Staphylococcus aureus Potentiates the Hemolytic Activity of Burkholderia cepacia Complex (Bcc) Bacteria.

Polymicrobial lung infections in individuals with Cystic Fibrosis (CF) contribute to the complexity of this disease and are a major cause of morbidity and mortality in the CF community. The microorganisms most commonly associated with severe airway infections in individuals with CF are the opportunistic pathogens S. aureus, P. aeruginosa and bacteria from the Burkholderia cepacia complex (Bcc), particularly B. cenocepacia and B. multivorans. Three Bcc strains, two S. aureus wild-type strains, and two derivative mutants were used to investigate the interplay between S. aureus and Bcc with a focus on the hemolytic activity of Bcc. Our results revealed that extracellular products from S. aureus potentiated the hemolysis of Bcc strains. Moreover, this effect was influenced by the composition of the medium in which S. aureus is grown. These findings contribute towards the understanding of the impact of interactions between S. aureus and Bcc and their possible implications in the context of co-infections by these pathogens in individuals with CF.

Distribution of Burkholderia cepacia complex species isolated from industrial processes and contaminated products in Argentina.

Burkholderia cepacia complex (Bcc) members have clinical relevance as opportunistic pathogens in patients with cystic fibrosis and are responsible of numerous nosocomial infections. These closely related bacteria are also reported as frequent contaminants of industrial products. In this retrospective study, we use PCR and recA gene sequence analysis to identify at species level Bcc isolates recovered from massive consumption products and industrial processes in Argentina during the last 25 years. The sequences obtained were also compared with recA sequences from clinical Bcc isolates deposited in GenBank database. We detected Bcc in purified water and preserved products from pharmaceutics, cosmetics, household cleaning articles, and beverages industries. B. contaminans (which is prevalent among people with cystic fibrosis in Argentina) was the most frequent Bcc species identified (42% of the Bcc isolates studied). B. cepacia (10%), B. cenocepacia (5%), B. vietnamiensis (16%), B. arboris (3%), and the recently defined B. aenigmatica (24%) were also detected. Rec A sequences from all B. cepacia and most B. contaminans industrial isolates obtained in this study displayed 100% identity with recA sequences from isolates infecting Argentinean patients. This information brings evidence for considering industrial massive consumption products as a potential source of Bcc infections. In addition, identification at species level in industrial microbiological laboratories is necessary for a better epidemiological surveillance. Particularly in Argentina, more studies are required in order to reveal the role of these products in the acquisition of B. contaminans infections.

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.

Biodiversity of cultivable Burkholderia species in Argentinean soils under no-till agricultural practices.

No-tillage crop production has revolutionized the agriculture worldwide. In our country more than 30 Mha are currently cultivated under no-till schemes, stressing the importance of this management system for crop production. It is widely recognized that soil microbiota is altered under different soil managements. In this regard the structure of Burkholderia populations is affected by soils management practices such as tillage, fertilization, or crop rotation. The stability of these structures, however, has not been evaluated under sustainable schemes where the impact of land practices could be less deleterious to physicochemical soils characteristics. In order to assess the structure of Burkholderia spp. populations in no-till schemes, culturable Burkholderia spp. strains were quantified and their biodiversity evaluated. Results showed that Burkholderia spp. biodiversity, but not their abundance, clearly displayed a dependence on agricultural managements. We also showed that biodiversity was mainly influenced by two soil factors: Total Organic Carbon and Total Nitrogen. Results showed that no-till schemes are not per se sufficient to maintain a richer Burkholderia spp. soil microbiota, and additional traits should be considered when sustainability of productive soils is a goal to fulfil productive agricultural schemes.

Draft Genome Sequences of Burkholderia contaminans FFI-28, a Strain Isolated from a Contaminated Pharmaceutical Solution.

Burkholderia contaminans is a species of the Burkholderia cepacia complex, a group of bacteria that can grow in pharmaceutical products and are capable of infecting the immunocompromised and people with cystic fibrosis. Here, we report draft genome sequences for Burkholderia contaminans FFI-28, a strain isolated from a contaminated pharmaceutical solution.

