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Louse-borne relapsing fever (LBRF) with high untreated mortality caused by spirochete Borrelia recurrentis is predominantly endemic to Sub-Saharan Africa and has re-emerged in parts of Eastern Europe, Asia and Latin America due to population migrations. Despite subtractive evolution of lice-borne pathogenic Borrelia spp. from tick-borne species, there has been no comprehensive report on conservation of protein targets across tick and lice-borne pathogenic Borrelia nor exploration of phytocompounds that are toxic to tick against lice. From the 19 available whole genomes including B. recurrentis, B. burgdorferi, B. hermsii, B. parkeri and B. miyamotoi, conservation of seven drug targets (80% domain identity) viz. 30 S ribosomal subunit proteins (RSP) S3, S7, S8, S14, S19, penicillin-binding protein-2 and 50 S RSP L16 were deciphered through multiple sequence alignments. Twelve phytocompounds (hydroxy-tyrosol, baicalein, cis-2-decanoic acid, morin, oenin, rosemarinic acid, kaempferol, piceatannol, rottlerin, luteolin, fisetin and monolaurin) previously explored against Lyme disease spirochete B. burgdorferi when targeted against LBRF-causing B. recurrentis protein targets revealed high multi-target affinity (2%-20% higher than conventional antibiotics) through molecular docking. However, based on high binding affinity against all target proteins, stable coarse-grained dynamics (fluctuations1 Å) and safe pharmacological profile, luteolin was prioritized. The study encourages experimental evaluation of the potent phytocompounds and similar protocols for investigating other emerging vector-borne diseases.

Anti-microbial resistance (AMR) creating healthcare concerns worldwide requires ardent exploration of therapeutic alternatives. Although anti-microbial peptides (AMP) are popular for broad-spectrum activity, recent evidence of increasing resistance to membrane-acting AMPs by ESKAPE pathogens has compelled us to design novel AMPs as therapeutic candidates. A library of 60 AMPs comprising natural AMPs and their mutants was constructed through in-silico methods. After physico-chemical property evaluations, each peptide in the library was subjected to flexible molecular docking against four major β-lactamases in Gram-negative ESKAPE pathogens. Among the potent AMP mutants, a Lactoferricin B-Mutant (M4) possessed uniformly high affinity with SHV1, OXA48, NDM1, and AmpC having energies -842.0Kcal/mol, -774.8Kcal/mol, -1103.3Kcal/mol, and -858.8Kcal/mol respectively. Coarse-grained clustering and flexibility analysis further accounted for the residue-level stable configurations of the protein-peptide complexes with high affinity. Highest affinity of Lactoferricin B_M4 was found with NDM1 due to H-bonds, salt-bridges, and hydrophobic interactions with the metallo-β-lactamase domain including crucial active-site residue Asp124. Molecular dynamics simulation further confirmed the stability of Lactoferricin B_M4-NDM1 complex having low residue-level root-mean square deviations (RMSD), atomic-level fluctuations, and radius of gyration (Rg). The study encourages experimental validations and similar methods to identify potential AMPs against drug-resistant pathogens.

Acinetobacter baumannii is an emerging nosocomial strain expressing extensive drug resistance (XDR). Whole-genome sequencing and molecular characterisation analysis revealed the presence of carbapenemase in 92.86% of studied Indian isolates having blaOXA-51, blaOXA-23, blaOXA-58, and blaNDM genes, with a few evidences of dual carbapenemase genes. As per the MLST scheme, IC2Oxf/CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of β-lactamases has rendered sulbactam (SUL) resistance (MIC: 16-256µg/ml) in all the studied isolates. The efficacy of novel durlobactam (DUR) in inhibiting β-lactamases and PBP2 was assessed through in-silico inter-molecular interaction analysis. Several non-synonymous single nucleotide polymorphisms (nsSNPs) were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein-backbone dynamics in active-site motifs of wild-type (WT) and mutants (MT), which correlated with the negligible binding energy fluctuations for PBP3-SUL (−5.85±0.04Kcal/mol) and PBP2-DUR (−5.16±0.66Kcal/mol) complexes. Furthermore, stronger binding affinities and low inhibition constants were noted in DUR complexed with OXA23 (−7.36Kcal/mol; 4.01µM), OXA58 (−6.44Kcal/mol; 19.07µM) and NDM (−6.82Kcal/mol; 10.01µM) when compared with conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR, can possibly restore SUL activity against both PBP3WT and PBP3MTs. The study establishes the efficacy of novel SUL-DUR combination as a successful treatment strategy to combat emerging XDR strains.

