GVF27, a human derived peptide with anti-infective properties against biofilm forming members of the Burkholderia cepacia complex

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the extremely promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, as Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by alternative microscopy approaches. Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS, were also performed. GVF27 exerts, at different stages of formation, meaningful anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from Burkholderia cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Multifrequency STD NMR Unveils the Interactions of Antibiotics With Burkholderia multivorans Biofilm Exopolysaccharide

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

Evaluation of antimicrobial susceptibility testing methods for Burkholderia cenocepacia and Burkholderia multivorans isolates from cystic fibrosis patients

The Burkholderia cepacia complex (BCC) is known for causing serious lung infections in people with cystic fibrosis (CF). These infections can require lung transplantation, eligibility for which may be guided by antimicrobial susceptibility testing (AST). While the Clinical and Laboratory Standards Institute recommends AST for BCC, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) does not due to poor method performance and correlation with clinical outcomes. Furthermore, limited data exists on the performance of automated AST methods for BCC. To address these issues, reproducibility and accuracy were evaluated for disk diffusion (DD), broth microdilution (BMD), and MicroScan WalkAway using 50 B. cenocepacia and 50 B. multivorans isolates collected from people with CF. The following drugs were evaluated in triplicate: chloramphenicol (CAM), ceftazidime (CAZ), meropenem (MEM), trimethoprim-sulfamethoxazole (TMP-SMX), minocycline (MIN), levofloxacin (LVX), ciprofloxacin (CIP) and piperacillin-tazobactam (PIP-TAZ). BMD reproducibility was ≥ 95% for MEM and MIN only, and MicroScan WalkAway reproducibility was similar to BMD. DD reproducibility was < 90% for all drugs tested when a 3 mm cut-off was applied. When comparing the accuracy of DD to BMD, only MEM met all acceptance criteria. TMP-SMX and LVX had high minor errors, CAZ had unacceptable very major errors (VME), and MIN, PIP-TAZ, and CIP had both unacceptable minor errors and VMEs. For MicroScan WalkAway, no drugs met acceptance criteria. Analyses also showed that errors were not attributed to one species. In general, our data agree with EUCAST recommendations that routine AST should not be performed for BCC CF isolates.

Activity of imipenem-relebactam against multi-drug and extensively-drug resistant Burkholderia cepacia complex and Burkholderia gladioli

The Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic pathogens that most commonly infect persons with cystic fibrosis or compromised immune systems. Members of the Burkholderia genus are intrinsically multidrug resistant (MDR), possessing both a PenA carbapenemase and an AmpC β-lactamase, which renders treatment of infection due to these species problematic. Here, we tested the β-lactam-β-lactamase inhibitor combination, imipenem-relebactam, against a panel of MDR Bcc and B. gladioli . The addition of relebactam to imipenem dramatically lowered the MICs for Bcc and B . gladioli with only 16% of isolates testing susceptible to imipenem vs. 71.3% being susceptible to the imipenem-relebactam combination. While ceftazidime-avibactam remained the most potent combination drug against this panel of Bcc and B. gladioli , imipenem-relebactam was active against 71.4% of the ceftazidime-avibactam-resistant isolates. Relebactam demonstrated potent inactivation of the Burkholderia multivorans PenA1 with a K i app value of 3.2 μM. Timed mass spectrometry revealed that PenA1 formed a very stable adduct with relebactam, without any detectable desulfation up to 24 hours. Based on our results, imipenem-relebactam may represent an alternative salvage therapy for Bcc and B . gladioli infection, especially in cases where the isolates are resistant to ceftazidime-avibactam.

Determination of Biodegradation Zone in Central Sumatra Basin

It is commonly known that heavy oil is mostly formed through biodegradation process within reservoir or on the surface both by aerobic and/or anaerobic bacteria that can live under specfi c temperature level(s). In order to investigate heavy oil occurences in Central Sumatra Basin, eff orts have been spent to determine the depths that represent the maximum temperature. By integrating the maximum viable temperature of typical bacteria and temperature gradient data, the depth of heavy oil zone is determined. The work is a combination of establishment of geothermal gradient map and laboratory analysis on fi eld sampled oil for determining types and temperature characteristics of microorganism living in the samples. Heavy oil sampling is made on seepages in areas nearby Minas fi eld. Subsequent laboratory analysis reveals Burkholderia multivorans ATCC BAA-247 as the predominant bacteria having maximum viabl temperature of 60° C. Based on the established geothermal gradient map, this maximum temperature correspond to average depth of 1818 ft (555.5 m). This average depth is used as the lower depth for the biodegradation zone over which investigation over presence of heavy oil bearing reservoirs/traps is made.

Antibiotic Cycling Reverts Extensive Drug Resistant Burkholderia multivorans

Antibiotic collateral sensitivity, in which acquired resistance to one drug leads to decreased resistance to a different drug, occurs in Burkholderia multivorans . Here we observed that treatment of extensive drug resistant variants from a CF sputum sample with either meropenem or sulfamethoxazole/trimethoprim, depending on past resistance phenotypes, resulted in increased sensitivity to five different classes of antibiotics. We further identified mutations that may be involved in this phenotype, including putative Resistance-Nodulation-Division efflux pump regulators and uncharacterized pumps in B. multivorans.

A histone-like nucleoid structuring protein regulates several virulence traits in Burkholderia multivorans

Burkholderia cepacia complex bacteria comprises opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring protein, a known global transcriptional repressor. This IS element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pili synthesis, type VI secretion, and chromosome associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line, reduced surface hydrophobicity, forms smaller cellular aggregates, but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid-structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, presenting a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein, is one out of eight orthologs present in B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.