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Draft Genome Sequences of Burkholderia contaminans, a Burkholderia cepacia Complex Species That Is Increasingly Recovered from Cystic Fibrosis Patients.

Burkholderia contaminans belongs to the Burkholderia cepacia complex (BCC), a group of bacteria that are ubiquitous in the environment and capable of infecting the immunocompromised and people with cystic fibrosis. We report here draft genome sequences for the B. contaminans type strain LMG 23361 and an Argentinian cystic fibrosis sputum isolate.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Gibbs energy of transfer processes for the antimicrobial agent Triclosan from water to some organic solvents at 25.0 °C

The thermodynamic function Gibbs energy for the dissolution processes of triclosan (TS) was calculated from solubility values obtained at 25.0 °C in organic solvents with different hydrogen-bonding capability. TS solubility was determined in ethanol, octanol, water-saturated octanol, isopropyl myristate, chloroform, and heptane. The excess Gibbs energy and the activity coefficients of the solute were also calculated. In addition, the corresponding Gibbs energies of the drug transfer process from water to the organic solvents under investigation were also calculated by means of previous reports. In all cases, this thermodynamic property comprised a negative value, indicating the preference of TS for all the organic media evaluated.

En este trabajo se presentan las energías de Gibbs para los procesos de disolución del triclosan (TS) en solventes orgánicos de diferente capacidad de formación de enlace de hidrógeno, las cuales fueron calculadas a partir de los valores de solubilidad a 25,0 °C. La solubilidad del TS se determinó en etanol, octanol, octanol saturado de agua, miristato de isopropilo, cloroformo, y heptano. Así mismo se calcularon las energías de Gibbs de exceso y los coeficientes de actividad del soluto en los mismos solventes. Adicionalmente, mediante el uso de valores previamente reportados en la literatura, se calcularon las energías de Gibbs de transferencia del TS desde el agua hasta los solventes orgánicos comprendidos en el estudio. En todos los casos, esta propiedad termodinámica fue negativa demostrando la preferencia del TS por los medios orgánicos evaluados.

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients.

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.

Triclosan-loaded poloxamine micelles for enhanced topical antibacterial activity against biofilm.

Our research group is interested in the study of different technological approaches to treat hospital biofilm as a means to constrain nosocomial-acquired infections. The present work investigated the effect of the incorporation of the antibacterial agent triclosan (TS) into polymeric micelles of poloxamine T1107 (MW=15 kDa, 70 wt% PEO). The aggregation phenomenon was primarily investigated by means of Critical Micellar Concentration in a broad range of pH. Then, the effect of the polymer concentration on the micellar size was evaluated by Dynamic Light Scattering. Solubility levels increased up to 4 orders of magnitude. The drug inclusion affected the micellization, resulting in size increase and micellar fusion. This phenomenon was only apparent in TS-saturated systems. TS-loaded aggregates proved to be active in vitro against a broad spectrum of bacteria but more importantly, also against two representative clinical pathogens: methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF). While the former was sensitive to even very low TS levels attainable in poloxamine-free aqueous media, the later was inhibited only when exposed to higher drug levels affordable exclusively using an inclusion system. These findings indicated the release of the drug from the reservoir. Finally, the activity of a TS-containing 5% poloxamine combination of pH 7.4 was assessed on biofilms of Staphylococcus epidermidis. Results showed a significant decrease (p<0.001) in the number of Colony-Formation Units when the biofilm was exposed to the TS/poloxamine as compared to the limited activity of the polymer-free TS control.

Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media.

The polyhydroxylated silane network of a sol-gel protected immobilised Saccharomyces cerevisiae against the effects of five organic solvents. The viability of immobilised yeast directly correlated with the logarithm of the partition coefficient of the solvent in an octanol/water two phase system increasing the decimal reduction time (D) and reaching the maximum with octanol, the most hydrophobic solvent assayed. The D value increased from 0.16 min for free yeast to 1.9 and to 22 min for immobilised yeast exposed to ethanol and 1-octanol respectively.