Cepacia Complex Organisms Research Articles

Development of a qPCR detection approach for pathogenic Burkholderia cenocepacia associated with fresh vegetables

Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg μl−1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.

Burkholderia contaminans Bacteriophage CSP3 Requires O-Antigen Polysaccharides for Infection.

The Burkholderia cepacia complex is a group of opportunistic pathogens that cause both severe acute and chronic respiratory infections. Due to their large genomes containing multiple intrinsic and acquired antimicrobial resistance mechanisms, treatment is often difficult and prolonged. One alternative to traditional antibiotics for treatment of bacterial infections is bacteriophages. Therefore, the characterization of bacteriophages infective for the Burkholderia cepacia complex is critical to determine their suitability for any future use. Here, we describe the isolation and characterization of novel phage, CSP3, infective against a clinical isolate of Burkholderia contaminans. CSP3 is a new member of the Lessievirus genus that targets various Burkholderia cepacia complex organisms. Single nucleotide polymorphism (SNP) analysis of CSP3-resistant B. contaminans showed that mutations to the O-antigen ligase gene, waaL, consequently inhibited CSP3 infection. This mutant phenotype is predicted to result in the loss of cell surface O-antigen, contrary to a related phage that requires the inner core of the lipopolysaccharide for infection. Additionally, liquid infection assays showed that CSP3 provides suppression of B. contaminans growth for up to 14 h. Despite the inclusion of genes that are typical of the phage lysogenic life cycle, we saw no evidence of CSP3's ability to lysogenize. Continuation of phage isolation and characterization is crucial in developing large and diverse phage banks for global usage in cases of antibiotic-resistant bacterial infections. IMPORTANCE Amid the global antibiotic resistance crisis, novel antimicrobials are needed to treat problematic bacterial infections, including those from the Burkholderia cepacia complex. One such alternative is the use of bacteriophages; however, a lot is still unknown about their biology. Bacteriophage characterization studies are of high importance for building phage banks, as future work in developing treatments such as phage cocktails should require well-characterized phages. Here, we report the isolation and characterization of a novel Burkholderia contaminans phage that requires the O-antigen for infection, a distinct phenotype seen among other related phages. Our findings presented in this article expand on the ever-evolving phage biology field, uncovering unique phage-host relationships and mechanisms of infection.

Quorum Sensing Inhibitors Increase the Susceptibility of Bacterial Biofilms to Antibiotics In Vitro and In Vivo

Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) has been proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although reductions were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.

In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria

The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells. The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods. Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms. Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria.

Microbiological and Epidemiological Features of Clinical Respiratory Isolates of Burkholderia gladioli

Burkholderia gladioli, primarily known as a plant pathogen, is involved in human infections, especially in patients with cystic fibrosis (CF). In the present study, the first respiratory isolates recovered from 14 French patients with CF and 4 French patients without CF, identified by 16S rRNA gene analysis, were tested for growth on B. cepacia selective media, for identification by commercial systems, and for their antimicrobial susceptibilities, and were compared by pulsed-field gel electrophoresis (PFGE). Patients' data were collected. All 18 isolates grew on oxidation-fermentation-polymyxin B-bacitracin-lactose medium and Pseudomonas cepacia agar, but only 13 grew on Burkholderia cepacia selective agar. API 20NE strips did not differentiate B. gladioli from B. cepacia, whereas Vitek 2 GN cards correctly identified 15 isolates. All isolates were susceptible to piperacillin, imipenem, aminoglycosides, and ciprofloxacin and were far less resistant to ticarcillin than B. cepacia complex organisms. Fifteen PFGE types were observed among the 18 isolates, but shared types were not identified among epidemiologically related patients. The microbiological follow-up of CF patients showed that colonization was persistent in 3 of 13 documented cases; B. gladioli was isolated from posttransplantation cultures of blood from 1 patient. Among the patients without CF, B. gladioli was associated with intubation (three cases) or bronchiectasis (one case). In summary, the inclusion of B. gladioli in the databases of commercial identification systems should improve the diagnostic capabilities of those systems. In CF patients, this organism is more frequently involved in transient infections than in chronic infections, but it may be responsible for complications posttransplantation; patient-to-patient transmission has not been demonstrated to date. Lastly, B. gladioli appears to be naturally susceptible to aminoglycosides and ciprofloxacin, although resistant isolates may emerge in the course of chronic infections.

