Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia

Background: Burkholderia cenocepacia is the most prominent species of the B. cepacia complex (Bcc), a group of nine closely related and difficult to identify bacteria that cause serious infections in patients with cystic fibrosis. Despite its clinical relevance, identification of B. cenocepacia as a single species is unavailable, as it splits by a widely used recA gene-based PCR identification method into discrete phylogenetic subgroups IIIA, IIIB, IIIC and IIID. With the aim of identifying gene targets suitable for unified detection of B. cenocepacia strains, we examined sequence polymorphisms in the repA and parB genes. These essential genes are involved in the replication and partitioning of bacterial replicons, hence we also had the opportunity for the first time to investigate the evolution of the multireplicon (three chromosome) structure of Bcc genomes. Results: Alignment of the repA and parB genes from publicly available Bcc genome sequences enabled the design of primers for their amplification and sequence analysis. Multilocus sequencing typing, a highly discriminatory method for Bcc species and strain discrimination, was used to select strains of unique sequence types (STs) that spanned the known Bcc genetic diversity. Sequence datasets of repA (83 isolates, 67 STs) and parB (120 isolates, 95 STs) genes from the second chromosome were aligned and examined phylogenetically to identify polymorphisms suitable for identification of B. cenocepacia. In contrast to parB, the Bcc repA sequences demonstrated distinct clustering of B. cenocepacia from other species, which enabled the design a species-specific multiplex PCR. The novel single-reaction B. cenocepacia detection method was tested on a panel of 142 different Bcc strains (142 STs) and distinguished recA groups IIIA, IIIB and IIID, from all other Bcc members with 100% sensitivity and 93% specificity. Conclusion: The repA-based multiplex PCR is a useful aid to the rapid identification of the most clinically relevant B. cenocepacia recA subgroups IIIA, IIIB and IIID. Phylogenetic analysis of repA and parB genes demonstrated that acquisition of the second and third replicons of Bcc genomes occurred prior to their differentiation into discrete species and that the sharing of replicons across species had not occurred

Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite

Background: Burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl. Results: The exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA. Conclusion: Our results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen

Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence

AbstractBurkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have the ability to spread between CF individuals. Of the 17 formally named species within the complex, Burkholderia multivorans and Burkholderia cenocepacia dominate in CF. Multilocus sequence typing has proven to be a very useful tool for tracing the global epidemiology of Bcc bacteria and has shown that B. cenocepacia strains with high transmissibility, such as the ET-12 strain (ST-28) and the Czech strain (ST-32), have spread epidemically within CF populations in Canada and Europe. The majority of research on the molecular pathogenesis of Bcc bacteria has focused on the B. cenocepacia ET-12 epidemic lineage, with gene mutation, genome sequence analysis and, most recently, global gene expression studies shedding considerable light on the virulence and antimicrobial resistance of this pathogen. These studies demonstrate that the ability of B. cenocepacia to acquire foreign DNA (genomic islands, insertion sequences and other mobile elements), regulate gene expression via quorum sensing, compete for iron during infection, and mediate antimicrobial resistance and inflammation via its membrane and surface polysaccharides are key features that underpin the virulence of different strains. With the wealth of molecular knowledge acquired in the last decade on B. cenocepacia strains, we are now in a much better position to develop strategies for the treatment of pathogenic colonization with Bcc and to answer key questions on pathogenesis concerning, for example, the factors that trigger the rapid clinical decline in CF patients

Epidemiology of Burkholderia cepacia Complex in Patients with Cystic Fibrosis, Canada

The Burkholderia cepacia complex is an important group of pathogens in patients with cystic fibrosis (CF). Although evidence for patient-to-patient spread is clear, microbial factors facilitating transmission are poorly understood. To identify microbial clones with enhanced transmissibility, we evaluated B. cepacia complex isolates from patients with CF from throughout Canada. A total of 905 isolates from the B. cepacia complex were recovered from 447 patients in 8 of the 10 provinces; 369 (83%) of these patients had genomovar III and 43 (9.6%) had B. multivorans (genomovar II). Infection prevalence differed substantially by region (22% of patients in Ontario vs. 5% in Quebec). Results of typing by random amplified polymorphic DNA analysis or pulsed-field gel electrophoresis indicated that strains of B. cepacia complex from genomovar III are the most potentially transmissible and that the B. cepacia epidemic strain marker is a robust marker for transmissibility

Over a Decade of recA and tly Gene Sequence Typing of the Skin Bacterium Propionibacterium acnes: What Have We Learnt?