Outbreaks of monkeypox virus infections have imposed major health concerns worldwide, with high morbidity threats to children and immunocompromised adults. Although repurposed drugs and vaccines are being used to curb the disease, the evolving traits of the virus, exhibiting considerable genetic dynamicity, challenge the limits of a targeted treatment. A pan-genome-based reverse vaccinology approach can provide fast and efficient solutions to resolve persistent inconveniences in experimental vaccine design during an outbreak-exigency. The approach encompassed screening of available monkeypox whole genomes (n = 910) to identify viral targets. From 102 screened viral targets, viral proteins L5L, A28, and L5 were finalized based on their location, solubility, and antigenicity. The potential T-cell and B-cell epitopes were extracted from the proteins using immunoinformatics tools and algorithms. Multiple vaccine constructs were designed by combining the epitopes. Based on immunological properties, chemical stability, and structural quality, a novel multi-epitopic vaccine construct, V4, was finalized. Flexible-docking and coarse-dynamics simulation portrayed that the V4 had high binding affinity towards human HLA-proteins (binding energy < −15.0 kcal/mol) with low conformational fluctuations (

In recent decades, antimicrobial resistance has been augmented as a global concern to public health owing to the global spread of multidrug-resistant strains from different ESKAPE pathogens. This alarming trend and the lack of new antibiotics with novel modes of action in the pipeline necessitate the development of non-antibiotic ways to treat illnesses caused by these isolates. In molecular biology, computational approaches have become crucial tools, particularly in one of the most challenging areas of multidrug resistance. The rapid advancements in bioinformatics have led to a plethora of computational approaches involving genomics, systems biology, and structural biology currently gaining momentum among molecular biologists since they can be useful and provide valuable information on the complex mechanisms of AMR research in ESKAPE pathogens. These computational approaches would be helpful in elucidating the AMR mechanisms, identifying important hub genes/proteins, and their promising targets together with their interactions with important drug targets, which is a crucial step in drug discovery. Therefore, the present review aims to provide holistic information on currently employed bioinformatic tools and their application in the discovery of multifunctional novel therapeutic drugs to combat the current problem of AMR in ESKAPE pathogens. The review also summarizes the recent advancement in the AMR research in ESKAPE pathogens utilizing the in silico approaches.

The present study reported a rare gentamicin-susceptible β-lactamase (PenA, OXA-57) expressing clinical Burkholderia pseudomallei isolate VB29710 from India. Whole-genome sequencing and structural analyses revealed the insertion of R962 and L963 into AmrB, the transmembrane-protein of the AmrAB-OprA efflux-pump that affected aminoglycoside-efflux through local alterations in backbone conformation.

Patients admitted to the hospital with coronavirus disease (COVID-19) are at risk for acquiring mycotic infections in particular Candidemia. Candida albicans (C. albicans) constitutes an important component of the human mycobiome and the most common cause of invasive fungal infections. Invasive yeast infections are gaining interest among the scientific community as a consequence of complications associated with severe COVID-19 infections. Early identification and surveillance for Candida infections is critical for decreasing the COVID-19 mortality. Our current study attempted to understand the molecular-level interactions between the human genes in different organs during systematic candidiasis. Our research findings have shed light on the molecular events that occur during Candidiasis in organs such as the kidney, liver, and spleen. The differentially expressed genes (up and down-regulated) in each organ will aid in designing organ-specific therapeutic protocols for systemic candidiasis. We observed organ-specific immune responses such as the development of the acute phase response in the liver; TGF-pathway and genes involved in lymphocyte activation, and leukocyte proliferation in the kidney. We have also observed that in the kidney, filament production, up-regulation of iron acquisition mechanisms, and metabolic adaptability are aided by the late initiation of innate defense mechanisms, which is likely related to the low number of resident immune cells and the sluggish recruitment of new effector cells. Our findings point to major pathways that play essential roles in specific organs during systemic candidiasis. The hub genes discovered in the study can be used to develop novel drugs for clinical management of Candidiasis.

Parkinson's disease (PD) has been designated as one of the priority neurodegenerative disorders worldwide. Although diagnostic biomarkers have been identified, early onset detection and targeted therapy are still limited. An integrated systems and structural biology approach were adopted to identify therapeutic targets for PD. From a set of 49 PD associated genes, a densely connected interactome was constructed. Based on centrality indices, degree of interaction and functional enrichments

