Modeling Microbial Community Networks: Methods and Tools for Studying Microbial Interactions.

Microbial interactions function as a fundamental unit in complex ecosystems. By characterizing the type of interaction (positive, negative, neutral) occurring in these dynamic systems, one can begin to unravel the role played by the microbial species. Towards this, various methods have been developed to decipher the function of the microbial communities. The current review focuses on the various qualitative and quantitative methods that currently exist to study microbial interactions. Qualitative methods such as co-culturing experiments are visualized using microscopy-based techniques and are combined with data obtained from multi-omics technologies (metagenomics, metabolomics, metatranscriptomics). Quantitative methods include the construction of networks and network inference, computational models, and development of synthetic microbial consortia. These methods provide a valuable clue on various roles played by interacting partners, as well as possible solutions to overcome pathogenic microbes that can cause life-threatening infections in susceptible hosts. Studying the microbial interactions will further our understanding of complex less-studied ecosystems and enable design of effective frameworks for treatment of infectious diseases.

A Comprehensive Bioinformatics Resource Guide for Genome-Based Antimicrobial Resistance Studies.

The use of high-throughput sequencing technologies and bioinformatic tools has greatly transformed microbial genome research. With the help of sophisticated computational tools, it has become easier to perform whole genome assembly, identify and compare different species based on their genomes, and predict the presence of genes responsible for proteins, antimicrobial resistance, and toxins. These bioinformatics resources are likely to continuously improve in quality, become more user-friendly to analyze the multiple genomic data, efficient in generating information and translating it into meaningful knowledge, and enhance our understanding of the genetic mechanism of AMR. In this manuscript, we provide an essential guide for selecting the popular resources for microbial research, such as genome assembly and annotation, antibiotic resistance gene profiling, identification of virulence factors, and drug interaction studies. In addition, we discuss the best practices in computer-oriented microbial genome research, emerging trends in microbial genomic data analysis, integration of multi-omics data, the appropriate use of machine-learning algorithms, and open-source bioinformatics resources for genome data analytics.

Nanoparticle-mediated amelioration of drought stress in plants: a systematic review.

Drought stress remains one of the most detrimental environmental constraints that hampers plant growth and development resulting in reduced yield and leading to economic losses. Studies have highlighted the beneficial role of carbon-based nanomaterials (NMs) such as multiwalled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), graphene, fullerene, and metal-based nanoparticles (NPs) (Ag, Au, Cu, Fe2O3, TiO2, and ZnO) in plants under unfavorable conditions such as drought. NPs help plants cope with drought by improving plant growth indices and enhancing biomass. It improves water and nutrient uptake and utilization. It helps retain water by altering the cell walls and regulating stomatal closure. The photosynthetic parameters in NP-treated plants reportedly improved with the increase in pigment content and rate of photosynthesis. Due to NP exposure, the activation of enzymatic and nonenzymatic antioxidants has reportedly improved. These antioxidants play a significant role in the defense system against stress. Studies have reported the accumulation of osmolytes and secondary metabolites. Osmolytes scavenge reactive oxygen species, which can cause oxidative stress in plants. Secondary metabolites are involved in the water retention process, thus improving plant coping strategies with stress. The deleterious effects of drought stress are alleviated by reducing malondialdehyde resulting from lipid peroxidation. Reactive oxygen species accumulation is also controlled with NP treatment. Furthermore, NPs have been reported to regulate the expression of drought-responsive genes and the biosynthesis of phytohormones such as abscisic acid, auxin, gibberellin, and cytokinin, which help plants defend against drought stress. This study reviewed 72 journal articles from 192 Google Scholar, ScienceDirect, and PubMed papers. In this review, we have discussed the impact of NP treatment on morphological, physio-biochemical, and molecular responses in monocot and dicot plants under drought conditions with an emphasis on NP uptake, transportation, and localization.

Emerging Pathogens in Planetary Health and Lessons from Comparative Genome Analyses of Three Clostridia Species.

