Biofilm antimicrobial susceptibility testing: where are we and where could we be going?

Our knowledge about the fundamental aspects of biofilm biology, including the mechanisms behind the reduced antimicrobial susceptibility of biofilms, has increased drastically over the last decades. However, this knowledge has so far not been translated into major changes in clinical practice. While the biofilm concept is increasingly on the radar of clinical microbiologists, physicians, and healthcare professionals in general, the standardized tools to study biofilms in the clinical microbiology laboratory are still lacking; one area in which this is particularly obvious is that of antimicrobial susceptibility testing (AST). It is generally accepted that the biofilm lifestyle has a tremendous impact on antibiotic susceptibility, yet AST is typically still carried out with planktonic cells. On top of that, the microenvironment at the site of infection is an important driver for microbial physiology and hence susceptibility; but this is poorly reflected in current AST methods. The goal of this review is to provide an overview of the state of the art concerning biofilm AST and highlight the knowledge gaps in this area. Subsequently, potential ways to improve biofilm-based AST will be discussed. Finally, bottlenecks currently preventing the use of biofilm AST in clinical practice, as well as the steps needed to get past these bottlenecks, will be discussed.

Effect of malate on the activity of ciprofloxacin against Pseudomonas aeruginosa in different in vivo and in vivo-like infection models.

The clinical significance of Pseudomonas aeruginosa infections and the tolerance of this opportunistic pathogen to antibiotic therapy makes the development of novel antimicrobial strategies an urgent need. We previously found that D,L-malic acid potentiates the activity of ciprofloxacin against P. aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium by increasing metabolic activity and tricarboxylic acid cycle activity. This suggested a potential new strategy to improve antibiotic therapy in P. aeruginosa infections. Considering the importance of the microenvironment on microbial antibiotic susceptibility, the present study aims to further investigate the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in physiologically relevant infection models, aiming to mimic the infection environment more closely. We used Caenorhabditis elegans nematodes, Galleria mellonella larvae, and a 3-D lung epithelial cell model to assess the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa. D,L-malate was able to significantly enhance ciprofloxacin activity against P. aeruginosa in both G. mellonella larvae and the 3-D lung epithelial cell model. In addition, ciprofloxacin combined with D,L-malate significantly improved the survival of infected 3-D cells compared to ciprofloxacin alone. No significant effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in C. elegans nematodes was observed. Overall, these data indicate that the outcome of the experiment is influenced by the model system used which emphasizes the importance of using models that reflect the in vivo environment as closely as possible. Nevertheless, this study confirms the potential of D,L-malate to enhance ciprofloxacin activity against P. aeruginosa-associated infections.

Adaptation of Pseudomonas aeruginosa biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes.

In the present study we evaluated the fitness, antimicrobial susceptibility, metabolic activity, gene expression, in vitro production of virulence factors and in vivo virulence of experimentally evolved Pseudomonas aeruginosa PAO1. These strains were previously evolved in the presence of tobramycin and the quorum sensing inhibitor furanone C-30 (C-30) and carried mutations in mexT and fusA1. Compared to the wild-type (WT), the evolved strains show a different growth rate and different metabolic activity, suggesting they have an altered fitness. mexT mutants were less susceptible to C-30 than WT strains; they also show reduced susceptibility to chloramphenicol and ciprofloxacin, two substrates of the MexEF-OprN efflux pump. fusA1 mutants had a decreased susceptibility to aminoglycoside antibiotics, and an increased susceptibility to chloramphenicol. The decreased antimicrobial susceptibility and decreased susceptibility to C-30 was accompanied by a changed metabolic activity profile during treatment. The expression of mexE was significantly increased in mexT mutants and induced by C-30, suggesting that MexEF-OprN exports C-30 out of the bacterial cell. The in vitro production of virulence factors as well as virulence in two in vivo models of the strains evolved in the presence of C-30 was unchanged compared to the virulence of the WT. Finally, the evolved strains were less susceptible towards tobramycin (alone and combined with C-30) in an in vivo mouse model. In conclusion, this study shows that mutations acquired during experimental evolution of P. aeruginosa biofilms in the presence of tobramycin and C-30, are accompanied by an altered fitness, metabolism, mexE expression and in vitro and in vivo antimicrobial susceptibility.

A systematic review of the use of bacteriophages for in vitro biofilm control.