Resistance of planktonic and biofilm-grown Burkholderia cepacia complex isolates to the transition metal gallium--authors' response

Sir, We thank Lechuga-Ballesteros et al. for their interest in our study and for their comments. We agree that the MIC values of gallium(III) reported in our study are of the same order of magnitude as those previously reported for some other Gram-negative bacteria. Nevertheless, the available data suggest that the MIC values of gallium(III) in an iron-depleted medium for the limited number of Burkholderia cepacia complex strains investigated so far ( 17 mg/L and not 4–18 mg/L as erroneously asserted by Lechuga-Ballesteros et al.) are markedly higher than those for the majority of Pseudomonas aeruginosa isolates recovered from cystic fibrosis (CF) patients [86% (81/94) of all isolates investigated by Kaneko et al. had an MIC 0.7 mg/L]. Whether this difference would hold up if a larger number of B. cepacia complex and P. aeruginosa isolates were investigated under identical conditions (e.g. in terms of growth media) remains to be determined. A second remark concerns our ‘simplistic view’ about the protective effect of Fe3þ. We completely agree with Lechuga-Ballesteros et al. that the in vitro evaluation of the competition by Fe3þ cannot be extrapolated to the in vivo situation, where the free Fe3þ concentration is low. However, the data in Table 1 of our publication clearly indicate that the addition of Fe3þ (final concentration: 50 mM) hardly affects the antimicrobial activity of gallium(III) towards Burkholderia multivorans and Burkholderia dolosa biofilms, while it abolishes this effect for Burkholderia cenocepacia biofilms. Indeed, treating 28 h old B. cenocepacia LMG 16656 or LMG 18828 biofilms with 32 or 64 mg/L Ga(NO3)3 in the presence of 50 mM Fe3þ did not result in meaningful reductions in the number of viable sessile cells (reductions between 0.1% and 10.3%). However, reductions in viable cell numbers in B. multivorans and B. dolosa biofilms in the presence of Fe3þ were hardly different from those in the absence of Fe3þ. While the relevance of these differences for the in vivo situation is at present unclear, they are real and cannot be ignored. In addition, the observation that B. cenocepacia can use ferritin as an iron source suggests that not only free Fe3þ concentrations should be taken into account, but also that iron bound to ferritin (and possibly also to other carriers) may play a role in countering the antimicrobial effect of gallium(III) for at least this B. cepacia complex species. The fact that most B. cepacia complex lineages infecting a large number of patients (including the ET12, PHDC and Midwest clones) belong to B. cenocepacia stresses the potential clinical relevance of these observations. A final remark concerns our conclusion that the added value of Ga(NO3)3 to treat CF patients infected with B. cepacia complex may be limited. We can assure Lechuga-Ballesteros et al. and the readers of JAC that we hope to be proved wrong; however, at this point, there are no published and peer-reviewed data supporting the claims (i) that gallium can be used to treat B. cepacia complex infections in an animal model and (ii) that high doses (400 mM) of gallium can be administered through nebulization without adverse effects. Until data on the safety and efficacy of nebulized Ga(NO3)3 to treat CF lung infections caused by B. cepacia complex organisms become available, it seems wiser to tone down speculations about its potential use to treat these infections in CF patients.

Production of exopolysaccharide by Burkholderia cenocepacia results in altered cell-surface interactions and altered bacterial clearance in mice.

Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B. cepacia complex organisms. We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343. Both isolates had identical random amplified polymorphic DNA patterns. C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as alpha -1,6-glucan. These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions. Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model. This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

Rapid characterization of the genomovars of the Burkholderia cepacia complex by PCR-single-stranded conformational polymorphism (PCR-SSCP) analysis

Four polymerase chain reaction (PCR) primer pairs (A–D), specific for the Burkholderia cepacia complex of organisms were designed to encompass the entire gene (,1300bp) from sequence alignments of the rec A operon of B. cepacia. Genomic bacterial DNA from type strains and wild-type B. cepacia complex isolates of previously determined genomovar status was amplified employing these four primer pairs, as well as a fifth primer set (E) already published. Primer sets B, C and E were successful in obtaining a PCR amplicon of correct estimated size of 598, 1107 and 1043bp, respectively. Subsequent single-stranded conformational polymorphism (SSCP) analysis of PCR amplicons demonstrated unique profiles for the five genomovar types for primer pairs B and E, but was unable to differentiate distinguishable profile types for primer pair C. SSCP analysis was demonstrated to be simple, rapid and cost effective and may prove a useful method of genomovar typing ofB. cepacia complex organisms from cystic fibrosis patients in busy diagnostic laboratories.