Clostridioides difficile (CD) is a major planetary health burden. A Gram-positive opportunistic pathogen, CD, colonizes the large intestine and is implicated in sepsis, pseudomembranous colitis, and colorectal cancer. C. difficile infection typically following antibiotic exposure results in dysbiosis of the gut microbiome, and is one of the leading causes of diarrhea in the elderly population. While several studies have focused on the toxigenic strains of CD, gut commensals such as Clostridium butyricum (CB) and Clostridium tertium (CT) could harbor toxin/virulence genes, and thus pose a threat to human health. In this study, we sequenced and characterized three isolates, namely, CT (MALS001), CB (MALS002), and CD (MALS003) for their antimicrobial, cytotoxic, antiproliferative, genomic, and proteomic profiles. Although in vitro cytotoxic and antiproliferative potential were observed predominantly in CD MALS003, genome analysis revealed pathogenic potential of CB MALS002 and CT MALS001. Pangenome analysis revealed the presence of several accessory genes typically involved in fitness, virulence, and resistance characteristics in the core genomes of sequenced strains. The presence of an array of virulence and antimicrobial resistance genes in CB MALS002 and CT MALS001 suggests their potential role as emerging pathogens with significant impact on planetary health.

How Can Omics Inform Diabetic Foot Ulcer Clinical Management? A Whole Genome Comparison of Four Clinical Strains of Staphylococcus aureus.

Foot ulcers and associated infections significantly contribute to morbidity and mortality in diabetes. While diverse pathogens are found in the diabetes-related infected ulcers, Staphylococcus aureus remains one of the most virulent and widely prevalent pathogens. The high prevalence of S. aureus in chronic wound infections, especially in clinical settings, is attributed to its ability to evolve and acquire resistance against common antibiotics and to elicit an array of virulence factors. In this study, whole genome comparison of four strains of S. aureus (MUF168, MUF256, MUM270, and MUM475) isolated from diabetic foot ulcer (DFU) infections showing varying resistance patterns was carried out to study the genomic similarity, antibiotic resistance profiling, associated virulence factors, and sequence variations in drug targets. The comparative genome analysis showed strains MUM475 and MUM270 to be highly resistant, MUF256 with moderate levels of resistance, and MUF168 to be the least resistant. Strain MUF256 and MUM475 harbored more virulence factors compared with other two strains. Deleterious sequence variants were observed suggesting potential role in altering drug targets and drug efficacy. This comparative whole genome study offers new molecular insights that may potentially inform evidence-based diagnosis and treatment of DFUs in the clinic.

Gut microbiota-derived metabolites and their importance in neurological disorders.

Microbial-derived metabolites are the intermediate or end products of bacterial digestion. They are one of the most important molecules for the gut to connect with the brain. Depending on the levels of specific metabolites produced in the host, it can exert beneficial or detrimental effects on the brain and have been linked to several neurodegenerative and neuropsychiatric disorders. However, the underlying mechanisms remain largely unexplored. Insight into these mechanisms could reveal new pathways or targets, resulting in novel treatment approaches targeting neurodegenerative diseases. We have reviewed selected metabolites, including short-chain fatty acids, aromatic amino acids, trimethylamine-N-oxide,  urolithin A, anthocyanins, equols, imidazole, and propionate to highlight their mechanism of action, underlying role in maintaining intestinal homeostasis and regulating neuro-immunoendocrine function. Further discussed on  how altered metabolite levels can influence the gut-brain axis could lead to new prevention strategies or novel treatment approaches to neural disorders.

Quorum-sensing regulation of virulence factors in bacterial biofilm.

