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

The hidden genomic diversity, specialised metabolite capacity, and revised taxonomy of Burkholderia sensu lato

Burkholderia sensu lato is a collection of closely related genera within the family Burkholderiaceae that includes species of environmental, industrial, biotechnological, and clinical importance. Multiple species within the complex are the source of diverse specialized metabolites, many of which have been identified through genome mining of their biosynthetic gene clusters (BGCs). However, the full, true genomic diversity of these species and genera, and their biosynthetic capacity have not been investigated. This study sought to cluster and classify over 4000 Burkholderia sensu lato genome assemblies into distinct genomic taxa representing named and uncharacterized species. We delineated 235 species groups by average nucleotide identity analyses that formed seven distinct phylogenomic clades, representing the genera of Burkholderia sensu lato: Burkholderia, Paraburkholderia, Trinickia, Caballeronia, Mycetohabitans, Robbsia, and Pararobbisa. A total of 137 genomic taxa aligned with named species possessing a sequenced type strain, while 93 uncharacterized species groups were demarcated. The 95% ANI threshold proved capable of delineating most genomic species and was only increased to resolve several closely related species. These analyses enabled the assessment of species classifications of over 4000 genomes, and the correction of over 400 genome taxonomic assignments in public databases into existing and uncharacterized genomic species groups. These species groups were genome mined for BGCs, their specialized metabolite capacity calculated per species and genus, and the number of distinct BGCs per species estimated through kmer-based de-replication. Mycetohabitans species dedicated a larger proportion of their relatively small genomes to specialized metabolite biosynthesis, while Burkholderia species harbored more BGCs on average per genome and possessed the most distinct BGCs per species compared to the remaining genera. Exploring the hidden genomic diversity of this important multi-genus complex contributes to our understanding of their taxonomy and evolutionary relationships, and supports future efforts toward natural product discovery

Discovery, mode of action and secretion of Burkholderia sensu lato key antimicrobial specialised metabolites

Burkholderia sensu lato bacteria have genomes rich in biosynthetic gene clusters (BGCs) encoding for multiple bioactive specialised metabolites. Diverse classes of antimicrobial natural products have been isolated from Burkholderia, including polyynes, shikimate pathway derivatives, polyketides, non-ribosomal peptides and hybrid polyketide non-ribosomal peptides. We highlight examples of Burkholderia metabolites, overviewing their biosynthesis, bioactivity, mechanisms of action and secretion

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

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/2027.42/116207/1/12864_2010_Article_3517.pd

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

A rapid screening method for the detection of specialised metabolites from bacteria: induction and suppression of metabolites from Burkholderia species

Screening microbial cultures for specialised metabolites is essential for the discovery of new biologically active compounds. A novel, cost-effective and rapid screening method is described for extracting specialised metabolites from bacteria grown on agar plates, coupled with HPLC for basic identification of known and potentially novel metabolites. The method allows the screening of culture collections to identify optimal production strains and metabolite induction conditions. The protocol was optimised on two Burkholderia species known to produce the antibiotics, enacyloxin IIa (B. ambifaria) and gladiolin (B. gladioli), respectively; it was then applied to strains of each species to identify high antibiotic producers. B. ambifaria AMMD and B. gladioli BCC0238 produced the highest concentrations of the respective antibiotic under the conditions tested. To induce expression of silent biosynthetic gene clusters, the addition of low concentrations of antibiotics to growth media was evaluated as known elicitors of Burkholderia specialised metabolites. Subinhibitory concentrations of trimethoprim and other clinically therapeutic antibiotics were evaluated and screened against a panel of B. gladioli and B. ambifaria. To enhance rapid strain screening with more antibiotic elicitors, antimicrobial susceptibility testing discs were included within the induction medium. Low concentrations of trimethoprim suppressed the production of specialised metabolites in B. gladioli, including the toxins, toxoflavin and bongkrekic acid. However, the addition of trimethoprim significantly improved enacylocin IIa concentrations in B. ambifaria AMMD. Rifampicin and ceftazidime significantly improved the yield of gladiolin and caryoynencin by B. gladioli BCC0238, respectively, and cepacin increased 2-fold with tobramycin in B. ambifaria BCC0191. Potentially novel metabolites were also induced by subinhibitory concentrations of tobramycin and chloramphenicol in B. ambifaria. In contrast to previous findings that low concentrations of antibiotic elicit Burkholderia metabolite production, we found they acted as both inducers or suppressors dependent on the metabolite and the strains producing them. In conclusion, the screening protocol enabled rapid characterization of Burkholderia metabolites, the identification of suitable producer strains, potentially novel natural products and an understanding of metabolite regulation in the presence of inducing or suppressing conditions