Genomic editing in Burkholderia multivorans by CRISPR/Cas9.

Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.

Acidic pH modulates Burkholderia cenocepacia antimicrobial susceptibility in the cystic fibrosis nutritional environment.

IMPORTANCE: Burkholderia cenocepacia causes severe infections in cystic fibrosis (CF) patients. CF patients are prone to reoccurring infections due to the accumulation of mucus in their lungs, where bacteria can adhere and grow. Some of the antibiotics that inhibit B. cenocepacia in the laboratory are not effective for CF patients. A major contributor to poor clinical outcomes is that antibiotic testing in laboratories occurs under conditions that are different from those of sputum. CF sputum may be acidic and have increased concentrations of iron and zinc. Here, we used a medium that mimics CF sputum and found that acidic pH decreased the activity of many of the antibiotics used against B. cenocepacia. In addition, we assessed susceptibility to more than 500 antibiotics and found four active compounds against B. cenocepacia. Our findings give a better understanding of the lack of a relationship between susceptibility testing and the clinical outcome when treating B. cenocepacia infections.

Identification of two distinct phylogenomic lineages and model strains for the understudied cystic fibrosis lung pathogen Burkholderia multivorans.

Burkholderia multivorans is the dominant Burkholderia pathogen recovered from lung infection in people with cystic fibrosis. However, as an understudied pathogen there are knowledge gaps in relation to its population biology, phenotypic traits and useful model strains. A phylogenomic study of B. multivorans was undertaken using a total of 283 genomes, of which 73 were sequenced and 49 phenotypically characterized as part of this study. Average nucleotide identity analysis (ANI) and phylogenetic alignment of core genes demonstrated that the B. multivorans population separated into two distinct evolutionary clades, defined as lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes). To examine the population biology of B. multivorans, a representative subgroup of 77 B. multivorans genomes (28 from the reference databases and the 49 novel short-read genome sequences) were selected based on multilocus sequence typing (MLST), isolation source and phylogenetic placement criteria. Comparative genomics was used to identify B. multivorans lineage-specific genes - ghrB_1 in lineage 1 and glnM_2 in lineage 2 - and diagnostic PCRs targeting them were successfully developed. Phenotypic analysis of 49 representative B. multivorans strains showed considerable inter-strain variance, but the majority of the isolates tested were motile and capable of biofilm formation. A striking absence of B. multivorans protease activity in vitro was observed, but no lineage-specific phenotypic differences were demonstrated. Using phylogenomic and phenotypic criteria, three model B. multivorans CF strains were identified, BCC0084 (lineage 1), BCC1272 (lineage 2a) and BCC0033 lineage 2b, and their complete genome sequences determined. B. multivorans CF strains BCC0033 and BCC0084, and the environmental reference strain, ATCC 17616, were all capable of short-term survival within a murine lung infection model. By mapping the population biology, identifying lineage-specific PCRs and model strains, we provide much needed baseline resources for future studies of B. multivorans.

Identification of Burkholderia cepacia Complex by PCR: A Simple Way.

United States Pharmacopeia (USP) General Chapter <60> for the detection of Burkholderia cepacia complex (Bcc) members in nonsterile products became official in December 2019. This isolation method requires confirmation of the identity of any growth found on Burkholderia cepacia Selective Agar (BCSA) by additional identification tests (refer to the Interpretation section). This article presents a singleplex polymerase chain reaction (PCR) method to rapidly confirm the membership of any microbial grown on BCSA (and other nutrient medium) in the Bcc group. This method is cost effective as it does not require expensive equipment or reagents; therefore, it can be easily adopted in the industry without an important investment. We validated this singleplex PCR Bcc identification method with previously published PCR primers with an expanded panel of 37 clinical and environmental Bcc isolates. The sources and repositories of these Bcc isolates include contaminated health products and medical devices, patients infected with cystic fibrosis, the National Microbiology Laboratory (NML) internal strain bank, and the American Type Culture Collection (ATCC). All 37 isolates that belong to the Bcc tested positive using our confirmatory identification method. Twenty-two negative controls including four isolates belonging to the genus Burkholderia tested negative as expected. Our work indicates that this singleplex PCR is an efficient confirmatory method for Bcc identification, and it can successfully supplement USP <60> for Bcc isolates identification found in pharmaceutical products.