Chronic polymicrobial wound infections are often characterized by the presence of bacterial biofilms. They show considerable structural and functional heterogeneity, which influences the choice of antimicrobial therapy and wound healing dynamics. The hallmarks of biofilm-associated bacterial infections include elevated antibiotic resistance and extreme pathogenicity. Biofilm helps bacteria to evade the host defense mechanisms and persist longer in the host. Quorum-sensing (QS)-mediated cell signaling primarily regulates biofilm formation in chronic infections and plays a major role in eliciting virulence. This review focuses on the QS mechanisms of two major bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa and explains how they interact in the wound microenvironment to regulate biofilm development and virulence. The review also provides an insight into the treatment modalities aimed at eradicating polymicrobial biofilms. This information will help us develop better diagnostic modalities and devise effective treatment regimens to successfully manage and overcome severe life-threatening bacterial infections.

Exploring the signature gut and oral microbiome in individuals of specific Ayurveda prakriti.

Diagnosis and treatment of various diseases in Ayurveda, the Indian system of medicine, relies on 'prakriti' phenotyping of individuals into predominantly three constitutions, kapha, pitta and vata. Recent studies propose that microbiome play an integral role in precision medicine. A study of the relationship between prakriti - the basis of personalized medicine in Ayurveda and that of gut microbiome, and possible biomarker of an individual's health, would vastly improve precision therapy. Towards this, we analyzed bacterial metagenomes from buccal (oral microbiome) and fecal (gut microbiome) samples of 272 healthy individuals of various predominant prakritis. Major bacterial genera from gut microbiome included Prevotella, Bacteroides and Dialister while oral microbiome included Streptococcus, Neisseria, Veilonella, Haemophilus, Porphyromonas and Prevotella. Though the core microbiome was shared across all individuals, we found prakriti specific signatures such as preferential presence of Paraprevotella and Christensenellaceae in vata individuals. A comparison of core gut microbiome of each prakriti with a database of 'healthy' microbes identified microbes unique to each prakriti with functional roles similar to the physiological characteristics of various prakritis as described in Ayurveda. Our findings provide evidence to Ayurvedic interventions based on prakriti phenotyping and possible microbial biomarkers that can stratify the heterogenous population and aid in precision therapy.

Photodynamic therapy to control microbial biofilms.

Microorganisms thrive in well-organized biofilm ecosystems. Biofilm-associated cells typically show increased resistance to antibiotics and contribute significantly to treatment failure. This has prompted investigations aimed at developing advanced and novel antimicrobial approaches that could effectively overcome the shortcomings associated with conventional antibiotic therapy. Studies are ongoing to develop effective curative strategies ranging from the use of peptides, small molecules, nanoparticles to bacteriophages, sonic waves, and light energy targeting various structural and physiological aspects of biofilms. In photodynamic therapy, a light source of a specific wavelength is used to irradiate non-toxic photosensitizers such as tetrapyrroles, synthetic dyes or, naturally occurring compounds to generate reactive oxygen species that can exert a lethal effect on the microbe especially by disrupting the biofilm. The photosensitizer preferentially binds to and accumulates in the microbial cells without causing any damage to the host tissue. Currently, photodynamic therapy is increasingly being used for the treatment of oral caries and dental plaque, chronic wound infections, infected diabetic foot ulcers, cystic fibrosis, chronic sinusitis, implant device-associated infections, etc. This approach is recognized as safe, as it is non-toxic and minimally invasive, making it a reliable, realistic, and promising therapeutic strategy for reducing the microbial burden and biofilm formation in chronic infections. In this review article, we discuss the current and future potential strategies of utilizing photodynamic therapy to extend our ability to impede and eliminate biofilms in various medical conditions.

ROS-dependent DNA damage and repair during germination of NaCl primed seeds.

