Changing facades of Vibrio cholerae: An enigma in the epidemiology of cholera.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged humanity from time immemorial. Although the disease can be treated using antibiotics along with administration of oral rehydration salts and controlled by good sanitation, cholera is known to have produced mayhems in ancient times when little was known about the pathogen. By the 21st century, ample information about the pathogen, its epidemiology, genetics, treatment and control strategies was revealed. However, there is still fear of cholera outbreaks in developing countries, especially in the wake of natural calamities. Studies have proved that the bacterium is mutating and evolving, out-competing all our efforts to treat the disease with previously used antibiotics and control with existing vaccines. In this review, the major scientific insights of cholera research are discussed. Considering the important role of biofilm formation in the V. cholerae life cycle, the vast availability of next-generation sequencing data of the pathogen and multi-omic approach, the review thrusts on the identification of suitable biofilm-inhibiting targets and the discovery of anti-biofilm drugs from nature to control the disease.

Lineage shift in Indian strains of Dengue virus serotype-3 (Genotype III), evidenced by detection of lineage IV strains in clinical cases from Kerala.

BACKGROUND: Local epidemiology of Dengue is defined by the genetic diversity of the circulating Dengue virus (DENV) strains. This important information is not available for the virus strains from most parts of the Indian subcontinent. The present study focused on the genetic diversity of the serotype 3 DENV strains (DENV-3) from India. RESULTS: A total of 22 DENV-3 strains identified by reverse-transcription PCR analysis of serum samples from 709 patients were studied. These samples were collected over a period of 4 years (2008-2011) from dengue fever suspected patients from Kerala, a dengue endemic state in South India. Comparison of a 1740bp nucleotide sequence of the viral Capsid-Pre-membrane-Envelope coding region of our strains and previously reported DENV-3 strains from India, South Asia and South America revealed non-synonymous substitutions that were genotype III-specific as well as sporadic. Evidence of positive selection was detected in the I81 amino acid residue of the envelope protein. Out of the 22 samples, three had I81A and 18 had I81V substitutions. In the phylogenetic analysis by maximum likelihood method the strains from Kerala clustered in two different lineages (lineage III and IV) within genotype III clade of DENV-3 strains. The ten strains that belonged to lineage IV had a signature amino acid substitution T219A in the envelope protein. Interestingly, all these strains were found to be closely related to a Singapore strain GU370053 isolated in 2007. CONCLUSIONS: Our study identifies for the first time the presence of lineage IV strains in the Indian subcontinent. Results indicate the possibility of a recent exotic introduction and also a shift from the existing lineage III strains to lineage IV. Lineage shifts in DENV-3 strains have been attributed to dramatic increase in disease severity in many parts of the world. Hence the present observation could be significant in terms of the clinical severity of future dengue cases in the region.

A Novel Brevinin2 HYba5 Peptide against Polymicrobial Biofilm of Staphylococcus aureus and Enterococcus faecalis.

BACKGROUND: Brevinin2 HYba5 (Peptide 29) is a novel cationic peptide identified from an endemic frog, Hydrophylax bahuvistara. Staphylococcus aureus and Enterococcus faecalis are troublesome biofilm-forming pathogens associated with nosocomial and community-acquired infections and contribute to the severity of infections associated with implanted devices and chronic wounds. Co-existence of both pathogens in biofilm mode contributes to an increased antibiotic resistance, treatment failure and hence persistent disease burden. Identifying a novel and stable, less toxic compound targeting multispecies biofilm with a lower probability of acquiring resistance in comparison to antibiotics is highly warranted. OBJECTIVE: Evaluate the activity of Brevinin2 HYba5 against S. aureus and E. faecalis mixed biofilm. METHODS: The anti-biofilm activity of peptide 29 was tested by Crystal violet assay, Confocal laser scanning Microscopy (CLSM) and MTT Assay. Cytotoxicity of the peptide was tested in RBC and L929 fibroblast cell line. Biofilm inhibitory activity of the peptide was evaluated at different temperatures, pH, serum and plasma concentrations. The antibiofilm potential of the peptide was tested against polymicrobial biofilm by Fluorescent in situ hybridisation (FISH) and plate counting on HiCromeTM UTI Agar media. RESULTS: The peptide 29 could inhibit biofilm formation of S. aureus and E. faecalis individually as well as in polymicrobial biofilm at 75 µM concentration. The peptide maintained its antibiofilm potential at different temperatures, serum and plasma concentrations. Activity of the peptide was high at acidic and neutral pH but found to get reduced towards alkaline pH. The peptide is nonhemolytic and does not exhibit significant cytotoxicity against the L929 fibroblast cell line (92.80% cell viability). CONCLUSION: The biofilm inhibition property makes peptide 29 a promising candidate for the management of S. aureus and E. faecalis biofilm, especially in catheter-associated devices to prevent the initial colonization and thus can ease the burden of pathogenic biofilm-associated infections.

