory cultures, exposures, and central line insertion procedures were reviewed. FINDINGS: B. cepacia was isolated from the blood cultures of 14 patients resulting from contamination of the gel applied to the ultrasound probe used to guide the insertion of a central venous catheter. Molecular pathogen typing using pulsed-field gel electrophoresis showed 95% similarity between the B. cepacia isolates from the blood of these patients and those isolated from the ultrasound gel. CONCLUSION: Ongoing surveillance and prompt investigation of unusual disease outbreaks are vital for identifying sources of contamination of B. cepacia and protecting at-risk patients. Sound epidemiological methods are very important for identifying the source of any hospital infection outbreak.

Evaluating the use of whole-genome sequencing for outbreak investigations in the lack of closely related reference genome.

Whole-genome sequencing (WGS) has emerged as a powerful molecular typing method for outbreak analysis enabling the rapid discrimination between outbreak and non-outbreak isolates. However, such analysis can be challenging in the absence of closely related reference genomes. In this study, we assessed the use of WGS in investigating an outbreak of a relatively understudied bacterial pathogen with no publicly available closely related reference genome. Eleven Burkholderia cepacia complex (Bcc) isolates (seven from patients and four from disposable dermal gloves packages) that were collected during an outbreak were sequenced using the Illumina MiSeq platform. Our results showed that mapping the 11 sequenced Bcc outbreak isolates against a genetically distant reference genome yield loses coverage (31.6-48.3%) and a high number of detected false single-nucleotide polymorphisms (SNPs) (1123-2139). Therefore, a reference genome consensus from an outbreak clinical isolate was generated by combining both de novo assembly and mapping approaches. Based on this approach, we were able to demonstrate that the Bcc outbreak isolates were closely related and were phylogenetically distinct from the 11 publically available Bcc genomes. In addition, the pairwise SNP distance analysis detected only 1 to 6 SNPs differences among the outbreak isolates, confirming that contaminated disposable dermal gloves were the cause of the outbreak.