Reactive oxygen species (ROS) generated during rehydration of seeds is a major source of cellular damage. Successful germination depends on maintaining the oxidative window and ability of the cells to repair the DNA damage accumulated during seed developmental process, maturational drying, and germination. We explored the role of DNA damage, repair, cell cycle progression and antioxidant machinery in germination of seeds of Solanum melongena L. primed with 0, 320, 640 and 1200 mM sodium chloride (NaCl). The expression of antioxidant genes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase2 (CAT2), and glutathione reductase (GR) was upregulated to maintain the oxidative window required for germination in seeds treated with 320 mM NaCl. ROS generated upon treatment with 320 mM NaCl resulted in minimal DNA damage and activated non-homologous end joining (NHEJ) and mismatch repair (MMR) pathway genes such as KU70 and mutS homolog 2 (MSH2) respectively. Treatment with higher concentrations of NaCl resulted in increased DNA damage despite lower ROS, without evoking DNA repair mechanisms. Uncontrolled rehydration resulted in higher levels of ROS and DNA damage, but activation of homologous recombination (HR) pathway gene, Nijmegen breakage syndrome 1 (NBS1), and genes involved in repairing oxidized guanine, such as oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA). In summary, controlled rehydration with 320 mM NaCl decreased the DNA damage, reactivated the antioxidant and DNA repair machinery, and cell cycle progression, thereby enhancing the seed germination.

DNA demethylation overcomes attenuation of colchicine biosynthesis in an endophytic fungus Diaporthe.

Fungal endophytes, a major component of the plant host microbiome, are known to synthesize plant-derived metabolites in vitro. However, attenuation of metabolite production upon repeated sub-culturing is a major drawback towards utilizing them as an alternative for plant-derived metabolites. In this study, we isolated Diaporthe perseae, a fungal endophyte from Gloriosa superba tubers, which showed the production of colchicine in axenic cultures. Mass spectrometry, Nuclear Magnetic Resonance spectroscopy, and tubulin polymerization assays confirmed the compound to be colchicine. Repeated sub-culturing of the endophyte for 10 generations led to a reduction in the yield of the metabolite from 55.25 µg/g to 2.32 µg/g of mycelial dry weight. Treatment of attenuated cultures with DNA methylation inhibitor 5-azacytidine resulted in increased metabolite concentration (39.68 µg/g mycelial dry weight) in treated samples compared to control (2.61 µg/g mycelial dry weight) suggesting that 5-azacytidine can induce demethylation of the fungal genome to overcome the phenomenon of attenuation of metabolite synthesis. Reduced levels of global methylation were observed upon 5-azacytidine treatment in attenuated cultures (0.41 % of total cytosines methylated) as compared to untreated control (0.78 % of total cytosines methylated). The results provide a significant breakthrough in utilizing fungal endophytes as a veritable source of plant-derived metabolites from critically endangered plants.

Pseudomonas aeruginosa virulence proteins pseudolysin and protease IV impede cutaneous wound healing.

The intricate biological process of cutaneous wound healing is achieved through precise and highly programmed events. Dermal fibroblasts and keratinocytes play a significant role in the process of reepithelialization during wound healing. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) may delay the proliferative phase of wound repair by secreting their proteins leading to delayed or impaired wound healing. We have analyzed three virulent strains of P. aeruginosa isolated from the wound environment which also differed in their ability to produce biofilms. Mass spectrometric analysis of differentially expressed secreted proteins by three virulent strains of P. aeruginosa revealed peptides from pseudolysin and protease IV expressed from lasB and prpL genes. Pseudolysin and protease IV recombinant proteins were tested for their ability to modulate wound healing in several cell types of wound microenvironment in in vitro and in vivo models. Both pseudolysin and protease IV inhibited migration and survival of fibroblasts, keratinocytes, and endothelial cells. In three dimensional spheroid endothelial models and matrigel assays these proteins impeded sprouting and tube formation. In a mouse model of excision wound, pseudolysin and protease IV treatment showed reduced collagen content, inhibited neovascularization and epithelialization, and delayed wound contraction. Furthermore, pseudolysin and protease IV treatment resulted in a significant increase in plasma IL-6 levels when compared to vehicle control and control, suggesting the induction of a state of prolonged inflammation. Taken together, our data indicate pseudolysin and protease IV secreted from biofilm producing and antibiotic resistant P. aeruginosa in wound microenvironment produce both local and systemic effects that is detrimental to the maintenance of tissue homeostasis. Hence, these proteins may serve as potential therapeutic targets toward better clinical management of wounds.

