Burkholderia Cepacia Complex Strains Research Articles

Development of a duplex droplet digital PCR assay for the detection of Burkholderia cepacia complex and Stenotrophomonas maltophilia in bloodstream infections.

Burkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are nosocomial pathogens that cause various infections and exhibit high resistance to multiple antimicrobial agents. In this study, we aimed to develop a duplex droplet digital PCR (ddPCR) assay for detecting BCC and S. maltophilia in bloodstream infections. We optimized the experimental conditions by setting the annealing temperature to 51°C and determining the optimal concentrations of primers and probes, as well as the thermal cycle numbers. The feasibility of the duplex ddPCR reaction system with the optimal conditions was established and verified through parallel reactions with reference strains of BCC and S. maltophilia. The specificity of the assay, tested with 33 reference strains, was found to be 100%. The duplex ddPCR assay demonstrated good repeatability and could detect as low as 5.35 copies/reaction of BCC and 7.67 copies/reaction of S. maltophilia. This level of sensitivity was consistent in the simulated blood and blood bottle samples. We compared nucleic acid extraction methods and found that the Chelex-100 boiling method and kit extraction method exhibited similar detection sensitivity, suggesting the potential application of the Chelex-100 boiling method in the ddPCR assay. In the clinical samples, the duplex ddPCR assay accurately detected BCC and S. maltophilia in 58 cases. In conclusion, our study successfully developed a duplex ddPCR assay that provides accurate and convenient detection of BCC and S. maltophilia in bloodstream infections.IMPORTANCEBurkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are implicated in a wide range of infections, including bloodstream infections (BSIs), pneumonia, and meningitis, and often exhibit high intrinsic resistance to multiple antimicrobial agents, limiting therapeutic options. The gold standard for diagnosing bloodstream infections remains blood culture. However, current blood culture detection and positivity rates do not meet the "rapid diagnosis" required for the diagnosis and treatment of critically ill patients with BSIs. The digital droplet PCR (ddPCR) method is a potentially more powerful tool in the diagnosis of BSIs compared to other molecular methods due to its greater sensitivity, specificity, accuracy, and reproducibility. In this study, a duplex ddPCR assay for the detection of BCC and S. maltophilia in BSIs was developed.

Specific biomarker mining and rapid detection of Burkholderia cepacia complex by recombinase polymerase amplification.

To mine specific proteins and their protein-coding genes as suitable molecular biomarkers for the Burkholderia cepacia Complex (BCC) bacteria detection based on mega analysis of microbial proteomic and genomic data comparisons and to develop a real-time recombinase polymerase amplification (rt-RPA) assay for rapid isothermal screening for pharmaceutical and personal care products. We constructed an automatic screening framework based on Python to compare the microbial proteomes of 78 BCC strains and 263 non-BCC strains to identify BCC-specific protein sequences. In addition, the specific protein-coding gene and its core DNA sequence were validated in silico with a self-built genome database containing 158 thousand bacteria. The appropriate methodology for BCC detection using rt-RPA was evaluated by 58 strains in pure culture and 33 batches of artificially contaminated pharmaceutical and personal care products. We identified the protein SecY and its protein-coding gene secY through the automatic comparison framework. The virtual evaluation of the conserved region of the secY gene showed more than 99.8% specificity from the genome database, and it can distinguish all known BCC species from other bacteria by phylogenetic analysis. Furthermore, the detection limit of the rt-RPA assay targeting the secY gene was 5.6 × 102 CFU of BCC bacteria in pure culture or 1.2 pg of BCC bacteria genomic DNA within 30 min. It was validated to detect <1 CFU/portion of BCC bacteria from artificially contaminated samples after a pre-enrichment process. The relative trueness and sensitivity of the rt-RPA assay were 100% in practice compared to the reference methods. The automatic comparison framework for molecular biomarker mining is straightforward, universal, applicable, and efficient. Based on recognizing the BCC-specific protein SecY and its gene, we successfully established the rt-RPA assay for rapid detection in pharmaceutical and personal care products.

Subtractive sequence analysis aided druggable targets mining in Burkholderia cepacia complex and finding inhibitors through bioinformatics approach.