Whole genome sequences of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and Salmonella enterica subspecies enterica serovar Paratyphi A (S. Paratyphi A) from pediatric settings were used to assess the emerging Antimicrobial Resistance (AMR). The high throughput sequences of twenty pediatric clinical isolates of S. Typhi and S. Paratyphi A were retrieved and were screened for prevalent Antimicrobial Resistance Genes (ARGs) and Virulent Factors (VF). The resistance data was compared with the reference strains of S. Typhi and S. Paratyphi A. AMR studies identified sul1, sul2, dfrA7, tem-1, AH(6)-Id and APH(3″)-Ib as common ARGs. VFs were identified to understand the level of pathogenicity. The most prevalent AMR genes in the sequenced genomes were detected in phenotypically azithromycin-resistant S. Typhi. Correlation with the global genomes projected a trend of concurrent resistance to macrolides, β lactams, fluoroquinolones (FQs), tetracyclines, ansamycins, and aminoglycosides. Traces of sulphonamide-resistance were observed indicating the emergence of a currently non-prevalent S. Typhi resistance that could be a future threat. Hence new antibiotic regimen to treat azithromycin-resistant S. Typhi should be formulated by avoiding the risks of aggravating sulphonamide resistance. The identified ARGs in genomes from paediatric isolates will aid future studies to design anti-bacterial compounds against S. Typhi and S. Paratyphi A.

Enteric fever caused by Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A remains one of the most common causes of community-acquired bloodstream infection among children in India. Multidrug resistance is emerging and is a cause of concern as it affects the choice of treatment in enteric fever.In this study, a 10-year analysis of resistance patterns was done along with in-silico homology modeling and molecular docking to understand the commonly occurring quinolone resistance.A total of 1010 cases of blood culture-confirmed enteric fevers (S. typhi n=849; S. paratyphi A n=161) were recorded at the study hospital during the period from 2011-2020. Multidrug resistance among cases of S. typhi was 2.12 %, whereas it was completely absent among cases of S. paratyphi A. Fluoroquinolone resistance was high (95%) throughout the study period. Resistance to ampicillin, chloramphenicol and co-trimoxacole was low (3%) among S. typhi cases. No deaths were observed among study participants. Molecular docking analysis showed that quinolone had less binding affinity to mutated gyrase A than to its wild type for both S. typhi and S. paratyphi A.Quinolone resistance was high among cases of enteric fever, whereas no resistance was observed among third-generation cephalosporins. In-silico studies indicated that a mutation in gyrase A might be the cause of the gradual increase in ciprofloxacin resistance over the study period.

Aim: The rapid emergence of β-lactam resistance in gram-negative bacteria is a major problem in the treatment of infections caused by pathogenic bacterial strains, in particular Klebsiella pneumoniae. In our study, we are presenting a systems biology approach to understand the role of SHV-11 gene in drug resistance mechanism patterns in K. pneumoniae strain. Results: From the results, we have observed that the SHV-11 gene has a role in drug resistance mechanism along with its functional partner genes gyrA, parC, glsA, osmE, yjhA, yhdT, rimL, pepB, KPN_00437, and KPN_01875. We have also observed that of 51 genes, 27 genes were enriched in various Gene Ontology terms such as DNA metabolic process, DNA repair, and response to stress. The genes gyrA, parC, gyrB, parE, recA, dnaA, polB, dnaK, mutS, and dnaN constitute >41% of the total interactions; thus, these genes can be considered as hub nodes in the network, and they can be used as the potential drug targets. Conclusions: Drug exposure leads to the DNA damage in bacterial spp. We observed that the SHV11 gene along with the functional partners help in maintaining the genomic integrity by withstanding the environmental stress by inducing DNA damage repair mechanism. Our results provide a detailed understanding on the role of SHV-11 gene in drug resistance mechanisms in K. pneumoniae, and we are of the opinion that our results will be useful for researchers exploring the antibiotic resistance mechanisms in pathogenic bacteria.

The principal causative agent of acute bacterial meningitis (ABM) in children and the elderly is Streptococcus pneumoniae, with a widespread increase in penicillin resistance. Resistance is due to non-synonymous single-nucleotide polymorphisms (nsSNPs) that alter the penicillin-binding proteins (PBPs), the targets for all β-lactam drugs. Hence, resistance against one β-lactam antibiotic may positively select another. Since meropenem is an alternative to cefotaxime in meningeal infections, we aim to identify whether nsSNPs in the PBPs causing penicillin and cefotaxime resistance can decrease the pneumococcal susceptibility to meropenem. Comparison of the nsSNPs in the PBPs between the cefotaxime-resistant Indian (n = 33) and global isolates (n = 28) revealed that nsSNPs in PBP1A alone elevated meropenem minimal inhibitory concentrations (MICs) to 0.12 μg/ml, and nsSNPs in both PBP2X and 2B combined with PBP1A increases MIC to ≥ 0.25 μg/ml. Molecular docking confirmed the decrease in the PBP drug binding affinity due to the nsSNPs, thereby increasing the inhibition potential and the MIC values, leading to resistance. Structural dynamics and thermodynamic stability pattern in PBPs as a result of mutations further depicted that the accumulation of certain nsSNPs in the functional domains reduced the drug affinity without majorly affecting the overall stability of the proteins. Restricting meropenem usage and promoting combination therapy with antibiotics having non-PBPs as targets to treat cefotaxime non-susceptible S. pneumoniae meningitis can prevent the selection of β-lactam resistance.