Virulence factors and clonal diversity of Staphylococcus aureus in colonization and wound infection with emphasis on diabetic foot infection.

Foot ulcer is a common complication in diabetic subjects and infection of these wounds contributes to increased rates of morbidity and mortality. Diabetic foot infections are caused by a multitude of microbes and Staphylococcus aureus, a major nosocomial and community-associated pathogen, significantly contributes to wound infections as well. Staphylococcus aureus is also the primary pathogen commonly associated with diabetic foot osteomyelitis and can cause chronic and recurrent bone infections. The virulence capability of the pathogen and host immune factors can determine the occurrence and progression of S. aureus infection. Pathogen-related factors include complexity of bacterial structure and functional characteristics that provide metabolic and adhesive properties to overcome host immune response. Even though, virulence markers and toxins of S. aureus are broadly similar in different wound models, certain distinguishing features can be observed in diabetic foot infection. Specific clonal lineages and virulence factors such as TSST-1, leukocidins, enterotoxins, and exfoliatins play a significant role in determining wound outcomes. In this review, we describe the role of specific virulence determinants and clonal lineages of S. aureus that influence wound colonization and infection with special reference to diabetic foot infections.

Species-specific genomic sequences for classification of bacteria.

Modern bacterial classification relies on genomic relatedness. Genetic variation in bacterial populations present a big challenge for taxonomic classification and recently several bacterial species have been reclassified based on the intra-species genome comparison. These were facilitated by next generation sequencing technologies and advances in genome comparison approaches which led to the rearrangement of diverse bacterial species and revolution in the microbial classification system. One of the outcome of these studies is the development of suitable DNA barcodes as reliable and cost-effective method for identifying various bacterial genera. Towards refining this further, we have applied a genome comparison approach in 1104 bacterial genome assemblies (excluding plasmids) to identify unique genomic segments among intra-species genome assemblies. Using extensive bioinformatics analysis, we have identified species-specific genomic regions and designed unique primers for 100 different species (belonging to 62 genera) which includes 62 pathogenic and 13 opportunistic pathogenic bacterial species and built a database (http://slsdb.manipal.edu/bact/). These species-specific genomic regions will have a major impact on in silico and molecular methods aimed at bacterial classification and identification. These may also serve as better DNA barcodes than the markers currently used for delineation of bacteria and may also find application in various translational research programs.

Prakriti phenotypes as a stratifier of gut microbiome: A new frontier in personalized medicine?

Ayurveda has a rich history and its significance woven deeply in the Indian culture. The concept of prakriti (a person's "nature" or constitutional type determined by the proportion of three doshas, namely - vata, pitta and kapha) in Ayurveda is deeply rooted in personalized health management. While the attributes of prakriti has been established to have a genomic basis, there is dearth of elaborate evidences linking prakriti with manifestation of diseases. Next generation sequencing studies have provided a causal link between variation in the gut microbiome and its effect on an individual's fitness. Separately, reports have identified gut microbial patterns associated with several host variables such as geography, age, diet and extreme prakriti phenotypes. Recently, few reports have identified a "core gut microbiome" consisting of Bacteroides, Faecalibacterium, Prevotella and Ruminococcus prevalent across the Indian population; however, a few bacterial genera were specifically enriched in certain prakritis. Hence, in this review we aim to analyse the role of prakriti variations on dysbiosis of the gut microbiome and concomitantly its effect on human health. We suggest that prakriti phenotyping can function as a potential stratifier of the gut microbiome in a given population and may provide evidence for the conceptual framework of personalized medicine in Ayurvedic system of medicine.

Microbial Community Distribution and Core Microbiome in Successive Wound Grades of Individuals with Diabetic Foot Ulcers.

