Qualitative and Quantitative Analysis of Siderophore Production from <em>Pseudomonas aeruginosa</em>

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

The Maharaja Sayajirao University of Baroda

To develop an infectious keratitis model using caprine (goat) corneas and to investigate the expression of virulence factors during infection.

 Caprine Model to Study Virulence Factors in Keratitis.

 (), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a "priority pathogen". Biofilm is a major virulence factor of  and yet the mechanism of strong biofilm formation in  is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of  on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of . High adhesion capacity and cell death seem to play a major role in formation of strong  biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in  .

With the advent of next-generation sequencing, large-scale initiatives for mining whole genomes and exomes have been employed to better understand global or population-level genetic architecture. India encompasses more than 17% of the world population with extensive genetic diversity, but is under-represented in the global sequencing datasets. This gave us the impetus to perform and analyze the whole genome sequencing of 1029 healthy Indian individuals under the pilot phase of the 'IndiGen' program. We generated a compendium of 55,898,122 single allelic genetic variants from geographically distinct Indian genomes and calculated the allele frequency, allele count, allele number, along with the number of heterozygous or homozygous individuals. In the present study, these variants were systematically annotated using publicly available population databases and can be accessed through a browsable online database named as 'IndiGenomes' http://clingen.igib.res.in/indigen/. The IndiGenomes database will help clinicians and researchers in exploring the genetic component underlying medical conditions. Till date, this is the most comprehensive genetic variant resource for the Indian population and is made freely available for academic utility. The resource has also been accessed extensively by the worldwide community since it's launch.

IndiGenomes: a comprehensive resource of genetic variants from over 1000 Indian genomes.

 Numerous drugs are being widely prescribed for COVID-19 treatment without any direct evidence for the drug safety/efficacy in patients across diverse ethnic populations.  We analyzed whole genomes of 1029 Indian individuals (IndiGen) to understand the extent of drug-gene (pharmacogenetic), drug-drug and drug-drug-gene interactions associated with COVID-19 therapy in the Indian population.  We identified 30 clinically significant pharmacogenetic variants and 73 predicted deleterious pharmacogenetic variants. COVID-19-associated pharmacogenes were substantially overlapped with those of metabolic disorder therapeutics. ,  and  are the most shared pharmacogenes. Fifteen COVID-19 therapeutics were predicted as likely drug-drug interaction candidates when used with four  inhibitor drugs.  Our findings provide actionable insights for future validation studies and improved clinical decisions for COVID-19 therapy in Indians.

Pharmacogenomic landscape of COVID-19 therapies from Indian population genomes.

Autoinflammatory disorders are the group of inherited inflammatory disorders caused due to the genetic defect in the genes that regulates innate immune systems. These have been clinically characterized based on the duration and occurrence of unprovoked fever, skin rash, and patient's ancestry. There are several autoinflammatory disorders that are found to be prevalent in a specific population and whose disease genetic epidemiology within the population has been well understood. However, India has a limited number of genetic studies reported for autoinflammatory disorders till date. The whole genome sequencing and analysis of 1029 Indian individuals performed under the IndiGen project persuaded us to perform the genetic epidemiology of the autoinflammatory disorders in India.

Genetic epidemiology of autoinflammatory disease variants in Indian population from 1029 whole genomes.

The rapid emergence of multidrug-resistant  is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected:  and two copies of  in the chromosome, and a second copy of  on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured . All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system.  CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal . Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Genomic evolution of the globally disseminated multidrug-resistant  clonal group 147.

In the present study, biofilm formation was quantified in UTI isolates of  ( = 22) using the crystal violet assay and was categorized into; strong ( = 16), weak ( = 4), and moderate ( = 2) biofilm producers. Further experiments were done using strong ( = 4) and weak ( = 4) biofilm producers. Biofilm formation was greater in Luria broth followed by natural urine and artificial urine on silicone and silicone-coated latex. Cell adhesion and twitching motility were greater in strong biofilm producers. The presence of thick biofilm with an increased number of dead and total number of cells of strong biofilm producers was observed using CLSM. The concentrations of exopolymeric substances (eDNA, protein, and pel polysaccharide) were high in strong biofilm producers. FEG-SEM visualization of biofilm produced by strong biofilm producers showed more cells encased in thick biofilm matrix than weak ones. Overall results provide evidence for increased cell adhesion and twitching motility in strong biofilm producers.

Cell adhesion and twitching motility influence strong biofilm formation in .