Synergistic Interactions among Burkholderia cepacia Complex-Targeting Phages Reveal a Novel Therapeutic Role for Lysogenization-Capable Phages.

Antimicrobial resistance is a danger to global public health and threatens many aspects of modern medicine. Bacterial species such as those of the Burkholderia cepacia complex (Bcc) cause life-threatening respiratory infections and are highly resistant to antibiotics. One promising alternative being explored to combat Bcc infections is phage therapy (PT): the use of phages to treat bacterial infections. Unfortunately, the utility of PT against many pathogenic species is limited by its prevailing paradigm: that only obligately lytic phages should be used therapeutically. It is thought that 'lysogenic' phages do not lyse all bacteria and can transfer antimicrobial resistance or virulence factors to their hosts. We argue that the tendency of a lysogenization-capable (LC) phage to form stable lysogens is not predicated exclusively on its ability to do so, and that the therapeutic suitability of a phage must be evaluated on a case-by-case basis. Concordantly, we developed several novel metrics-Efficiency of Phage Activity, Growth Reduction Coefficient, and Stable Lysogenization Frequency-and used them to evaluate eight Bcc-specific phages. Although these parameters vary considerably among Bcc phages, a strong inverse correlation (R2 = 0.67; P < 0.0001) exists between lysogen formation and antibacterial activity, indicating that certain LC phages with low frequency of stable lysogenization may be therapeutically efficacious. Moreover, we show that many LC Bcc phages interact synergistically with other phages in the first reported instance of mathematically defined polyphage synergy, and that these interactions result in the eradication of in vitro bacterial growth. Together, these findings reveal a novel therapeutic role for LC phages and challenge the current paradigm of PT. IMPORTANCE The spread of antimicrobial resistance is an imminent threat to public health around the world. Particularly concerning are species of the Burkholderia cepacia complex (Bcc), which cause life-threatening respiratory infections and are notoriously resistant to antibiotics. Phage therapy is a promising alternative being explored to combat Bcc infections and antimicrobial resistance in general, but its utility against many pathogenic species, including the Bcc, is restricted by the currently prevailing paradigm of exclusively using rare obligately lytic phages due to the perception that 'lysogenic' phages are therapeutically unsuitable. Our findings show that many lysogenization-capable phages exhibit powerful in vitro antibacterial activity both alone and through mathematically defined synergistic interactions with other phages, demonstrating a novel therapeutic role for LC phages and therefore challenging the currently prevailing paradigm of PT.

Identification of Burkholderia cenocepacia non-coding RNAs expressed during Caenorhabditis elegans infection.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematode Caenorhabditis elegans was used as an infection model by the epidemic CF strain B. cenocepacia J2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated under C. elegans infection conditions, was shown to directly affect B. cenocepacia virulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with the dusA and pyrC messengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved in B. cenocepacia virulence. KEY POINTS: • 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified • The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility • RIT11b directly binds to and regulates dusA and pyrC mRNAs.

The Burkholderia contaminans prevalent phenotypes as possible markers of poor clinical outcomes in chronic lung infection of children with cystic fibrosis.

Burkholderia contaminans, a species of the Burkholderia cepacia complex-prevalent in certain Latin-American and European countries-can cause chronic pulmonary infection in persons with cystic fibrosis. Our aim was to gain insights into long-term lung infections with a focus on correlating how bacterial phenotypic traits in the chronic infection impact on patients' clinical outcome. Genotypic characteristics of 85 B. contaminans isolates recovered from 70 patients were investigated. For 16 of those patients, the clinical status and bacterial phenotypic characteristics, e.g. several virulence factors, phenotypic variants, and the antimicrobial susceptibility pattern, were evaluated. Two clones were found in the whole bacterial population: (i) the multiresistant ST 872 PCR-recA-RFLP-HaeIII-K-pattern clone, which carries a pathogenic island homologous to BcenGI11 of B. cenocepacia J2315, and (ii) the ST 102 PCR-recA-RFLP-HaeIII-AT-pattern clone. The emergence of certain bacterial phenotypes in the chronic infection such as the nonmucoid phenotype, small colony variants, brownish pigmented colonies, and hypermutators, proved to be, together with coinfection with Pseudomonas aeruginosa, the possible markers of more challenging infections and poor prognosis. The presence of cocolonizers and the bacterial phenotypes that are especially adapted to persist in long-term respiratory tract infections have a crucial role in patients' clinical outcomes.

