Burkholderia cenocepacia ST-250 in cystic fibrosis patients in Switzerland: Genomic investigation of transmission routes.

This report describes the characterization of Burkholderia cenocepacia isolates belonging to sequence type (ST)-250, detected in eight patients with cystic fibrosis (CF) in Switzerland. We retrospectively analyzed 18 isolates of B. cenocepacia ST-250 isolated between 2003 and 2015 by whole-genome sequencing and evaluated clinical and epidemiological data. Single nucleotide polymorphism analysis of the B.°cenocepacia ST-250 lineage showed that the isolates from all patients cluster tightly, suggesting that this cluster has a recent common ancestor. Epidemiological investigations showed that six out of eight patients acquired B.°cenocepacia ST-250 in the years 2001-2006, where participation in CF summer camps was common. Two patients were siblings. Genomic relatedness of the B. cenocepacia ST-250 isolates supported transmission by close contact, however, a common source or nosocomial routes cannot be excluded. With respect to the fatal outcome in six patients, our study shows the importance of infection control measurements in CF patients with B.°cenocepacia.

Cloaking antibodies are prevalent in Burkholderia cepacia complex infection and their removal restores serum killing.

Introduction: The Burkholderia cepacia complex encompasses a group of gram-negative opportunistic pathogens that cause chronic lung infections in people with cystic fibrosis. Distinct from other respiratory pathogens, Burkholderia causes a unique clinical disease in a subset of patients known as 'cepacia syndrome', fulminant pneumonia accompanied by bacteraemia and sepsis with a mortality rate of up to 75%. Due to the bacteraemia associated with this disease, the mechanisms that allow Burkholderia to resist the bactericidal effects of serum complement-depending killing are vital. Antibodies usually promote serum killing; however, we have described 'cloaking antibodies', specific for lipopolysaccharides that paradoxically protect serum-sensitive bacteria from complement-mediated lysis. Cloaking antibodies that protect Pseudomonas aeruginosa have been found in 24%-41% of patients with chronic lung diseases. The presence of these antibodies is also associated with worse clinical outcomes. Here, we sought to determine the relevance of cloaking antibodies in patients with Burkholderia infection. Methods: Twelve Burkholderia spp. were isolated from nine pwCF and characterised for susceptibility to healthy control serum. Patient serum was analysed for the titre of the cloaking antibody. The ability of the patient serum to prevent healthy control serum (HCS) killing of its cognate isolates was determined. Results: We found that several of the Burkholderia strains were shared between patients. Ten of the 12 isolates were highly susceptible to HCS killing. Four of nine (44%) patients had cloaking antibodies that protected their cognate strain from serum killing. Depleting cloaking antibodies from patient serum restored HCS killing of Burkholderia isolates. Discussion: Cloaking antibodies are prevalent in patients with Burkholderia pulmonary infection and protect these strains from serum killing. Removal of cloaking antibodies via plasmapheresis, as previously described for individuals with life-threatening Pseudomonas infection, may be a useful new strategy for those with serious and life-threatening Burkholderia infection.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B12) Confer Tolerance to Bactericidal Antibiotics in Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

Outbreak of Burkholderia cenocepacia in an intensive care unit of a tertiary care hospital identified by pulsed-field gel electrophoresis.

Nosocomial outbreaks of Burkholderia cepacia complex, transmitted through contaminated medical surfaces or equipment have been reported. Pulsed-field Gel Electrophoresis (PFGE) is recognized as the "gold standard" for molecular subtyping, yet studies on clonal relationships in India are limited. PFGE was used to study the clonal relationships of 22 isolates of Burkholderia cenocepacia from 12 patients admitted to a critical care unit during 2 months (November and December 2021). PFGE revealed three different profiles with 15 isolates belonging to a single cluster suggesting a common source within the hospital, emphasizing the need for preventive measures to control B. cenocepacia transmission.

Epidemiology and Clinical Features of Burkholderia Bacteremia at a Hospital in India.