Diabetic foot ulcer (DFU) is a major complication of diabetes with high morbidity and mortality rates. The pathogenesis of DFUs is governed by a complex milieu of environmental and host factors. The empirical treatment is initially based on wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time-consuming. Hence, a thorough and rapid analysis of the microbial landscape is a major requirement toward devising evidence-based interventions. Toward this, 122 wound (100 diabetic and 22 nondiabetic) samples were sampled for their bacterial community structure using both culture-based and next-generation 16S rRNA-based metagenomics approach. Both the approaches showed that the Gram-negative microbes were more abundant in the wound microbiome. The core microbiome consisted of bacterial genera, including Alcaligenes, Pseudomonas, Burkholderia, and Corynebacterium in decreasing order of average relative abundance. Despite the heterogenous nature and extensive sharing of microbes, an inherent community structure was apparent, as revealed by a cluster analysis based on Euclidean distances. Facultative anaerobes (26.5%) were predominant in Wagner grade 5, while strict anaerobes were abundant in Wagner grade 1 (26%). A nonmetric dimensional scaling analysis could not clearly discriminate samples based on HbA1c levels. Sequencing approach revealed the presence of major culturable species even in samples with no bacterial growth in culture-based approach. Our study indicates that (i) the composition of core microbial community varies with wound severity, (ii) polymicrobial species distribution is individual specific, and (iii) antibiotic susceptibility varies with individuals. Our study suggests the need to evolve better-personalized care for better wound management therapies.IMPORTANCE Chronic nonhealing diabetic foot ulcers (DFUs) are a serious complication of diabetes and are further exacerbated by bacterial colonization. The microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance, and virulence. Treatment involves empirical decisions until definitive results on the causative wound pathogens and their antibiotic susceptibility profiles are available. Hence, there is a need for rapid and accurate detection of these polymicrobial communities for effective wound management. Deciphering microbial communities will aid clinicians to tailor their treatment specifically to the microbes prevalent in the DFU at the time of assessment. This may reduce DFUs associated morbidity and mortality while impeding the rise of multidrug-resistant microbes.

Deciphering biophysical signatures for microbiological applications.

Identification and classification of microbes are vital for maintenance of normal and altered state of human health and have applications in pharmaceutical industries, food processing, clinical analysis, and treatment. Development of methods aimed towards achieving these goals must be rapid and reliable. Conventional physiochemical and morphology-based methods of identification are often ambiguous, while newer molecular methods such as flow cytometry and polymerase chain reaction, though reliable, are time and resource intensive. Spectroscopic methods provide advantages over conventional methods as these can be fast, non-destructive, and highly specific. Surface charge of bacteria is an important parameter which can reveal composition of cell wall and is attributed to the presence of carboxyl and phosphoryl groups. Interaction of the cell with the solvent and response to various stresses can hence be measured by the changes in surface charge. In this study, we have obtained auto-fluorescence spectra (tryptophan) and dynamic light scattering (DLS) measurements from common pathogenic strains of Pseudomonas aeruginosa and Staphylococcus aureus. Fluorescence emission spectra were obtained in the range of 300-550 nm at excitation wavelength of 280 nm and DLS measurements comprised zeta potential and size parameters. Both types of measurements were performed in physiological and stress-induced conditions such as heat, sonication, and antibiotic treatment with vancomycin and cetylpyridinium chloride. Effects of these antibiotics on membrane integrity and cell viability, as obtained by DLS measurements, were statistically significant and comparable with conventional methods. Multivariate analysis enabled clustering of 83% of the samples at the genera level, based on variances from auto-fluorescence and DLS measurements.

Genome-based taxonomic classification.

