Increased tolerance to commonly used antibiotics in a Pseudomonas aeruginosa ex vivo porcine keratitis model.

Introduction. Bacterial keratitis, particularly caused by Pseudomonas aeruginosa, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo.Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.Methodology. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model.Results. Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l-1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l-1) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.

Phage-based therapy against biofilm producers in gram-negative ESKAPE pathogens.

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5-1 µg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803.

Cyanobacterial alternative sigma factors are crucial players in environmental adaptation processes, which may involve bacterial responses related to maintenance of cell envelope and control of secretion pathways. Here, we show that the Group 3 alternative sigma factor F (SigF) plays a pleiotropic role in Synechocystis sp. PCC 6803 physiology, with a major impact on growth and secretion mechanisms, such as the production of extracellular polysaccharides, vesiculation and protein secretion. Although ΔsigF growth was significantly impaired, the production of released polysaccharides (RPS) increased threefold to fourfold compared with the wild-type. ΔsigF exhibits also impairment in formation of outer-membrane vesicles (OMVs) and pili, as well as several other cell envelope alterations. Similarly, the exoproteome composition of ΔsigF differs from the wild-type both in amount and type of proteins identified. Quantitative proteomics (iTRAQ) and an in silico analysis of SigF binding motifs revealed possible targets/pathways under SigF control. Besides changes in protein levels involved in secretion mechanisms, our results indicated that photosynthesis, central carbon metabolism and protein folding/degradation mechanisms are altered in ΔsigF. Overall, this work provided new evidences about the role of SigF on Synechocystis physiology and associates this regulatory element with classical and non-classical secretion pathways.

Enumeration of sulphate-reducing bacteria for assessing potential for hydrogen sulphide production in urban drainage systems.

Urban drainage structures have increasing demands which can lead to increasing hydrogen sulphide related problems forming in places where they have not previously been prevalent. This puts pressure on the methods currently used to monitor and diagnose these problems and more sophisticated methods may be needed for identifying the origin of the problems. Molecular microbiological techniques, such as quantitative polymerase chain reaction, offer a potential alternative for identifying and quantifying bacteria likely to be causing the production of hydrogen sulphide, information that, when combined with an appropriate sampling programme, can then be used to identify the potentially most effective remediation technique. The application of these methods in urban drainage systems is, however, not always simple, but good results can be achieved. In this study bacteria producing hydrogen sulphide were quantified in three small combined sewer overflow storage tanks. Bacterial counts were compared between wastewater, biofilms and sediments. Similar numbers were found in the wastewater and biofilms, with the numbers in the sediments being lower. If remediation methods for hydrogen sulphide are deemed necessary in the tanks, methods that target both the wastewater and the biofilms should therefore be considered.

Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.

Development of wastewater treatment system based on cascade dielectric barrier discharge plasma atomizers.

A novel design for a cascade dielectric barrier discharge (DBD) atomizer was applied for treating samples of water containing biological and organic contaminants. Several experimental investigations were conducted on artificial samples and real sample (digested sludge collected from a wastewater treatment plant, WWTP). The artificial water samples were prepared by using different concentrations of E. coli for biological samples, whereas acetic acid was used to prepare the organic samples. The biological samples were subjected to the plasma effect for different treatment periods, and the growth curves of E. coli were generated for 24 h after treatment. Moreover, the viable cells were counted after each treatment period and the change in E. coli morphology was monitored. The results showed that a significant reduction in the viable cell number, by 3 orders of magnitude, occurred for an artificial biological sample after only 5-min treatment. The treatment of organic samples for 10 min showed a significant reduction in the concentration of acetic acid by 50%. In consequence, treatment of real wastewater sample for 10 min resulted in more than 70% reduction in BOD5 and 30% reduction in COD.

Bottom-up construction and screening of algae-bacteria consortia for pollutant biodegradation.