Burkholderia cepacia complex (BCC) is a group of gram-negative bacteria composed of at least 20 different species that cause diseases in plants, animals as well as humans (cystic fibrosis and airway infection). Here, we analyzed the proteomic data of 47 BCC strains by classifying them in three groups. Phylogenetic analyses were performed followed by individual core region identification for each group. Comparative analysis of the three individual core protein fractions resulted in 1766 ortholog/proteins. Non-human homologous proteins from the core region gave 1680 proteins. Essential protein analyses reduced the target list to 37 proteins, which were further compared to a closely related out-group, Burkholderia gladioli ATCC 10,248 strain, resulting in 21 proteins. 3D structure modeling, validation, and druggability step gave six targets that were subjected to further target prioritization parameters which ultimately resulted in two BCC targets. A library of 12,000 ZINC drug-like compounds was screened, where only the top hits were selected for docking orientations. These included ZINC01405842 (against Chorismate synthase aroC) and ZINC06055530 (against Bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/Glucosamine-1-phosphate acetyltransferase glmU). Finally, dynamics simulation (200ns) was performed for each ligand-receptor complex, followed by ADMET profiling. Of these targets, details of their applicability as drug targets have not yet been elucidated experimentally, hence making our predictions novel and it is suggested that further wet-lab experimentations should be conducted to test the identified BCC targets and ZINC scaffolds to inhibit them.

Rapid detection of Burkholderia cepacia complex carrying the 16S rRNA gene in clinical specimens by recombinase-aided amplification.

The Burkholderia cepacia complex (BCC) is a group of opportunistic pathogens, including Burkholderia cepacia, Burkholderia multivorans, Burkholderia vietnamiensis and Burkholderia ambifaria, which can cause severe respiratory tract infections and lead to high mortality rates among humans. The early diagnosis and effective treatment of BCC infection are therefore crucial. In this study, a novel and rapid recombinase-aided amplification (RAA) assay targeting the 16S rRNA gene was developed for BCC detection. The protocol for this RAA assay could be completed in 10 min at 39°C, with a sensitivity of 10 copies per reaction and no cross-reactivity with other pathogens. To characterize the effectiveness of the RAA assay, we further collected 269 clinical samples from patients with bacterial pneumonia. The sensitivity and specificity of the RAA assay were 100% and 98.5%, respectively. Seven BCC-infected patients were detected using the RAA assay, and three BCC strains were isolated from the 269 clinical samples. Our data showed that the prevalence of BCC infection was 2.60%, which is higher than the 1.40% reported in previous studies, suggesting that high sensitivity is vital to BCC detection. We also screened a patient with B. vietnamiensis infection using the RAA assay in clinic, allowing for appropriate treatment to be initiated rapidly. Together, these data indicate that the RAA assay targeting the 16S rRNA gene can be applied for the early and rapid detection of BCC pathogens in patients with an uncharacterized infection who are immunocompromised or have underlying diseases, thereby providing guidance for effective treatment.

Biosynthesis of Ditropolonyl Sulfide, an Antibacterial Compound Produced by Burkholderia cepacia Complex Strain R-12632.

Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 μg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role. IMPORTANCEBurkholderia bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products.

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

Evaluation of susceptibility testing methods for Burkholderia cepacia complex: a comparison of broth microdilution, agar dilution, gradient strip and EUCAST disc diffusion

ObjectivesTo evaluate the accuracy and reproducibility of antimicrobial susceptibility testing methods in Burkholderia cepacia complex (BCC). MethodsMinocycline, ciprofloxacin, trimethoprim/sulphamethoxazole, meropenem, ceftazidime and chloramphenicol were tested against 155 BCC strains using broth microdilution at 35 ± 1°C (BMD35) in triplicate, then BMD at 30 ± 1°C (BMD30), agar dilution at 30°C and 35°C (AD30 and AD35), gradient strip (GS) and EUCAST standardized disc diffusion (DD) testing methods once. ResultsBMD35 reproducibility ranged from 70% to 84.5% for all agents. Correlations of MICs from BMD35 with BMD30 ranged from 63% to 85%, with AD35 from 32.9% to 87% and with GS methods from 36% to 83.9%. Essential agreement (EA) of MICs by GS with BMD35 ranged from 62.6% (trimethoprim-sulphamethoxazole) to 83.9% (minocycline). EA of EUCAST DD zone diameters using CLSI breakpoint criteria was between 85.8% and 97.4%, however Very Major Errors (VME) for trimethoprim/sulphamethoxazole were 31%. ConclusionsBMD at 35 ± 1°C was poorly reproducible for most agents and no method showed acceptable performance. Of particular concern were the GS results. Although this is the most commonly used method for determining MICs in laboratories, there was poor correlation with BMD35 for meropenem and trimethoprim/sulphamethoxazole. EUCAST DD correlated poorly with BMD35 MICs. This study confirms that no susceptibility method is capable of providing reproducible and accurate MICs when testing BCC.