Bacterial populations are routinely characterized based on microscopic examination, colony formation, and biochemical tests. However, in the recent past, bacterial identification, classification, and nomenclature have been strongly influenced by genome sequence information. Advances in bioinformatics and growth in genome databases has placed genome-based metadata analysis in the hands of researchers who will require taxonomic experience to resolve intricacies. To achieve this, different tools are now available to quantitatively measure genome relatedness within members of the same species, and genome-wide average nucleotide identity (gANI) is one such reliable tool to measure genome similarity. A genome assembly with a gANI score of <95% at the intraspecies level is generally considered indicative of a separate species. In this study, we have analysed 300 whole-genome sequences belonging to 26 different bacterial species available in the NCBI Genome database and calculated their similarity at the intraspecies level based on gANI score. At the intraspecies level, nine bacterial species showed less than 90% gANI and more than 10% of unaligned regions. We suggest the appropriate use of available bioinformatics resources after genome assembly to arrive at the proper bacterial identification, classification, and nomenclature to avoid erroneous species assignments and disparity due to diversity at the intraspecies level.

Severity of drug resistance and co-existence of Enterococcus faecalis in diabetic foot ulcer infections.

The genes encoding aminoglycoside resistance in Enterococcus faecalis may promote collateral aminoglycoside resistance in polymicrobial wounds. We studied a total of 100 diabetic foot ulcer samples for infection and found 60 samples to be polymicrobial, 5 to be monomicrobial, and 35 samples to be culture negative. A total of 65 E. faecalis isolates were screened for six genes coding for aminoglycoside resistance, antibiotic resistance patterns, and biofilm production. Infectious Diseases Society of America/International Working Group on the Diabetic Foot system was used to classify the wound ulcers. Majority of the subjects with culture-positive wound were recommended conservative management, while 14 subjects underwent amputation. Enterococcal isolates showed higher resistance for erythromycin, tetracycline, and ciprofloxacin. Isolates from grade 3 ulcer showed higher frequency of aac(6')-Ie-aph(2″)-Ia, while all the isolates were negative for aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id. The isolates from grade 3 ulcers showed higher resistance to aminoglycosides as well as teicoplanin and chloramphenicol. All the 39 biofilm producers were obtained from polymicrobial wound and showed higher resistance when compared to biofilm non-producers. Higher frequency of isolates carrying aac(6')-Ie-aph(2″)-Ia in polymicrobial community showing resistance to key antibiotics suggests widespread distribution of aminoglycoside-resistant E. faecalis and their role in worsening diabetic foot ulcers.

Virulence determinants in clinical Staphylococcus aureus from monomicrobial and polymicrobial infections of diabetic foot ulcers.

Antibiotic resistance in Staphylococcus aureus is a major public health concern, and methicillin-resistant S. aureus has emerged as an important pathogen. We characterized S. aureus isolates from monomicrobial and polymicrobial wound infections from 200 diabetic individuals with foot ulcers to understand their underlying diversity and pathogenicity. Staphylococcal cassette chromosome mec typing was performed, and genes coding for production of biofilm, Panton-Valentine leukocidin, toxic shock syndrome toxin and leukotoxins DE and M were screened. Biofilm production was also quantified by the tissue culture plate method. Strains were genotyped using multilocus sequence typing, multiple-locus variable number tandem repeat analysis and repetitive sequence PCR methods. Polymicrobial infections were present in 115 samples, 61 samples showed monomicrobial infection and 24 samples were culture negative. Polymicrobial infections were significantly higher in patients with previous amputation history. Of the 86 samples infected with S. aureus, virulence genes were found in 81 isolates, and 41 isolates possessed more than one virulence gene. Strains which contained pvl gene alone or luk-DE alone were significantly higher in polymicrobial wounds. Based on biofilm production, 18.6 % of isolates were classified as high, 24.4 % as moderate and 57 % as low biofilm producers. Genotyping of 30 strains revealed 10 different sequence types with a strong association among sequence types, specific virulence markers and antibiotic resistance profiles. Moreover, isolates from monomicrobial and polymicrobial wounds differed significantly in their virulence potential and the sequence types to which they belonged, and these are helpful in mapping the evolution of the identified strains of S. aureus.