Microbial communities have been used as important biological tools for a variety of purposes associated with agriculture, the food industry and human health. Artificial engineering of microbial communities is an emerging field of research motivated by finding stable and efficient microbial systems. However, the successful design of microbial communities with desirable functions not only requires profound understanding of microbial activities, but also needs efficient approaches to piece together the known microbial traits to give rise to more complex systems. This study demonstrates the bottom-up integration of environmentally isolated phototrophic microalgae and chemotrophic bacteria as artificial consortia to bio-degrade selected volatile organic compounds (VOCs). A high throughput screening method based on 96-well plate format was developed for discovering consortia with bioremediation potential. Screened exemplar consortia were verified for VOCs degradation performance, among these, certain robust consortia were estimated to have achieved efficiencies of 95.72% and 92.70% and near 100% removal (7 days) of benzene, toluene, and phenol, respectively, with initial concentrations of 100 mg/L. VOCs degradation by consortia was mainly attributed to certain bacteria including Rhodococcus erythropolis, and Cupriavidus metallidurans, and directly contributed to the growth of microalgae Coelastrella terrestris (R = 0.82, p < 0.001). This work revealed the potential of converting VOCs waste into algal biomass by algae-bacteria consortia constructed through a bottom-up approach. The screening method enables rapid shortlisting of consortia combinatorial scenarios without prior knowledge about the individual strains or the need for interpreting complex microbial interactions.

Integrating molecular microbial methods to improve faecal pollution management in rivers with designated bathing waters.

Rivers are at risk from a variety of pollution sources. Faecal pollution is of particular concern since it disperses pathogenic microorganisms in the aquatic environment. Currently, faecal pollution levels in rivers is monitored using faecal indicator bacteria (FIB) that do not offer information about pollution sources and associated risks. This study used a combined molecular approach, along with measurements of water quality, to gain information on pollution sources, and risk levels, in a newly designated recreational bathing site in the River Wharfe (UK). Physico-chemical parameters were monitored in situ, with water quality multiparameter monitoring sondes installed during the 2021 bathing season. The molecular approach was based on quantitative PCR (qPCR)-aided Microbial Source Tracking (MST) and 16S rRNA gene metabarcoding to obtain a fingerprint of bacterial communities and identify potential bioindicators. The analysis from the water quality sondes showed that ammonium was the main parameter determining the distribution of FIB values. Lower faecal pollution levels were detected in the main river when compared to tributaries, except for samples in the river located downstream of a wastewater treatment plant. The faecal pollution type (anthropogenic vs. zoogenic) changed the diversity and the structure of bacterial communities, giving a distinctive fingerprint that can be used to inform source. DNA-based methods showed that the presence of human-derived bacteria was associated with Escherichia coli spikes, coinciding with higher bacterial diversity and the presence of potential pathogenic bacteria mainly of the genus Mycobacterium, Aeromonas and Clostridium. Samples collected after a heavy rainfall event were associated with an increase in Bacteroidales, which are markers of faecal pollution, including Bacteroides graminisolvens, a ruminant marker associated with surface run-off from agricultural sources. The combined use of qPCR and 16S rRNA sequencing was able to identify pollution sources, and novel bacterial indicators, thereby aiding decision-making and management strategies in recreational bathing rivers.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG.

BACKGROUND: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. RESULTS: The cells were grown at pH 5, 5.5, 6 (temperature 37°C) and at pH 6.5 (temperature 25°C, 30°C and 37°C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37°C). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. CONCLUSIONS: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

Using a multi-faceted approach to determine the changes in bacterial cell surface properties influenced by a biofilm lifestyle.

Biofilm formation is a developmental process in which initial reversible adhesion is governed by physico-chemical forces, whilst irreversible adhesion is mediated by biological changes within a cell, such as the production of extracellular polymeric substances. Using two bacteria, E. coli MG1655 and B. cereus ATCC 10987, this study establishes that the surface of the bacterial cell also undergoes specific modifications, which result in biofilm formation and maintenance. Using various surface characterisation techniques and proteomics, an increase in the surface exposed proteins on E. coli cells during biofilm formation was demonstrated, along with an increase in hydrophobicity and a decrease in surface charge. For B. cereus, an increase in the surface polysaccharides during biofilm formation was found as well as a decrease in hydrophobicity and surface charge. This work therefore shows that surface modifications during biofilm formation occur and understanding these specific changes may lead to the formulation of effective biofilm control strategies in the future.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen's ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

Macromolecular fingerprinting of sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis.

Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ~3.4 and ~1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development.

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins.

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.

"Biofilmology": a multidisciplinary review of the study of microbial biofilms.