Systematic review of healthcare-associated Burkholderia cepacia complex outbreaks: presentation, causes and outbreak control

SummaryBackgroundOver the past decades, the Burkholderia cepacia complex (BCC) has been linked to multiple healthcare-associated outbreaks. No systematic analysis of these outbreaks has been carried out to date. The aim of this study was to conduct a systematic review of reports on nosocomial BCC outbreaks.MethodsPublished studies from 1971 until 9/12/2019 presenting nosocomial BCC outbreaks were identified using Embase, Pubmed and abstracts from professional meetings.ResultsWe identified a total of 111 outbreak reports. Thirty-two percent of the affected institutions were academic hospitals and 43.8% community hospitals. The average outbreak duration was 198.6 ± 604.4 days. A total of 240 deaths (10% of the 2390 case patients) were reported but only 28 (1.2% of the 2390 case patients and 11.7% of the 240 deaths) were directly attributable to BCC. The source could be identified in 73.9% of the outbreaks; 53.2% were caused by contaminated medical solutions and medications, 12% were due to a contaminated disinfectant. In 28.2% of the outbreaks intrinsic product contamination was reported. Multidrug resistance was noted in 26.1% of the BCC strains. PFGE was the most frequently used typing method (43.2%) in the context of outbreak work-up.ConclusionMedical products are the most frequent source of BCC outbreaks, representing over half of the identified sources, with 12% of the outbreaks caused by disinfectant products. Intrinsic product contamination was detected frequently, suggesting a need for stricter regulation. While BCC-related mortality was low, our systematic review revealed significant heterogeneity in both investigations and reporting of BCC outbreaks.

Genome-based classification of Burkholderia cepacia complex provides new insight into its taxonomic status

BackgroundAccurate classification of different Burkholderia cepacia complex (BCC) species is essential for therapy, prognosis assessment and research. The taxonomic status of BCC remains problematic and an improved knowledge about the classification of BCC is in particular needed.MethodsWe compared phylogenetic trees of BCC based on 16S rRNA, recA, hisA and MLSA (multilocus sequence analysis). Using the available whole genome sequences of BCC, we inferred a species tree based on estimated single-copy orthologous genes and demarcated species of BCC using dDDH/ANI clustering.ResultsWe showed that 16S rRNA, recA, hisA and MLSA have limited resolutions in the taxonomic study of closely related bacteria such as BCC. Our estimated species tree and dDDH/ANI clustering clearly separated 116 BCC strains into 36 clusters. With the appropriate reclassification of misidentified strains, these clusters corresponded to 22 known species as well as 14 putative novel species.ConclusionsThis is the first large-scale and systematic study of the taxonomic status of the BCC and could contribute to further insights into BCC taxonomy. Our study suggested that conjunctive use of core phylogeny based on single-copy orthologous genes, as well as pangenome-based dDDH/ANI clustering would provide a preferable framework for demarcating closely related species.ReviewerThis article was reviewed by Dr. Xianwen Ren.

Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains, opportunistic pathogens of Cystic Fibrosis patients

Burkholderia cepacia complex bacteria (Bcc) represent a serious threat for immune-compromised patient affected by Cystic Fibrosis (CF) since they are resistant to many substances and to most antibiotics. For this reason, the research of new natural compounds able to inhibit the growth of Bcc strains has raised new interest during the last years. A source of such natural compounds is represented by medicinal plants and, in particular, by bacterial communities associated with these plants able to produce molecules with antimicrobial activity. In this work, a panel of 151 (endophytic) bacteria isolated from three different compartments (rhizospheric soil, roots, and stem/leaves) of the medicinal plant Echinacea purpurea were tested (using the cross-streak method) for their ability to inhibit the growth of 10 Bcc strains. Data obtained revealed that bacteria isolated from the roots of E. purpurea are the most active in the inhibition of Bcc strains, followed by bacteria isolated from the rhizospheric soil, and endophytes from stem/leaf compartment. At the same time, Bcc strains of environmental origin showed a higher resistance toward inhibition than the Bcc strains with clinical (i.e. CF patients) origin. Differences in the inhibition activity of E. purpurea-associated bacteria are mainly linked to the environment −the plant compartment- rather than to their taxonomical position.

English

Burkholderia cepacia complex (BCC) is gram negative bacteria which causes different types of human infections including catheter-associated infections and respiratory tract infections especially in immunocompromised patients. This study was conducted to decide the source, to prevent the possible hospital outbreak of BCC and to determine the epidemiological source. Eleven cartridge of liquid soaps were purchased by Near East University Hospital, Nicosia. Liquid soaps were cultured on 5% sheep blood agar and Eosine Methilene Blue Agar at 35°C in 24 h. Colonies which suspected were confirmed by Phoenix 100. Patient blood was cultured in BD Bactec Plus Aerobic culture bottles. BCC strains and patient blood sample were identified by using two different polymerase chain reaction (PCR) methods. The liquid soaps were contaminated with BCC. A few days later, in the blood culture of the 35-year-old Turkish Cyptiot male patient, BCC was identified. The molecular results indicated that the BCC strain isolated from the patient blood culture was identical with the strain isolated from the liquid soaps. The molecular epidemiological studies showed that the patient blood culture strain and the liquid soap strain was in the same cluster. It was suggested that the liquid soaps should always be analyzed and then distributed to the hospital departments. Key words: Burkholderia cepacia complex (BCC), liquid soap, prevent.

Survival and susceptibility of Burkholderia cepacia complex in chlorhexidine gluconate and benzalkonium chloride.

The Burkholderia cepacia complex (BCC) includes opportunistic pathogenic bacteria that have occasionally been recovered from various pharmaceutical products, including antiseptics and disinfectants. Plausible reasons for the contamination include intrinsic sources, such as inadequate process controls, especially for water or equipment used during product manufacture, or extrinsic sources, such as improper handling and dilution or distribution in contaminated containers. Because the survival of BCC in antiseptics is a concern to the public health and pharmaceutical industry, we determined minimum inhibitory concentrations (MICs) of 36 BCC strains against the antiseptics, following exposure to chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions (1-500 µg/ml for each chemical). Susceptibility to CHX and BZK varied across the BCC strains and was recorded as mean 90.3 and 111.1 µg/ml, respectively, at initial inoculation, which was significantly higher than the 46.4 and 61.1 µg/ml levels measured for BCC incubated in water for 40 days. After determining antiseptic MICs of individual BCC strains, BCC recovery was measured on Tryptic Soy Agar (TSA), Reasoner's Second Agar (R2A) and diluted preparations of these media under their sub-MICs. The survival of BCC was monitored for 14 days (336 h) in sub-MICs diluted to less than their antiseptic susceptible concentration value. Diluted TSA and R2A media exhibited greater efficiency of recovery for most BCC strains from the CHX and BZK solutions than full strength TSA or R2A. For BCC survival in antiseptic solutions, the cell number of BCC decreased rapidly within the first 20 min in both antiseptics, but after this, recovery remained constant in CHX and increased in BZK over the 14 day incubation period. The results indicate that BCC in water can remain viable with low susceptibility to antiseptics for 14 days, which suggests the necessity for improved detection methods and control measures to monitor BCC contamination in pharmaceutical products.