Eukaryotic cells respond to environmental cues through mitogen activated protein kinase (MAPK) signaling pathways. Each MAPK cascade is specific to particular stimuli and mediates specialized responses through activation of transcription factors. In the budding yeast, Saccharomyces cerevisiae, the pheromone-induced mating pathway and the starvation-responsive invasive growth/filamentation pathway generate their distinct outputs through the transcription factors Ste12 and Tec1, respectively. In this study, we report the functional characterization of these transcription factors in the closely related human opportunistic pathogenic yeast Candida glabrata. Two homologues each for S. cerevisiae TEC1 and STE12 were identified in C. glabrata. Both C. glabrata Tec1 proteins contain the N-terminal TEA DNA-binding domain characteristic of the TEA/ATTS transcription factor family. Similarly, the DNA-binding homeodomain shared by members of the highly conserved fungal Ste12 transcription factor family is present in N-terminus of both C. glabrata Ste12 transcription factors. We show that both C. glabrata STE12 genes are at least partial functional orthologues of S. cerevisiae STE12 as they can rescue the mating defect of haploid S. cerevisiae ste12 null mutant. Knockout of one of the STE12 genes (ORF CAGL0H02145g) leads to decreased biofilm development; a stronger biofilm-impaired phenotype results from loss of both CgSTE12 genes in the double deletion mutant (Cgste12ΔΔ). The transcript levels of one of the TEC1 genes (ORF CAGL0M01716g) were found to be upregulated upon exposure to low pH; its deletion causes slightly increased sensitivity to higher concentrations of acetic acid. Heat shock leads to increase in mRNA levels of one of the STE12 genes (ORF CAGL0M01254g). These findings suggest a role of Tec1 and Ste12 transcription factors in the regulation of some traits (biofilm formation, response to low pH stress and elevated temperature) that contribute to C. glabrata's ability to colonize various host niches and to occasionally cause disease.

Tec1 and Ste12 transcription factors play a role in adaptation to low pH stress and biofilm formation in the human opportunistic fungal pathogen Candida glabrata.

Persister cell (PC) is dormant, tolerant to antibiotics, and a transient reversible phenotype. These phenotypes are observed in P. aeruginosa and cause bacterial chronic infection as well as recurrence of biofilm-mediated infection. PC formation requires stringent response and toxin-antitoxin (TA) modules. This study shows the P. aeruginosa PC formation in planktonic and biofilm stages on ceftazidime, gentamicin, and ciprofloxacin treatments. The PC formation was studied using persister assay, flow cytometry using Redox Sensor Green, fluorescence as well as Confocal Laser Scanning Microscopy, and gene expression of stringent response and TA genes. In the planktonic stage, ceftazidime showed a high survival fraction, high redox activity, and elongation of cells was observed followed by ciprofloxacin and gentamicin treatment having redox activity and rod-shaped cells. The gene expression of stringent response and TA genes were upregulated on gentamicin followed by ceftazidime treatment and varied among the isolates. In the biofilm stage, gentamicin and ciprofloxacin showed the biphasic killing pattern, redox activity, gene expression level of stringent response and TA varied across the isolates. Ceftazidime treatment showed higher persister cells in planktonic growth while all three antibiotics were able to induce persister cell formation in the biofilm stage.

Pseudomonas aeruginosa persister cell formation upon antibiotic exposure in planktonic and biofilm state.

 (Kp) has gained prominence in the last two decades due to its global spread as a multidrug-resistant (MDR) pathogen. Further, carbapenem-resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of -lactamases, especially carbapenemases, in Kp isolates from India, and (2) determine the most prevalent sequence type (ST) and plasmids, and their association with -lactamases. Clinical samples of  ( = 65) were collected from various pathology labs, and drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was performed for  = 22 resistant isolates, including multidrug-resistant (MDR) ( = 4), extensively drug-resistant (XDR) ( = 15), and pandrug-resistant (PDR) ( = 3) categories, and genomic analysis was performed using various bioinformatics tools. Additional Indian MDRKp genomes ( = 187) were retrieved using the Pathosystems Resource Integration Center (PATRIC) database. Detection of -lactamase genes, location (on chromosome or plasmid), plasmid replicons, and ST of genomes was carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST, respectively. All data were analyzed and summarized using the iTOL tool. ST231 was highest, followed by ST147, ST2096, and ST14, among Indian isolates.  was detected as the most prevalent gene, followed by  and . Among carbapenemase genes,  was prevalent and associated with ST231, ST2096, and ST14, which was followed by , which was observed to be prevalent in ST147, ST395, and ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col(BS512) was abundantly present in ST147 genomes. One Kp isolate with a novel MLST profile was identified, which carried , and . ST16 and ST14 are mostly dual-producers of carbapenem and ESBL genes and could be emerging high-risk clones in India.

Devarshi Gajjar

Department of Microbiology and Biotechnology Centre,
Faculty of Science,
Department of Microbiology and Biotechnology Centre, Faculty of Science

The Maharaja Sayajirao University of Baroda

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Devarshi Gajjar

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology and Biotechnology Centre,Faculty of Science,Department of Microbiology and Biotechnology Centre, Faculty of Science

The Maharaja Sayajirao University of Baroda

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Department of Microbiology and Biotechnology Centre,
Faculty of Science,
Department of Microbiology and Biotechnology Centre, Faculty of Science