A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies.

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Potential effectiveness and adverse implications of repurposing doxycycline in COVID-19 treatment.

Introduction: COVID-19 infection with no known-specific drugs or vaccines has impacted mankind and has become beyond precedence. Currently, re-purposing of existing drugs is the only therapeutic option for managing COVID-19 symptoms and associated co-infections to reduce mortality. Antimicrobials as varied as antiparasitic, antiviral, and antibiotics are under various stages of evaluation.Areas covered: Recently, doxycycline, a broad-spectrum antibiotic that has also reported antiviral and anti-inflammatory properties was widely investigated in clinical trials, either alone or in combination with other drugs, and repurposed for COVID-19 treatment. In the review, the potential therapeutic applications of doxycycline in COVID-19 treatment and its potential adverse implications with respect to antimicrobial resistance bestowed by repurposing the antibiotic have been expounded.Expert opinion: 'Fighting disease with already existing antibiotics' and 'antimicrobial resistance progression' are like two arms of a balance that has to be carefully equilibrated. Any imbalance by the inappropriate or indiscriminate use of the repurposed drugs would cause a disastrous increase in antimicrobial resistance (AMR). Hence, cautious parallel assessment of potential long-term consequences of AMR is of great importance to mankind as its impacts would prevail even after the current pandemic.

Genome sequence of a multidrug-resistant Klebsiella pneumoniae ST78 with high colistin resistance isolated from a patient in India.

OBJECTIVES: Multidrug-resistant (MDR) Klebsiella pneumoniae isolates with colistin resistance are a major concern in healthcare settings. This study aimed to evaluate the genome-wide distribution of antimicrobial resistance genes in K. pneumoniae CRKP I with high colistin resistance isolated from a patient in India. METHODS: The whole genome of K. pneumoniae CRKP I was sequenced on an Illumina MiSeq platform. De novo genome assembly was performed using SPAdes v.3.0.0, and the genome sequence was analysed using bioinformatics tools available from the Center for Genomic Epidemiology. RESULTS: The genome of K. pneumoniae CRKP I is 5.1 Mb in size and contains different classes of antimicrobial resistance genes. The isolate is highly resistant to colistin owing to a point mutation in mgrB gene, encoding a negative regulator of the PhoP/PhoQ two-component system. Multilocus sequence typing (MLST) showed that K. pneumoniae CRKP I belongs to ST78. CONCLUSION: These data provide useful information for comparative genomic analysis regarding the dissemination of antimicrobial resistance genes in K. pneumoniae. To our knowledge, this is the first report of a MDR K. pneumoniae with high colistin resistance belonging to ST78 causing infection in a human.

Correlation of phylogenetic clade diversification and in vitro infectivity differences among Cosmopolitan genotype strains of Chikungunya virus.

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.

Middle East Respiratory Syndrome Corona Virus (MERS CoV): The next steps.

Developing countries are at risk of importing Middle East Respiratory Syndrome Corona Virus (MERS CoV) from the Middle East. Hospitals in the Middle East currently reporting the disease are staffed by immigrants. In the current hot spots for MERS CoV a sizeable portion of the population is from other countries, but many of these countries have yet to detect any importation of MERS CoV. To assess the disease transmission in these countries, supplemental surveillance strategies are urgently needed beyond the currently recommended measures. A few strategies to address the situation are: (i) improving preparedness with enhanced surveillance in particular regions; (ii) targeting certain sentinel groups for surveillance in hot spots; and (iii) limited use of serosurveillance. Recovered, immune patients can be employed to give patient care during outbreaks.

Development of broad-spectrum antibiofilm drugs: strategies and challenges.

The severity of many chronic bacterial infections is mainly due to the biofilm mode of life adapted by pathogenic bacteria. The bacteria in biofilm-stage exhibit high resistance to host immune responses and antimicrobials, which complicates the treatment process and results in life threatening conditions. Most of the chronic infections are polymicrobial in nature. In order to combat the polymicrobial biofilm infections and to increase the efficiency of antimicrobials, there is an urgent need for broad-spectrum antibiofilm drugs. This review discusses the clinical needs and current status of broad-spectrum antibiofilm drugs with special emphasis on prospective strategies and hurdles in the process of new drug discovery.