Bacteriophages (phages) are very promising biological agents for the prevention and control of bacterial biofilms. However, little is known about the parameters that can influence the efficacy of phages on biofilms. This systematic review provides a summary and analysis of the published data about the use of phages to control pre-formed biofilms in vitro, suggesting recommendations for future experiments in this area. A total of 68 articles, containing data on 605 experiments addressing the efficacy of phages to control biofilms in vitro were included, after a search conducted in Web of Science, Embase, and Medline (PubMed). The data collected from each experiment included information about biofilm growth conditions, phage characteristics, treatment conditions and biofilm reduction. In most cases, biofilms were formed in the surface of microtiter plates (82.5%); the median time for biofilm formation was 24 h, as is the median treatment duration. Quantification of biofilm biomass (52.6%), viable cells (25.5%) and metabolic activity (17.9%) were the most common biofilm assessment methods. Correlation analysis revealed that some phage parameters can influence the treatment outcome: higher phage concentrations were strongly associated with improved biofilm control, leading to higher levels of biofilm reduction, and phages with higher burst sizes and shorter latent periods seem to be the best candidates to control biofilms in vitro. However, the great variability of the methodologies used prompts the need for the development of standardized in vitro methodologies to characterize phage/biofilm interactions and to assess the efficacy of phages to control biofilms.

Pellicle Biofilm Formation in Burkholderia cenocepacia J2315 is Epigenetically Regulated through WspH, a Hybrid Two-Component System Kinase-Response Regulator.

The chemosensory signal transduction system Wsp regulates biofilm formation and related phenotypes by influencing cyclic-di-GMP (c-di-GMP) levels in bacterial cells. This is typically achieved by activation of the diguanylate cyclase WspR, through phosphorylation of its phosphoreceiver domain. The Wsp system of Burkholderia cenocepacia J2315 is in one operon with the hybrid response regulator/histidine kinase wspH, but lacks the diguanylate cyclase wspR which is located in a different operon. The expression of wspH, the first gene in the B. cenocepacia Wsp operon as well as pellicle biofilm formation are epigenetically regulated in B. cenocepacia J2315. To investigate whether WspH regulates pellicle biofilm formation, several mutants with altered expression of wspH were constructed. Mutants with increased expression of wspH showed accelerated pellicle biofilm formation, reduced swimming motility and increased c-di-GMP levels. This was independent of WspR phosphorylation, showing that WspR is not the cognate response receiver for histidine kinase WspH. IMPORTANCE Biofilms are surface-attached or suspended aggregates of cells, that are problematic in the context of bacterial infections, as they provide protection from antibiotic treatment. Burkholderia cenocepacia can colonize the lung of immunocompromised patients and forms biofilms that increase its recalcitrance to antibiotic treatment. Pellicles are biofilms which form at an air-liquid interface to take advantage of the higher oxygen concentrations in this environment. How quickly pellicles are formed is crucial for the fitness of obligate aerobic bacteria such as B. cenocepacia. Cyclic-di-GMP (c-di-GMP) levels determine the transition between planktonic and biofilm lifestyle, and WspH controls c-di-GMP production. WspH is therefore important for the fitness of B. cenocepacia in environments with gradients in oxygen concentration, such as the human lung.

Organic Acids and Their Salts Potentiate the Activity of Selected Antibiotics against Pseudomonas aeruginosa Biofilms Grown in a Synthetic Cystic Fibrosis Sputum Medium.

The failure of antibiotic therapy in respiratory tract infections in cystic fibrosis is partly due to the high tolerance observed in Pseudomonas aeruginosa biofilms. This tolerance is mediated by changes in bacterial metabolism linked to growth in biofilms, opening up potential avenues for novel treatment approaches based on modulating metabolism. The goal of the present study was to identify carbon sources that increase the inhibiting and/or eradicating activity of tobramycin, ciprofloxacin, and ceftazidime against P. aeruginosa PAO1 biofilms grown in a synthetic cystic fibrosis sputum medium (SCFM2) and to elucidate their mode of action. After screening 69 carbon sources, several combinations of antibiotics + carbon sources that showed markedly higher anti-biofilm activity than antibiotics alone were identified. d,l-malic acid and sodium acetate could potentiate both biofilm inhibiting and eradicating activity of ciprofloxacin and ceftazidime, respectively, while citric acid could only potentiate biofilm inhibitory activity of tobramycin. The mechanisms underlying the increased biofilm eradicating activity of combinations ciprofloxacin/d,l-malic acid and ceftazidime/sodium acetate are similar but not identical. Potentiation of ceftazidime activity by sodium acetate was linked to increased metabolic activity, a functional TCA cycle, increased ROS production, and high intracellular pH, whereas the latter was not required for d,l-malic acid potentiation of ciprofloxacin. Finally, our results indicate that the potentiation of antibiotic activity by carbon sources is strain dependent.