Genomic analysis of three sponge-associated Arthrobacter Antarctic strains, inhibiting the growth of Burkholderia cepacia complex bacteria by synthesizing volatile organic compounds

In this work we analyzed the ability of three Arthrobacter strains (namely TB23, TB26 and CAL618), which were isolated from the Antarctic sponges Haliclonissa verrucosa and Lyssodendrix nobilis, to specifically inhibit the growth of a panel of 40 Burkholderia cepacia complex strains, representing a major cause of infections in patients that are affected by Cystic Fibrosis. The inhibitory activity was due to the synthesis of antimicrobial compounds, very likely volatile organic compounds (VOCs), and was partially dependent on the growth media that were used for Antarctic strains growth. The phylogenetic analysis revealed that two of them (i.e. CAL 618 and TB23) were very close and very likely belonged to the same Arthrobacter species, whereas the strain TB26 was placed in a distant branch. The genome of the strains TB26 and CAL618 was also sequenced and compared with that of the strain TB23. The analysis revealed that TB23 and CAL618 shared more genomic properties (GC content, genome size, number of genes) than with TB26. Since the three strains exhibited very similar inhibition pattern vs Bcc strains, it is quite possible that genes involved in the biosynthesis of antimicrobial compounds very likely belong to the core genome.

Characterization of genotypes for Burkholderia cepacia complex strains isolated from patients in hospitals of the Russian federation

Eighty-eight cultures of microorganisms classed as part of the Burkholderia cepacia complex (Bcc) during initial identification were analyzed by multilocus sequencing (multilocus sequence typing, MLST). Thirteen genotypes (sequence type, ST) were detected; nine of them (708, 709, 710, 711, 712, 714, 727, 728, 729) were identified for the first time. Two new alleles for the gene trpB (357, 358) and one of the genes atpD (306) and gltB 9352 were detected and registered. It was found that strains of two genotypes (711, 712) belong to the species B. multivorans, one (ST102) to B. contaminans, one (ST51) to B. stabilis, and one (ST729) to B. vietnamiensis. Most strains of the sample representing eight genotypes (208, 241, 728, 727, 708, 709, 710, 714) belong to the species B. cenocepacia. The identified genotypes differ in global extent: four genotypes (51, 102, 208, 241) have intercontinental distribution, and one (712) has intracontinental distribution. It is shown that strains causing nosocomial infections in most cases refer to genotypes 728 and 708. Genotype 709 detected in strains isolated from patients in seven federal districts (FDs) of Russia should be recognized as epidemiologically significant for patients with cystic fibrosis. The Bcc strains of genotypes 241 (B. cenocepacia) and 729 (B. vietnamiensis) were isolated from the patients of the Far Eastern FD. They are not typical for the other FDs of Russia. The possibility of concomitant infection in cystic fibrosis patient with two genotypes (709, 708), that is, are epidemiologically significant and nosocomial was indicated. The longterm persistence of a single genotype strain in the organism of patients with cystic fibrosis was shown both for Bcc species B. cenocepacia (ST 709) and for B. multivorans (ST712). The possibility of transfection of the Bcc strain typical for a nosocomial environment to patients with cystic fibrosis during surgery was observed.

Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives

The Burkholderia cepacia complex (BCC) is a group of 17 closely related opportunistic pathogens that are able to infect the respiratory tract of cystic fibrosis patients. BCC bacteria are intrinsically resistant to many antibiotics and are therefore difficult to eradicate. Fosmidomycin could be a new therapeutic agent to treat BCC infections as it inhibits 1-deoxy- d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in the non-mevalonate pathway essential in BCC bacteria for isoprenoid synthesis. In this study, the antimicrobial activity of fosmidomycin and eight fosmidomycin derivatives towards 40 BCC strains was investigated. All BCC strains were resistant to fosmidomycin, although addition of glucose-6-phosphate reduced the minimum inhibitory concentration values of FR900098, the fosmidomycin acetyl derivative, from 512 mg/L to 64 mg/L for Burkholderia multivorans and B. cepacia. This enhanced activity was linked to increased expression of the genes involved in glycerol-3-phosphate transport, which appears to be the only route for fosmidomycin import in BCC bacteria. Furthermore, upregulation of a fosmidomycin resistance gene ( fsr) encoding an efflux pump was observed during fosmidomycin and FR900098 treatment. These results strongly suggest that the observed resistance in BCC bacteria is due to insufficient uptake accompanied by fosmidomycin and FR900098 efflux.

