Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z’ factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six “best in class” compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Disinfectant, Soap or Probiotic Cleaning? Surface Microbiome Diversity and Biofilm Competitive Exclusion

This study extends probiotic cleaning research to a built environment. Through an eight-month cleaning trial, we compared the effect of three cleaning products (disinfectant, plain soap, and a probiotic cleaner containing a patented Bacillus spore consortium), and tap water as the control, on the resident microbiome of three common hospital surfaces (linoleum, ceramic, and stainless steel). Pathogens, Escherichia coli and Staphylococcus aureus, were deposited and desiccated, and competitive exclusion was assessed for each microbiome. Cell survival was shown to be an incomplete tool for measuring microbial competitive exclusion. Biofilm competition offered a fuller understanding of competitive dynamics. A test for culturable cell survival showed that both plain soap and probiotic cleaner regimes established a surface microbiome that outcompeted the two pathogens. A different picture emerged when observing biofilms with a deposited and desiccated GFP-labeled pathogen, Pseudomonas aeruginosa. Competitive exclusion was again demonstrated. On surfaces cleaned with disinfectant the pathogen outcompeted the microbiomes. On surfaces cleaned with plain soap, the microbiomes outcompeted the pathogen. However, on surfaces cleaned with probiotic cleaner, despite the exponentially higher surface microbial loads, the microbiome did not completely outcompete the pathogen. Thus, the standard culturable cell test for survival on a surface confirmed the competitive advantage that is typically reported for probiotic cleaners. However, observation of competition in biofilms showed that the more diverse microbiome (according to alpha and beta indices) established on a surface cleaned with plain soap had a better competitive advantage than the monoculture established by the probiotic cleaner. Therefore, microbial diversity appears to be as critical to the competitive exclusion principle as cell numbers. The study showed that both plain soap and probiotic cleaner fostered competitive exclusion far more effectively than disinfectant. Probiotic cleaners with microbial diversity could be worth considering for hospital cleaning.

Drug screening to identify compounds to act as co-therapies for the treatment of pathogenic Burkholderia

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics, and is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to most currently available antibiotics. The most efficacious treatment for melioidosis requires at least two weeks of intravenous treatment with ceftazidime or meropenem. This places a large treatment burden on the predominantly middle income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z’ factor of 0.68. We screened a library of 61,250 compounds, and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six “best in class” compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, preliminary data also demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy against these bacteria.

Cryptococcus neoformansresist to drastic conditions by switching to viable but non-culturable cell phenotype

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce C. neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy inCryptococcus neoformans.We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC.All together, these data show that we were able to generate for the first time VBNC phenotype inCryptococcocus neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

Salmonella entericaFilamentation Induced by Pelargonic Acid Is a Transient Morphotype

ABSTRACTUnder stressful conditions,Salmonella entericaforms multinucleated elongated filaments. The triggers and outcomes of filamentation are not well characterized.S. entericaserotypes Newport, Javiana, and Typhimurium were evaluated for their ability to form filaments upon exposure to 20 mM pelargonic acid.S.Newport was used as a model to investigate the progression and fate of filamentation via culturable population size, cell length, and viability assays. All serotypes displayed filament formation after 16 h of incubation. Pelargonic acid amendment of tryptic soy broth (TSBpel) produced a 5-log CFU reduction compared to TSB after 24 h (P < 0.05), and the growth rate decreased (P < 0.02). Cell elongation started within 12 h, peaked at 16 h, and was followed by filament disintegration at 20 to 24 h. The ratio of filaments to regular-sized cells (F/R) in TSBpel was 3.87 ± 0.59 at 16 h, decreasing to 0.23 ± 0.04 and 0.03 ± 0.01 (P < 0.05) at 20 and 24 h, respectively. Mg2+supplementation repressed filamentation (F/R = 0.25 ± 0.11) and enhanced culturable cell counts (P < 0.05). Continued exposure to pelargonic acid inhibited growth in TSB and M9 compared to that in unamended media (P < 0.05). However, in M9 medium without Mg2+amended with 20 mM pelargonic acid (M9pel), filament fragmentation progressed independently of pelargonic acid or Mg2+. When cells were pretreated with pelargonic acid to induce filamentation and then transferred to fresh medium, a positive effect of Mg2+was noted under nutrient-deficient conditions, with higher live/dead cell ratios in M9 supplemented with 5 mM Mg2+(M9Mg) than in M9 (P < 0.05). No change was observed when pelargonic acid was also added. Filamentation was ubiquitous in all serotypes tested, transient, and sensitive to Mg2+. Fragmentation, but not recovery, progressed irrespective of antimicrobial or Mg2+presence.IMPORTANCESome bacteria form elongated multinucleated structures, or filaments, when exposed to stress. The filamentous form of foodborne bacterial pathogens can interfere with food protection practices and diagnostic testing. Filamentation inSalmonella entericaNewport was investigated in response to pelargonic acid, a compound naturally found in several fruit and vegetables, and also used commercially as an herbicide.Salmonellareadily formed filaments when exposed to pelargonic acid. Filaments were not stable, however, and fragmented to individual cells even when the fatty acid was still present, recovering fully when the stress was alleviated. A deeper exploration of the molecular mechanisms regulating filamentation and the conditions that induce it in agriculture and the food supply chain is needed to devise strategies that curb this response.

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

ABSTRACT The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.