The Gram-positive, anaerobic bacterium Propionibacterium acnes forms part of the normal microbiota on human skin and mucosal surfaces. While normally associated with skin health, P. acnes is also an opportunistic pathogen linked with a range of human infections and clinical conditions. Over the last decade, our knowledge of the intraspecies phylogenetics and taxonomy of this bacterium has increased tremendously due to the introduction of DNA typing schemes based on single and multiple gene loci, as well as whole genomes. Furthermore, this work has led to the identification of specific lineages associated with skin health and human disease. In this review we will look back at the introduction of DNA sequence typing of P. acnes based on recA and tly loci, and then describe how these methods provided a basic understanding of the population genetic structure of the bacterium, and even helped characterize the grapevine-associated lineage of P. acnes, known as P. acnes type Zappe, which appears to have undergone a host switch from humans-to-plants. Particular limitations of recA and tly sequence typing will also be presented, as well as a detailed discussion of more recent, higher resolution, DNA-based methods to type P. acnes and investigate its evolutionary history in greater detail

Mannitol promotes adherence of an outbreak strain of Burkholderia multivorans via an exopolysaccharide-independent mechanism that is associated with upregulation of newly identified fimbrial and afimbrial adhesins

© Society for General Microbiology, 2013. Post-print version of article deposited in accordance with SHERPA RoMEO guidelinesBurkholderia multivorans, a member of the Burkholderia cepacia complex (Bcc), is an important pathogen of the cystic fibrosis (CF) lung. Mannitol, approved as an inhaled osmolyte therapy for use in CF patients, promotes exopolysaccharide (EPS) production by the Bcc. In the present study, we investigated the role of mannitol-induced EPS in the adherence of B. multivorans. We report that mannitol promoted adherence of two representative B. multivorans strains. However, whilst this enhanced adherence was largely EPS-dependent in an environmental isolate, it was EPS-independent within a CF outbreak strain, suggesting strain-to-strain variation in adhesins. Genome sequencing of the outbreak strain enabled the identification of two distinct loci encoding putative fimbrial and afimbrial adhesins. The putative fimbriae-encoding locus was found to be widely distributed amongst clinical and environmental B. multivorans. In contrast, the locus encoding the putative afimbrial adhesin (of the filamentous haemagglutinin family, FHA) was restricted to clinical isolates. Both loci contributed to biofilm formation and mucin adherence. Furthermore, we report that mannitol promoted expression of both loci, and that the locus encoding the putative FHA-family adhesin is a key determinant of the enhanced adherence observed following growth in mannitol. Our studies provide the first characterization, to our knowledge, of B. multivorans adhesins, and in so doing highlight the strain-dependent role of EPS in the Bcc and the difficulties in assigning phenotypic traits to Bcc EPS due to the wider response to mannitol. Our observations also highlight the need to monitor the microbiological effects of inhaled mannitol therapy in Bcc-infected CF patients

The domestication of the probiotic bacterium Lactobacillus acidophilus

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population

Understanding the challenges of non-food industrial product contamination

Preventing microbial contamination of non-food products is a major area of industrial microbiology where preservatives are used to stop microbial growth. However, microorganisms occasionally overcome product preservation, causing recalls and the implementation of multiple procedures to prevent further contamination. Correct reporting of microbial contamination in non-food industrial products is vital, especially if spoilage organisms are antimicrobial resistant and pose a health threat. Gram-negative bacteria such as Pseudomonas, Burkholderia, and Enterobacteriaceae are frequently reported as non-food product contaminants, including species that overlap current antimicrobial resistance priorities. Historical analysis of recall databases highlighted that for greater than 15% of contamination incidents, the causative microbial agents are reported as unidentified. Here we review the current antimicrobial resistant bacterial species associated with non-food product contamination and evaluate recall reporting in Europe from 2005 to 2018. Our review shows that 49% of microbial contaminants are reported as unidentified despite frequent detection of antimicrobial resistant pathogens; in contrast, 98% of food-related microbial contaminants are classified. Recommendations to fill this microbial identification gap in non-food product recalls are made. Overall, reporting standards for microbial contamination in non-food products must be improved to enable surveillance and for understanding the risks associated with antimicrobial resistant microorganism

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

<p>Abstract</p> <p>Background</p> <p>The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two <it>Lactobacillus </it>strains, and also tracked the fate of these two administered strains using a RAPD technique.</p> <p>Results</p> <p>A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two <it>Lactobacillus </it>strains (<it>Lactobacillus salivarius </it>NCIMB 30211 and <it>Lactobacillus acidophilus </it>NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The <it>L. salivarius </it>strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the <it>L. acidophilus </it>NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the <it>Lactobacillus </it>capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both <it>Lactobacillus </it>strains during capsule consumption was statistically significant (p < 0.05).</p> <p>Conclusion</p> <p>We have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials.</p

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis

Abstract Background Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. Results Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. Conclusions Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.http://deepblue.lib.umich.edu/bitstream/2027.42/134738/1/12864_2010_Article_3517.pd