Study of class I integron in a Burkholderia cepacia complex strain isolated from blood colture

The Burkholderia cepacia complex (Bcc) consists of several species that cause lung infections in patients with cystic fibrosis but are also capable to colonize immunocompromised patients. Once established, the infection is usually difficult to eradicate, as Bcc is intrinsically resistant to many antibiotics. Besides, the acquisition of additional resistance determinants by horizontal gene transfer makes very difficult the therapeutic approach to these infections. Among horizontally acquired DNAs, integrons have been frequently reported in many Gramnegative bacteria that affect human health, but they have not been found frequently in Burkholderia isolates until now. In the present work we report on a Bcc isolate, recovered from the blood of an immunocompromised patient, that carries a 2.3 kb class I integron already described in a Salmonella enterica isolate eight years ago, coding for aacA4, aadA1 and catB2 in its cassette array.

Successful cessation of transmitting healthcare-associated inpections due to Burkholderia cepacia complex in a neonatal intensive care unit in a japanese children's hospital

BackgroundBurkholderia cepacia strains have been known to possess the capability to cause serious infections especially in neonatal intensive care units (NICUs), and their multi-drug resistances become a severe threat in hospital settings. The aim of this investigation was to evaluate the B. cepacia complex infections in the NICU in Nagano Children's Hospital, Azumino 399-8288, Japan, and to report the intervention leading to the successful cessation of the outbreak.MethodologyThe incidence of isolation and antimicrobial susceptibilities of nosocomial Burkholderia cepacia complex strains during a four-year period were retrospectively examined by clinical microbiological records, and by pulsed-field gel electrophoresis analyses along with the bacteriological verification of disinfectant device itself and procedures for its maintenance routinely used in the NICU.ResultsDuring the period surveyed between 2007 and 2009, only an isolate per respective year of B. cepacia complex was recovered from each neonate in the NICU. However, in 2010, the successive 6 B. cepacia complex isolates were recovered from different hospitalized neonates. Among them, an isolate was originated from peripheral blood of a neonate, apparently giving rise to systemic infection. In addition, the hospitalized neonate with bacteremia due to B. cepacia complex also exhibited positive cultures from repeated catheterized urine samples together with tracheal aspirate secretions. However other 5 isolates were considered as the transients or contaminants having little to do with infections. Moreover, the 5 isolates between July and October in 2010 revealed completely the same electrophoresis patterns by means of pulsed-field gel electrophoresis analyses, strongly indicating that they were infected through the same medical practices, or by transmission of the same contaminant.ConclusionsA small outbreak due to B. cepacia complex was brought about in the NICU in 2010, which appeared to be associated with the same genomovar of B. cepacia complex. The source or the rout of infection was unknown in spite of the repeated epidemiological investigation. It is noteworthy that no outbreak due to B. cepacia complex was noted in the NICU after extensive surveillance intervention.

In Vitro Activity of Doripenem against Burkholderia cepacia Complex Isolates from Non-Cystic Fibrosis Patients