Burkholderia spp. are opportunistic pathogens that cause infection in patients with disrupted immunity. The study intended to demonstrate the epidemiology and clinical features associated with Burkholderia spp. bacteremia. This retrospective study was performed to assess the clinical and laboratory characteristics of patients whose blood cultures were growing Burkholderia spp. and, based on their underlying comorbidities, were subjected to survival analysis from January 2022 to December 2022 at a university hospital in northern India. Three hundred patients with Burkholderia spp. bacteremia were included in this study conducted over 1 year. The mean age of the patients was 33.86 years with a male predominance of 56.67% (170/300, 56.67%). Underlying malignancies (207/300, 69.0%) were the most common clinical diagnosis, and catheter in situ (300/300, 100.0%) was the most common risk factor. Burkholderia cenocepacia (244/300, 81.33%) was the most common Burkholderia spp. isolated. All isolates were highly susceptible to minocycline. Kidney disease (P = 0.029), hypertension (P = 0.005), type 2 diabetes mellitus (P = 0.039), and respiratory disease (P <0.001) in patients were significantly associated with death owing to Burkholderia spp. bacteremia, whereas patients with malignancies (P <0.001) and undergoing treatment were significantly associated with a better outcome when the microorganism was susceptible to empirical antibiotics. The presence of indwelling devices, mechanical ventilation (P <0.001), and a hemodialysis catheter (P = 0.026) were statistically significant risk factors associated with poor outcomes.

Case Report: The Conundrum of What to Pick? Antibiotic Susceptibility Variability in Burkholderia cenocepacia in Cystic Fibrosis: Implications for Antibiotic Susceptibility Testing and Treatment.

Within cystic fibrosis microbiology, there is often mismatch between the antibiotic susceptibility result of an isolated bacterial pathogen and the clinical outcome, when the patient is treated with the same antibiotic. The reasoning for this remains largely elusive. Antibiotic susceptibility to four antibiotics (ceftazidime, meropenem, minocycline and trimethoprim-sulfamethoxazole) was determined in consecutive isolates (n = 11) from an adult cystic fibrosis patient, over a 63 month period. Each isolate displayed its own unique resistotype. The first isolate was sensitive to all four antibiotics, in accordance with Clinical and Laboratory Standards Institute methodology and interpretative criteria. Resistance was first detected at four months, showing resistance to ceftazidime and meropenen and intermediate resistance to minocycline and trimethoprim-sulfamethoxazole. Pan resistance was first detected at 18 months (resistotype IV), with three resistotypes (I, II and III) preceding this complete resistotype. The bacterium continued to display further antibiotic susceptibility heterogeneity for the next 45 months, with the description of an additional seven resistotypes (resistotypes V-XI). The Relative Resistance Index of this bacterium over the 63 month period showed no relationship between the development of antibiotic resistance and time. Adoption of mathematical modelling employing multinomial distribution demonstrated that large numbers of individual colony picks (>40/sputum), would be required to be 78% confident of capturing all 11 resistotypes present. Such a requirement for large numbers of colony picks combined with antibiotic susceptibility-related methodological problems creates a conundrum in biomedical science practice, in providing a robust assay that will capture antibiotic susceptibility variation, be pragmatic and cost-effective to deliver as a pathology service, but have the reliability to help clinicians select appropriate antibiotics for their patients. This study represents an advance in biomedical science as it demonstrates potential variability in antibiotic susceptibility testing with Burkholderia cenocepacia. Respiratory physicians and paediatricians need to be made aware of such variation by biomedical scientists at the bench, so that clinicians can contextualise the significance of the reported susceptibility result, when selecting appropriate antibiotics for their cystic fibrosis patient. Furthermore, consideration needs to be given in providing additional guidance on the laboratory report to highlight this heterogeneity to emphasise the potential for misalignment between susceptibility result and clinical outcome.

Mutation of hmgA, encoding homogentisate 1,2-dioxygenase, is responsible for pyomelanin production but does not impact the virulence of Burkholderia cenocepacia in a chronic granulomatous disease mouse lung infection.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Burkholderia semiarida as Cause of Recurrent Pulmonary Infection in Immunocompetent Patient, China.

Burkholderia semiarida was previously identified solely as a plant pathogen within the Burkholderia cepacia complex. We present a case in China involving recurrent pneumonia attributed to B. semiarida infection. Of note, the infection manifested in an immunocompetent patient with no associated primary diseases and endured for >3 years.

Evaluation of CHROMagar™ B. cepacia agar for the detection of Burkholderia cepacia complex species from sputum samples of patients with cystic fibrosis.