Phage-antibiotic synergy reduces Burkholderia cenocepacia population.

BACKGROUND: Burkholderia cenocepacia is an opportunistic pathogen that can cause acute and chronic infections in patients with weakened immune systems and in patients with cystic fibrosis. B. cenocepacia is resistant to many antibiotics making treatment challenging. Consequently, there is a critical need for alternative strategies to treat B. cenocepacia infections such as using bacteriophages and/or bacteriophages with subinhibitory doses of antibiotic called phage-antibiotic synergy. RESULTS: We isolated a bacteriophage, KP1, from raw sewage that infects B. cenocepacia. Its morphological characteristics indicate it belongs in the family Siphoviridae, it has a 52 Kb ds DNA genome, and it has a narrow host range. We determined it rescued infections in Lemna minor (duckweed) and moderately reduced bacterial populations in our artificial sputum medium model. CONCLUSION: These results suggest that KP1 phage alone in the duckweed model or in combination with antibiotics in the ASMDM model improves the efficacy of reducing B. cenocepacia populations.

Molecular Research Comparing the Probabilities of Burkholderia Cepacia Bacterium Diagnosis Procedures.

Burkholderia cepacia is found as part of the B. cepacia complex (Bcc), a collection of highly pathogenic organisms. The Bcc is present almost everywhere in nature; however, it is most prevalent in damp settings, plant roots, and soils. Moreover, Bcc is a major source of morbidity and death in patients due to its high intrinsic antibiotic resistance. The present study aims to isolate and identify gram-negative aerobic bacteria from clinical samples derived from a variety of pathological diseases and investigate the bacterium's virulence factors and genes. The current study included 250 specimens collected from patients suffering from diabetic foot ulcers, urine, burn, wound, sputum, and discharge from the eyes. The samples were collected from both sexes with the age range of 1-75 years. The recorded data showed that males had a higher frequency of infection (79.2%) than females (52%). The results revealed that 7.6% of infected females were between 1-15 years old, while 22% of infected males were aged between 31-45 years. In addition, 26.8% of infected patients (both males and females) were aged between 31-45 years.

The Genetic relatedness of Burkholderia contaminans clinical isolates from cystic fibrosis and non-cystic fibrosis patients in Argentina.

INTRODUCTION: The Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that cause nosocomial infections and are especially dangerous for cystic fibrosis (CF) patients. Burkholderia contaminans is an emerging BCC species isolated from CF patients that also occurs as a contaminant in pharmaceutical and personal care products, sometimes linking it with outbreaks. METHODOLOGY: A total of 55 B. contaminans isolates from CF and non-CF patients in Argentina were identified by recA sequencing and MALDI TOF MS. A standardized Pulsed Field Gel Electrophoresis (PFGE) protocol was set up in order to assess genetic diversity, outbreak investigations, and possible clone persistence. RESULTS: All isolates were identified as B. contaminans by both MALDI-TOF MS and recA sequence analysis. PFGE has enabled us to compare and determine the genetic relationship between B. contaminans isolates. Isolates were distributed in different PFGE clusters with evidence of the presence and persistence of a clone, over a period of 3 years, in the same hospital. This large hospital outbreak involved CF and non-CF patients. Moreover, PFGE results showed a good correlation between sporadic or outbreak-related isolates and the available epidemiological information. CONCLUSIONS: These findings highlight the importance of B. contaminans in Argentina and provide evidence for encouraging the surveillance of highly transmissible clones. The study also contributes to global knowledge about B. contaminans infections.

The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Evaluating the alginate oligosaccharide (OligoG) as a therapy for Burkholderia cepacia complex cystic fibrosis lung infection.

