Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative ß-Lactamases.

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients.

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-ß-lactam ß-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major ß-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 µM-1 s-1 and a slow koff of (2 ± 1) × 10-3 s-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA-avibactam revealed several interactions that stabilized the acyl-enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime-avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime-avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime-avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacilli that are ubiquitous in the environment and have emerged over the past 30 years as opportunistic pathogens in immunocompromised populations, specifically individuals with cystic fibrosis (CF) and chronic granulomatous disease. This complex of at least 18 distinct species is phenotypically and genetically diverse. One phenotype observed in a subset of Burkholderia cenocepacia (a prominent Bcc pathogen) isolates is the ability to produce a melanin-like pigment. Melanins have antioxidant properties and have been shown to act as virulence factors allowing pathogens to resist killing by the host immune system. The melanin-like pigment expressed by B. cenocepacia is produced through tyrosine catabolism, specifically through the autoxidation and polymerization of homogentisate. Burkholderia cenocepacia J2315 is a CF clinical isolate that displays a pigmented phenotype when grown under normal laboratory conditions. We examined the amino acid sequences of critical enzymes in the melanin synthesis pathway in pigmented and non-pigmented Bcc isolates, and found that an amino acid substitution of glycine for arginine at amino acid 378 in homogentisate 1,2-dioxygenase correlated with pigment production; we identify this as one mechanism for expression of pigment in Bcc isolates.

The tyrosine kinase BceF and the phosphotyrosine phosphatase BceD of Burkholderia contaminans are required for efficient invasion and epithelial disruption of a cystic fibrosis lung epithelial cell line.

Bacterial tyrosine kinases and their cognate protein tyrosine phosphatases are best known for regulating the biosynthesis of polysaccharides. Moreover, their roles in the stress response, DNA metabolism, cell division, and virulence have also been documented. The aim of this study was to investigate the pathogenicity and potential mechanisms of virulence dependent on the tyrosine kinase BceF and phosphotyrosine phosphatase BceD of the cystic fibrosis opportunistic pathogen Burkholderia contaminans IST408. The insertion mutants bceD::Tp and bceF::Tp showed similar attenuation of adhesion and invasion of the cystic fibrosis lung epithelial cell line CFBE41o- compared to the parental strain B. contaminans IST408. In the absence of bceD or bceF genes, B. contaminans also showed a reduction in the ability to translocate across polarized epithelial cell monolayers, demonstrated by a higher transepithelial electrical resistance, reduced flux of fluorescein isothiocyanate-labeled bovine serum albumin, and higher levels of tight junction proteins ZO-1, occludin, and claudin-1 present in monolayers exposed to these bacterial mutants. Furthermore, bceD::Tp and bceF::Tp mutants induced lower levels of interleukin-6 (IL-6) and IL-8 release than the parental strain. In conclusion, although the mechanisms of pathogenicity dependent on BceD and BceF are not understood, these proteins contribute to the virulence of Burkholderia by enhancement of cell attachment and invasion, disruption of epithelial integrity, and modulation of the proinflammatory response.

In vitro activity of ceftazidime+NXL104 against Pseudomonas aeruginosa and other non-fermenters.

BACKGROUND: NXL104 potentiates ceftazidime and ceftaroline against Enterobacteriaceae with extended-spectrum, AmpC, KPC and OXA ß-lactamases. We examined whether similar potentiation was obtained against non-fermenters, which are less permeable than Enterobacteriaceae and have more potent efflux. METHODS: MICs of ceftazidime+NXL104 (with NXL104 at 4 mg/L) and comparators were determined by CLSI agar dilution for: (i) Pseudomonas aeruginosa AmpC mutants and extended-spectrum ß-lactamase (ESBL)-producing transconjugants; (ii) clinical P. aeruginosa isolates with AmpC enzymes, ESBLs or up-regulated efflux; (iii) P. aeruginosa and Burkholderia cepacia complex isolates from cystic fibrosis patients; and (iv) Acinetobacter baumannii with OXA carbapenemases, which also compromise ceftazidime. RESULTS: NXL104 reversed AmpC-mediated ceftazidime resistance in P. aeruginosa, reducing MICs for fully derepressed mutants and isolates to ≤ 8 mg/L. NXL104 also reversed ceftazidime resistance caused by the ESBL PER-1, but not that due to OXA ESBLs or VEB-1 enzyme. Efflux-mediated resistance was unaffected. Resistance to ceftazidime in isolates of P. aeruginosa and the B. cepacia complex from patients with cystic fibrosis was variably overcome, generally to greater effect for B. cepacia. NXL104 had little effect on MICs of ceftazidime for A. baumannii isolates with OXA carbapenemases. CONCLUSIONS: The potentiation of ceftazidime against many ß-lactamase-producing P. aeruginosa and B. cepacia complex strains confirms that NXL104 penetrates these organisms. The utility of the combination against these pathogens will depend on the local prevalence of strains with ß-lactamase- versus efflux-mediated resistance. The lack of potentiation against A. baumannii may reflect failure of NXL104 to penetrate these bacteria to inhibit relevant (OXA-23, -40, -51 and -58) carbapenemases.

Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate.

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H2O2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Role of lipase in Burkholderia cepacia complex (Bcc) invasion of lung epithelial cells.

The Burkholderia cepacia complex (Bcc) is a group of ten closely related species associated with life-threatening infection in cystic fibrosis (CF). These bacteria are highly antibiotic resistant, with some strains transmissible, and in a subgroup of patients, they can cause a rapid and fatal necrotising pneumonia. The Bcc organisms produce a range of exoproducts with virulence potential, including exopolysaccharide, proteases and lipases. Many members of the Bcc are also capable of epithelial cell invasion, although the mechanism(s) involved are poorly understood. This study investigates a role for Bcc lipase in epithelial cell invasion by Bcc strains. Lipase activity was measured in eight species of the Bcc. Strains that produced high levels of lipase were predominantly from the B. multivorans and B. cenocepacia species. Pre-treatment of two epithelial cell lines with Bcc lipase significantly increased invasion by two B. multivorans strains and one B. cenocepacia strain and did not affect either plasma membrane or tight junction integrity. Inhibition of Bcc lipase production by the lipase inhibitor Orlistat significantly decreased invasion by both B. multivorans and B. cenocepacia strains in a concentration-dependent manner. This study demonstrates the extent of lipase production across the Bcc and establishes a potential role for lipase in Bcc epithelial cell invasion.

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia.

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo.

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.