INTRODUCTION: Burkholderia cepacia complex (BCC) are non-fermenting Gram-negative bacteria that can chronically colonize the lungs of people with cystic fibrosis (pwCF), causing a severe and progressive respiratory failure, post-transplant complications and epidemic outbreaks. Therefore, rapid and accurate identification of these bacteria is relevant for pwCF, in order to facilitate early eradication and prevent chronic colonization. However, BCCs are often quite difficult to detect on culture media as they have a slow growth rate and can be hidden by other fast-growing microorganisms, including Pseudomonas aeruginosa and filamentous fungi. MATERIAL AND METHODS: We evaluated the sensitivity of CHROMagar™ B. cepacia agar using 11 isolates from a well-characterized BCC collection, using BCA agar (Oxoid, UK) as a gold standard. We also studied 180 clinical sputum samples to calculate positive (PPV) and negative (NPV) predictive values. Furthermore, we used three of the well-characterized BCC isolates to determine the limit of detection (LOD). RESULTS: Eleven isolates grew on CHROMagar™ B. cepacia at 37ºC after 48 h. The NPV and PPV of CHROMagar™ B. cepacia were 100% and 87.5%, respectively. The LOD of CHROMagar™ B. cepacia was around 1 × 103 CFU/ml, requiring a ten-fold dilution lower bacterial load than BCA for BCC detection. CONCLUSION: CHROMagar™ B. cepacia agar proved to have a very good sensitivity and specificity for the detection of clinical BCCs. Moreover, the chromogenic nature of the medium allowed us to clearly differentiate BCC from other Gram-negative species, filamentous fungi and yeasts, thereby facilitating the identification of contaminants.

Species distribution and antimicrobial susceptibility of Burkholderia cepacia complex isolates in clinical infections: Experience from a tertiary care hospital, Southern India.

PURPOSE: Burkholderia cepacia complex (Bcc) is a diverse group of environmental bacteria associated with opportunistic infections. The identification of Bcc using conventional methods poses challenges. Bcc infections are difficult to treat due to intrinsic antibiotic resistance. The study aimed to investigate the species distribution and antimicrobial susceptibility of clinical Bcc isolates. METHODS: A total of 153 Bcc isolates obtained from clinical samples were analysed. Species identification was carried out using automated methods, including MALDI-TOF MS and VITEK2. Antimicrobial susceptibility testing was performed using the disc diffusion method. RESULTS: Burkholderia cenocepacia (70.5%) emerged as the most prevalent species, followed by Burkholderia contaminans (9.8%) and Burkholderia cepacia (7.2%). Ventilator-associated pneumonia (38.6%) was the most common infection, followed by sepsis (28.1%). Co-existence of Bcc with other pathogens in many cases suggested potential co-infection scenarios. Antimicrobial susceptibility revealed that ceftazidime, co-trimoxazole and meropenem were the most effective drugs, while levofloxacin proved to be the least effective. Moderate susceptibility was noted to minocycline, with 4.6% of isolates exhibiting multi-drug resistance. CONCLUSION: This study provides valuable insights into the prevalence, clinical associations, and antibiotic susceptibility of Bcc in India. It highlights the importance of Bcc as a nosocomial pathogen, especially in vulnerable patient populations. The findings contribute to understanding Bcc infections, their distribution, and emphasize the necessity for accurate identification methods in clinical settings.

Isolation of a PRD1-like phage uncovers the carriage of three putative conjugative plasmids in clinical Burkholderia contaminans.

The Burkholderia cepacia complex (Bcc) is a group of increasingly multi-drug resistant opportunistic bacteria. This resistance is driven through a combination of intrinsic factors and the carriage of a broad range of conjugative plasmids harbouring virulence determinants. Therefore, novel treatments are required to treat and prevent further spread of these virulence determinants. In the search for phages infective for clinical Bcc isolates, CSP1 phage, a PRD1-like phage was isolated. CSP1 phage was found to require pilus machinery commonly encoded on conjugative plasmids to facilitate infection of Gram-negative bacteria genera including Escherichia and Pseudomonas. Whole genome sequencing and characterisation of one of the clinical Burkholderia isolates revealed it to be Burkholderia contaminans. B. contaminans 5080 was found to contain a genome of over 8 Mbp encoding multiple intrinsic resistance factors, such as efflux pump systems, but more interestingly, carried three novel plasmids encoding multiple putative virulence factors for increased host fitness, including antimicrobial resistance. Even though PRD1-like phages are broad host range, their use in novel antimicrobial treatments shouldn't be dismissed, as the dissemination potential of conjugative plasmids is extensive. Continued survey of clinical bacterial strains is also key to understanding the spread of antimicrobial resistance determinants and plasmid evolution.

Burkholderia thailandensis Isolated from Infected Wound, Southwest China, 2022.

We report a clinical isolate of Burkholderia thailandensis 2022DZh obtained from a patient with an infected wound in southwest China. Genomic analysis indicates that this isolate clusters with B. thailandensis BPM, a human isolate from Chongqing, China. We recommend enhancing monitoring and surveillance for B. thailandensis infection in both humans and livestock.

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.

Genomic insights into an extensively drug-resistant and hypervirulent Burkholderia dolosa N149 isolate of a novel sequence type (ST2237) from a Vietnamese patient hospitalised for stroke.

OBJECTIVES: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke. METHODS: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary. RESULTS: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains. CONCLUSIONS: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa.

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.