The observation of biofilm formation is not a new phenomenon. The prevalence and significance of biofilm and aggregate formation in various processes have encouraged extensive research in this field for more than 40 years. In this review, we highlight techniques from different disciplines that have been used to successfully describe the extracellular, surface and intracellular elements that are predominant in understanding biofilm formation. To reduce the complexities involved in studying biofilms, researchers in the past have generally taken a parts-based, disciplinary specific approach to understand the different components of biofilms in isolation from one another. Recently, a few studies have looked into combining the different techniques to achieve a more holistic understanding of biofilms, yet this approach is still in its infancy. In order to attain a global understanding of the processes involved in the formation of biofilms and to formulate effective biofilm control strategies, researchers in the next decade should recognise that the study of biofilms, i.e. biofilmology, has evolved into a discipline in its own right and that mutual cooperation between the various disciplines towards a multidisciplinary research vision is vital in this field.

Adapting the algal microbiome for growth on domestic landfill leachate.

We aimed to improve algal growth rate on leachate by optimising the algal microbiome. An algal-bacterial consortium was enriched from landfill leachate and subjected to 24 months of adaptive laboratory evolution, increasing the growth rate of the dominant algal strain, Chlorella vulgaris, almost three-fold to 0.2 d-1. A dramatic reduction in nitrate production suggested a shift in biological utilisation of ammoniacal-N, supported by molecular 16S rRNA taxonomic analyses, where Nitrosomonas numbers were not detected in the adapted consortium. A PICRUSt approach predicted metagenomic functional content and revealed a high number of sequences belonging to bioremediation pathways, including degradation of aromatic compounds, benzoate and naphthalene, as well as pathways known to be involved in algal-bacterial symbiosis. This study enhances our understanding of beneficial mechanisms in algal-bacterial associations in complex effluents, and ultimately enables the bottom-up design of optimised algal microbiomes for exploitation within industry.

Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Influence of phosphate dosing on biofilms development on lead in chlorinated drinking water bioreactors.

Phosphate dosing is used by water utilities to prevent plumbosolvency in water supply networks. However, there is a lack of knowledge regarding biofilm formation on lead and plastic materials when phosphate concentrations are modified in drinking water systems. In this study, biofilms were grown over lead coupons and PVC tubes in bioreactors supplied with local drinking water treated to provide different phosphate doses (below 1, 1 and 2 mg/L) over a period of 28 days. A range of commercial iron pellets (GEH104 and WARP) were tested aiming to maintain phosphate levels below the average 1 mg/L found in drinking water. Changes in biofilm community structure in response to three different phosphate treatments were characterised by Illumina sequencing of the 16S rRNA gene for bacteria and the ITS2 gene for fungi. Scanning electron microscopy was used to visualise physical differences in biofilm development in two types of materials, lead and PVC. The experimental results from the kinetics of phosphate absorption showed that the GEH104 pellets were the best option to, in the long term, reduce phosphate levels while preventing undesirable turbidity increases in drinking water. Phosphate-enrichment promoted a reduction of bacterial diversity but increased that of fungi in biofilms. Overall, higher phosphate levels selected for microorganisms with enhanced capabilities related to phosphorus metabolism and heavy metal resistance. This research brings new insights regarding the influence of different phosphate concentrations on mixed-species biofilms formation and drinking water quality, which are relevant to inform best management practices in drinking water treatment.

Characterization of Ocular Clinical Isolates of Pseudomonas aeruginosa from Non-Contact Lens Related Keratitis Patients from South India.

P. aeruginosa is the most common Gram-negative organism causing bacterial keratitis. Pseudomonas utilizes various virulence mechanisms to adhere and colonize in the host tissue. In the present study, we examined virulence factors associated with thirty-four clinical P. aeruginosa isolates collected from keratitis patients seeking care at L V Prasad Eye Institute, Hyderabad. The virulence-associated genes in all the isolates were genotyped and characteristics such as antibiotic susceptibility, biofilm formation, swarming motility, pyoverdine production and cell cytotoxicity were analyzed. All the isolates showed the presence of genes related to biofilm formation, alkaline proteases and elastases; however, there was a difference in the presence of genes related to the type III secretion system (T3SS). A higher prevalence of exoU+ genotype was noted in the drug-resistant isolates. All the isolates were capable of forming biofilms and more than 70% of the isolates showed good swarming motility. Pyoverdine production was not associated with the T3SS genotype. In the cytotoxicity assay, the presence of exoS, exoU or both resulted in higher cytotoxicity compared to the absence of both the genes. Overall, our results suggest that the T3SS profile is a good indicator of P. aeruginosa virulence characteristics and the isolates lacking the effector genes may have evolved alternate mechanisms of colonization in the host.