OligoG has previously shown potentiation of aztreonam against Burkholderia cepacia complex (Bcc) through biofilm disruption. A randomized, double-blind, placebo-controlled cross-over design was used to evaluate safety and efficacy of inhaled OligoG as a therapy for Bcc-infected CF patients taking aztreonam. Subjects received OligoG (1050 mg daily) or matching placebo for 28-days. Of 14 subjects completing the study, 8 showed a mean decrease in total bacterial CFU's (0.82 log10) after OligoG treatment. There was a reduction in mean Bcc CFU's (2.19 log10) after OligoG treatment but this was not statistically significant. Rheology analysis showed improvements in phase-angle after OligoG, but there was no statistically significant improvement in lung function parameters. Six out of 12 QoL summary scores showed relative improvement after OligoG treatment compared to placebo. There was a favourable safety profile for OligoG. Potential for reducing Bcc warrants further investigation of OligoG for the treatment of infection in CF.

The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. B. cenocepacia is strongly associated with cepacia syndrome, making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of B. cenocepacia in lung infections and cepacia syndrome remain to be uncovered. B. cenocepacia is primarily an intracellular pathogen and encodes the type VI secretion system (T6SS) effector TecA, which is translocated into host phagocytes. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines, such as interleukin-1ß, from macrophages. Previous work with the B. cenocepacia clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second B. cenocepacia clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and mice engineered to have a CF genotype. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a B. cenocepacia virulence factor that exacerbates lung inflammation, weight loss, and lethality in mouse infection models. IMPORTANCE B. cenocepacia is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (1). Prior to the current study, virulence factors of B. cenocepacia important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of B. cenocepacia exacerbates inflammatory cell recruitment and weight loss and is required for lethality and, thus, acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecA's role as a virulence factor and in investigating ways to prevent or treat B. cenocepacia infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.

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

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

Evolution towards Virulence in a Burkholderia Two-Component System.

Bacteria in the Burkholderia cepacia complex (BCC) are significant pathogens for people with cystic fibrosis (CF) and are often extensively antibiotic resistant. Here, we assess the impacts of clinically observed mutations in fixL, which encodes the sensor histidine kinase FixL. FixL along with FixJ compose a two-component system that regulates multiple phenotypes. Mutations in fixL across two species, B. dolosa and B. multivorans, have shown evidence of positive selection during chronic lung infection in CF. Herein, we find that BCC carrying the conserved, ancestral fixL sequence have lower survival in macrophages and in murine pneumonia models than mutants carrying evolved fixL sequences associated with clinical decline in CF patients. In vitro phosphotransfer experiments found that one evolved FixL protein, W439S, has a reduced ability to autophosphorylate and phosphorylate FixJ, while LacZ reporter experiments demonstrate that B. dolosa carrying evolved fixL alleles has reduced fix pathway activity. Interestingly, B. dolosa carrying evolved fixL alleles was less fit in a soil assay than those strains carrying the ancestral allele, demonstrating that increased survival of these variants in macrophages and the murine lung comes at a potential expense in their environmental reservoir. Thus, modulation of the two-component system encoded by fixLJ by point mutations is one mechanism that allows BCC to adapt to the host infection environment. IMPORTANCE Infections caused by members of the Burkholderia cepacia complex (BCC) are a serious concern for patients with cystic fibrosis (CF) as these bacteria are often resistant to many antibiotics. During long-term infection of CF patients with BCC, mutations in genes encoding the FixLJ system often become prevalent, suggesting that these changes may benefit the bacteria during infection. The system encoded by fixLJ is involved in sensing oxygen and regulating many genes in response and is required for full virulence of the bacteria in a murine pneumonia model. Evolved fixL mutations seen later in infection improve bacterial persistence within macrophages and enhance infection within mice. However, these adaptations are short sighted because they reduce bacterial fitness within their natural habitat, soil.

Presence of the Hmq System and Production of 4-Hydroxy-3-Methyl-2-Alkylquinolines Are Heterogeneously Distributed between Burkholderia cepacia Complex Species and More Prevalent among Environmental than Clinical Isolates.