The anti-virulence activity of the non-mevalonate pathway inhibitor FR900098 towards Burkholderia cenocepacia is maintained during experimental evolution.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the ß-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several ß-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia. Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

BACKGROUND: Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood. METHODS: We hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples. RESULTS: We show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1ß) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes. CONCLUSIONS: These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

Explaining the working mechanism of laser-activated irrigation and its action on microbial biofilms: A high-speed imaging study.

AIM: Laser-activated irrigation (LAI) using pulsed erbium lasers has been studied with regard to canal cleaning, but its working mechanism remains poorly understood. This study sought to unravel the method of action of LAI and to assess its effect on bacterial biofilms in a root canal model, by means of high-speed imaging. METHODOLOGY: A root canal model consisting of dentine and glass walls was used. Visualization of the canal space during activation was achieved with a high-speed camera, capturing 20-s activation series at 50 000 frames per second. Recordings were made of canal models filled with water, models filled with water containing glass microspheres, and models with a biofilm (an undefined biofilm originating from oral samples, a 1-week-old Enterococcus faecalis biofilm or a 11-day-old multispecies biofilm) grown on the dentine walls. LAI parameters were 2940 nm, 15 Hz, 50 µs, 20 mJ and 400 µm conical tip held at orifice level. Quantitative (measurement of size, life time and timing of cavitation bubbles; velocity and amplitude of root canal content movement) and qualitative (descriptive) analysis of the intracanal events was performed using imaging software. RESULTS: During the implosion of the primary bubble, smaller cavitation bubbles emerged throughout the entire canal. This process began in the coronal canal part and continued in the apical direction. Expansion of these bubbles was followed by an implosion, and this volumetric change over a time span of a few 100 µs resulted in a very rapid vertical movement of the canal content with a mean amplitude of 900 µm. The succession of these movements with every pulse, resulted in biofilm detachment from the root canal walls and the gradual displacement of fragments coronally, until their complete removal. The pattern of the biofilm removal was the same for all groups. LAI was able to remove biofilm from the root canal models. CONCLUSIONS: The hydrodynamic effect of LAI is based on the generation of small cavitation bubbles throughout the entire canal, far from the primary bubble. Their volumetric oscillation results in a small yet very fast vertical movement of the root canal content and local liquid streaming on each pulse, resulting in biofilm detachment and coronal displacement.

The Quorum-Sensing Inhibitor Furanone C-30 Rapidly Loses Its Tobramycin-Potentiating Activity against Pseudomonas aeruginosa Biofilms during Experimental Evolution.

The use of quorum-sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop, although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the in vivo situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin would be "evolution-proof" when used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm-eradicating activity of the tobramycin/furanone C-30 combination already decreased after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in mexT, fusA1, and parS, genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in mexT was also observed. Our data indicate that furanone C-30 is not "evolution-proof" and quickly becomes ineffective as a tobramycin potentiator.

Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria.

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.

Biosynthesis of Ditropolonyl Sulfide, an Antibacterial Compound Produced by Burkholderia cepacia Complex Strain R-12632.

Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 µg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role. IMPORTANCEBurkholderia bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.

Model system parameters influence the sodium hypochlorite susceptibility of endodontic biofilms.