Burkholderia cepacia complex (BCC), a group of nonfermenting Gram-negative bacilli (NFGNB), is emerging as an important nosocomial pathogen. It causes a wide variety of infections, such as pneumonia (especially in patients with cystic fibrosis), meningitis, and septicemia. These infections are often difficult to treat due to high intrinsic antimicrobial resistance in BCC and the lack of effective antibiotics. BCC is resistant to aminoglycosides, antipseudomonal penicillins, and cephalosporins (4). Hence, the need for new agents active against this pathogen has become critical for patient care. In recent years, carbapenems have assumed a greater therapeutic role for multidrug-resistant (MDR) isolates. Doripenem is a new broad-spectrum intravenous 1-β-methyl carbapenem. It has high activity against many Gram-positive and -negative organisms, including NFGNB. Against Pseudomonas aeruginosa, doripenem exhibits rapid bactericidal activity, with 2- to 4-fold-lower MICs than those of meropenem. However, the activity of doripenem against BCC has not been reported much, and present susceptibility reports are mainly from isolates of cystic fibrosis patients (6). Isolation of BCC from blood of non-CF septicemic patients is on the rise in our region (3), and the in vitro activity of doripenem against these isolates has been studied. Fifty blood isolates of BCC obtained between May 2007 and January 2010 were included in this study. These isolates were identified using standard biochemical tests and confirmed using recA PCR-based restriction fragment length polymorphism (RFLP) analysis as described by Mahenthiralingam et al. to identify species (5). B. cenocepacia (genomovar IIIA) was the most prevalent species of BCC obtained. Pure cultures of the isolates were lyophilized and stored at 4°C. The meropenem susceptibilities of these 50 isolates were determined by the disk diffusion method (Astra Zeneca Pvt. Ltd., India) according to CLSI guidelines, with 8 isolates (16%) being meropenem resistant. In vitro susceptibility to doripenem (Ranbaxy Intensiva, India) was determined by the Etest. Etest strips (bioMerieux India Pvt. Ltd.) were used per the instructions of the manufacturer. In addition, Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were tested for quality control, and MICs obtained for these organisms were within the ranges recommended by the CLSI (2a). A distribution of the MICs of doripenem determined by the Etest for 50 strains of BCC is shown in Fig. ​Fig.11. FIG. 1. Distribution of the MICs of doripenem determined by Etest for 50 strains of BCC. Black bars represent meropenem-resistant isolates. The MIC values classifying isolates as susceptible or resistant to doripenem suggested by Bhavnani et al. (2) and Andes et al. (1) indicate susceptibility at ≤4 mg/liter and resistance at ≥16 mg/liter, with intermediate resistance being inferred as 8 mg/liter. Since there are no FDA- or CLSI-approved breakpoints at this time for doripenem, the MIC values obtained were interpreted based on the above criteria, with all 50 isolates (100%) of BCC being susceptible to doripenem (MIC ≤ 4 mg/liter). A greater proportion of strains among meropenem-resistant BCC were inhibited by doripenem at 2 mg/liter (Fig. ​(Fig.11). Therefore, the in vitro susceptibility profile of BCC to doripenem is comparable to those of BCC to meropenem, and it can be an effective therapy for BCC strains resistant to meropenem.

Case-Crossover Study of Burkholderia cepacia Complex Bloodstream Infection Associated with Contaminated Intravenous Bromopride

To investigate an outbreak of healthcare-associated Burkholderia cepacia complex (BCC) primary bloodstream infections (BCC-BSI). Case-crossover study in a public hospital, a university hospital and a private hospital in Rio de Janeiro, Brazil, from March 2006 to May 2006. Twenty-five patients with BCC-BSI. After determining the date BCC-BSI symptoms started for each patient, 3 time intervals of data collection were defined, each one with a duration of 3 days: the case period, starting just before BCC-BSI symptoms onset; the control period, starting 6 days before BCC-BSI symptoms onset; and the washout period, comprising the 3 days between the case period and the control period. Exposures evaluated were intravascular solutions and invasive devices and procedures. Potential risk factors were identified by using the McNemar chi(2) adjusted test. Cultures of samples of potentially contaminated solutions were performed. BCC strain typing was performed by pulsed-field gel electrophoresis using SpeI. The statistical analysis revealed that the use of bromopride and dipyrone was associated with BCC-BSI. A total of 21 clinical isolates from 17 (68%) of the 25 patients and an isolate obtained from the bromopride vial were available for strain typing. Six pulsotypes were detected. A predominant pulsotype (A) accounted for 11 isolates obtained from 11 patients (65%) in the 3 study hospitals. Our investigation, using a case-crossover design, of an outbreak of BCC-BSI infections concluded it was polyclonal but likely caused by infusion of contaminated bromopride. The epidemiological finding was validated by microbiological analysis. After recall of contaminated bromopride vials by the manufacturer, the outbreak was controlled.

Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity

BackgroundAcinetobacter baumannii and species within the Burkholderia cepacia complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections.MethodsThe aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds.ResultsOnly three crude extracts (0.9%) had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 μg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 μg/ml), and a weak MIC (128 μg/ml) against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml.ConclusionThe small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 μg/ml) for both A. baumannii and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.