The Burkholderia cepacia complex (Bcc) comprises several species of closely related, versatile bacteria. Some Bcc strains produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously estimated that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intraspecies variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 312 Bcc strains (222 of clinical and 90 of environmental origins) belonging to 18 Bcc species. We confirmed that this operon is not distributed evenly among Bcc species. Among the 30% of strains bearing the hmqABCDEFG operon, we found that 92% of environmental isolates and 82% of clinically isolated Bcc strains produce levels of HMAQs detectable by liquid chromatography-mass spectrometry in at least one of the tested culture conditions. Among the hmqABCDEFG-positive but HMAQ-negative strains, none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g., Burkholderia ambifaria and Burkholderia cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g., Burkholderia cenocepacia and Burkholderia multivorans), suggesting a role for the Hmq system in niche adaptation. We investigated the impact of the Hmq system on plant growth promotion and found that Pisum sativum root development by B. ambifaria required a functional HMAQ system. IMPORTANCE Environmental bacteria belonging to the various closely related species forming the Burkholderia cepacia complex (Bcc) can infect plants and animals, including humans. Their pathogenicity is regulated by intercellular communication, or quorum sensing, allowing them to collaborate instead of acting individually. Bcc organisms generally exploit interacting quorum sensing systems based on N-acyl-homoserine lactones as signaling molecules. Several Bcc strains also carry an hmqABCDEFG operon responsible for the biosynthesis of 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), molecules analogous to the Pseudomonas quinolone signal (PQS) system of P. aeruginosa. Our finding that the prevalences of the Hmq system and HMAQ production are very different between various Bcc species suggests a key role in niche adaptation or pathogenicity. This is supported by a significant reduction in plant growth promotion in the absence of HMAQ production for a beneficial Bcc strain.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

Clinico-microbiological profile of Burkholderia cepacia keratitis: a case series.

BACKGROUND: Burkholderia cepacia, an opportunistic pathogen mainly affecting patients with cystic fibrosis or immunocompromised, has rarely been documented as a cause of corneal infection. The clinical and microbiological profiles of B. cepacia keratitis are reported herein. METHODS: We retrospectively reviewed the medical record of 17 patients with culture-proven B. cepacia keratitis, treated between 2000 and 2019 at Chang Gung Memorial Hospital, Taiwan. Our data included predisposing factors, clinical presentations, treatments, and visual outcomes of B. cepacia keratitis as well as the drug susceptibility of the causative agent. RESULTS: The most common predisposing factor for B. cepacia keratitis was preexisting ocular disease (seven, 41.2%), particularly herpetic keratitis (five). Polymicrobial infection was detected in seven (41.2%) eyes. All B. cepacia isolates were susceptible to ceftazidime. Main medical treatments included levofloxacin or ceftazidime. Surgical treatment was required in five (29.4%) patients. Only four (23.5%) patients exhibited final visual acuity better than 20/200. CONCLUSIONS: B. cepacia keratitis primarily affects patients with preexisting ocular disease, particularly herpetic keratitis, and responds well to ceftazidime or fluoroquinolones. However, the visual outcomes are generally poor.

Current Advances in Burkholderia Vaccines Development.

The genus Burkholderia includes a wide range of Gram-negative bacterial species some of which are pathogenic to humans and other vertebrates. The most pathogenic species are Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). B. mallei and B. pseudomallei, the cause of glanders and melioidosis, respectively, are considered potential bioweapons. The Bcc comprises a subset of Burkholderia species associated with respiratory infections in people with chronic granulomatous disease and cystic fibrosis. Antimicrobial treatment of Burkholderia infections is difficult due to the intrinsic multidrug antibiotic resistance of these bacteria; prophylactic vaccines provide an attractive alternative to counteract these infections. Although commercial vaccines against Burkholderia infections are still unavailable, substantial progress has been made over recent years in the development of vaccines against B. pseudomallei and B. mallei. This review critically discusses the current advances in vaccine development against B. mallei, B. pseudomallei, and the Bcc.

Burkholderia vietnamiensis causing a non-lactational breast abscess in a non-cystic fibrosis patient in Tamil Nadu, India.

Burkholderia cepacia complex is a Gram-negative opportunistic pathogen usually found in people with an immunocompromised condition such as cystic fibrosis (CF). In a tropical country like India, this organism has been associated with a number of hospital-acquired infections including sepsis. We present here a report of a case of Burkholderia vietnamiensis causing a non-lactational breast abscess in a non-CF patient. The pathogen was identified as B. cepacia using Vitek system and matrix-assisted laser desorption ionisation-time of flight. This was confirmed by polymerase chain reaction (PCR) using recA genus-specific gene and sequencing of the PCR amplicons. recA-restriction fragment length polymorphism and recA gene sequencing revealed that the isolate is B. vietnamiensis. This is the first description of B. vietnamiensis isolated from a clinical case from India.