AIM: To evaluate in a laboratory setting the influence of several model system parameters on the sodium hypochlorite (NaOCl) susceptibility of endodontic biofilms. Based on these findings, a relevant in vitro endodontic biofilm model is proposed. METHODOLOGY: In vitro biofilms were cultured, varying the following experimental model parameters: biofilm composition (monospecies Enterococcus faecalis and a multispecies biofilm including E. faecalis, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), incubation time (24 h or 11 days), incubation atmosphere (aerobically or anaerobically) and biofilm substrate (polystyrene microtiter plate wells, hydroxyapatite or dentine). Biofilms were subjected to treatment with NaOCl (0.025%, 0.1%, 0.5%, 2.5%) for 1 min, control groups included treatment with purified water. Biofilms were harvested and the number of surviving cells was determined by plate counting using general (monospecies biofilms) or selective (multispecies biofilms) media. A two-way ANOVA was used to explore the effect of the model parameters on biofilm eradication. Finally, the most physiologically relevant biofilm model (11-day-old multispecies biofilm grown anaerobically on dentine discs) was characterized by selective media plate counting, NaOCl susceptibility testing, scanning and transmission electron microscopy. RESULTS: There was no difference in NaOCl eradication between the anaerobically and aerobically grown E. faecalis biofilms. One-day-old biofilms of E. faecalis were more susceptible to most tested NaOCl concentrations than 11-day-old biofilms (p < .05). When grown in a multispecies biofilm, E. faecalis was significantly less susceptible to NaOCl treatment than in a monospecies biofilm (p < .05). E. faecalis in a multispecies biofilm grown in a MTP was more susceptible to NaOCl (0.025% and 0.1%) than when grown on hydroxyapatite or dentine. No difference in biofilm NaOCl susceptibility was seen between hydroxyapatite and dentine. The multispecies biofilm proved to be a reproducible model with high NaOCl resistance, complex structure and organization. CONCLUSION: The parameters biofilm age, biofilm composition and substrate had a significant influence on the NaOCl susceptibility of E. faecalis biofilms. Older biofilms, multispecies biofilms and biofilms grown on dentine and hydroxyapatite had reduced NaOCl susceptibility. These findings emphasize the importance of selecting relevant parameters when designing a laboratory biofilm model system for the evaluation of antimicrobial treatments.

Dynamic Constitutional Frameworks as Antibacterial and Antibiofilm Agents.

Dynamic constitutional frameworks (DCFs) were synthesized and screened for biofilm inhibition or disruption. They are composed of a trialdehyde core reversibly linked to a diamine PEG connector and to a variety of neutral, anionic, or cationic heads, to generate a library of DCFs to generate multivalent dendritic architectures in the presence of Pseudomonas aeruginosa and Staphylococcus aureus. The best DCFs were always polycationic and the nature of the cationic heads significantly impact the antibiofilm activity. The best antibiofilm activity was observed for DCF3B, displaying a polyethyleneimine head. A simple inactive guanidinium functional head strongly inhibited biofilm growth when assayed as a multivalent DCF3C. Using a more advanced in vitro biofilm model of chronic wound infection, DCF3C was found significantly superior than all other DCFs. These results demonstrate the versatility and effectiveness of DCFs as low cost and efficient systems for antibiofilm disruption.

First Report of Candidemia Clonal Outbreak Caused by Emerging Fluconazole-Resistant Candida parapsilosis Isolates Harboring Y132F and/or Y132F+K143R in Turkey.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2 AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios.

PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection.

C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms.

Small RNA NcS27 co-regulates utilization of carbon sources in Burkholderia cenocepacia J2315.

Small non-coding sRNAs have versatile roles in regulating bacterial metabolism. Four short homologous Burkholderia cenocepacia sRNAs strongly expressed under conditions of growth arrest were recently identified. Here we report the detailed investigation of one of these, NcS27. sRNA NcS27 contains a short putative target recognition sequence, which is conserved throughout the order Burkholderiales. This sequence is the reverse complement of the Shine-Dalgarno sequence of a large number of genes involved in transport and metabolism of amino acids and carbohydrates. Overexpression of NcS27 sRNA had a distinct impact on growth, attenuating growth on a variety of substrates such as phenylalanine, tyrosine, glycerol and galactose, while having no effect on growth on other substrates. Transcriptomics and proteomics of NcS27 overexpression and silencing mutants revealed numerous predicted targets changing expression, notably of genes involved in degradation of aromatic amino acids phenylalanine and tyrosine, and in transport of carbohydrates. The conserved target recognition sequence was essential for growth phenotypes and gene expression changes. Cumulatively, our data point to a role of NcS27 in regulating the shutdown of metabolism upon nutrient deprivation in B. cenocepacia. We propose Burkholderiadouble-hairpin sRNA regulator bdhR1 as designation for ncS27.