Docking scores and simulation parameters to study the potency of natural compounds against protein targets in

The delayed diagnosis of pancreatic cancer has resulted in rising mortality rate and low survival rate that can be circumvented using potent theranostics biomarkers. The treatment gets complicated with delayed detection resulting in lowered 5-year relative survival rate. In our present study, we employed systems biology approach to identify central genes that play crucial roles in tumor progression. Pancreatic cancer genes collected from various databases were used to construct a statistically significant interactome with 812 genes that was further analysed thoroughly using topological parameters and functional enrichment analysis. The significant genes in the network were then identified based on the maximum degree parameter. The overall survival analysis indicated through hazard ratio [HR] and gene expression [log Fold Change] across pancreatic adenocarcinoma revealed the critical role of FN1 [HR 1.4; log2(FC) 5.748], FGA [HR 0.78; log2(FC) 1.639] FGG [HR 0.9; log2(FC) 1.597], C3 [HR 1.1; log2(FC) 2.637], and QSOX1 [HR 1.4; log2(FC) 2.371]. The functional significance of the identified hub genes signified the enrichment of integrin cell surface interactions and proteoglycan syndecan-mediated cell signaling. The differential expression, low overall survival and functional significance of FN1 gene implied its possible role in controlling metastasis in pancreatic cancer. Furthermore, alternate splice variants of FN1 gene showed 10 protein coding transcripts with conserved cell attachment site and functional domains indicating the variants' potential role in pancreatic cancer. The strong association of the identified hub-genes can be better directed to design potential theranostics biomarkers for metastasized pancreatic tumor.

The novel coronavirus SARS-CoV-2 since its emergence at Wuhan, China in December 2019 has been creating global health turmoil despite extensive containment measures and has resulted in the present pandemic COVID-19. Although the virus and its interaction with the host have been thoroughly characterized, effective treatment regimens beyond symptom-based care and repurposed therapeutics could not be identified. Various countries have successfully developed vaccines to curb the disease-transmission and prevent future outbreaks. Vaccination-drives are being conducted on a war-footing, but the process is time-consuming, especially in the densely populated regions of the world. Bioinformaticians and computational biologists have been playing an efficient role in this state of emergency to escalate clinical research and therapeutic development. However, there are not many reviews available in the literature concerning COVID-19 and its management. Hence, we have focused on designing a comprehensive review on in-silico approaches concerning COVID-19 to discuss the relevant bioinformatics and computational resources, tools, patterns of research, outcomes generated so far and their future implications to efficiently model data based on epidemiology; identify drug targets to design new drugs; predict epitopes for vaccine design and conceptualize diagnostic models. Artificial intelligence/machine learning can be employed to accelerate the research programs encompassing all the above urgent needs to counter COVID-19 and similar outbreaks.

BackgroundThe incidence of hypervirulent (hv) carbapenem-resistant (CR)Klebsiella pneumoniae(Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.MethodsTwenty-seven CRKp isolates were identified to possessrmpA2by whole-genome sequencing; and resistance and virulence determinants were characterized. Byin silicoprotein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.ResultsThe CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncatedrmpAandrmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones.In silicoanalysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.ConclusionIncreasing incidence of convergence of AMR and virulence is observed amongK. pneumoniaeglobally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

Streptococcus pneumoniae is the principal etiological agent of acute bacterial meningitis (ABM) which has fatal outcome in children and elderly. Due to poor blood-brain barrier (BBB) permeation, conventional β-lactam antibiotics fail to establish the requisite bactericidal concentration in central nervous system leading to resistance in meningeal infections. The present study intended to identify potential therapeutic alternatives against Streptococcal meningitis.Virtual screening, pharmacokinetics/pharmacodynamics (PK/PD) and anti-bacterial evaluations were employed to screen potential drugs. Molecular docking and structural dynamics simulations were performed to analyze the binding affinity and interaction stability of the drugs against the conventional Penicillin binding protein (PBP) targets. Screened drugs were also checked for interactions with other possible Streptococcal targets and relevant host targets.Non-steroidal anti-inflammatory drugs (NSAIDs) ketorolac and etodolac exhibiting high BBB-permeation and anti-bacterial potency were identified. Ketorolac and etodolac possessed uniform binding affinities against PBP1A, PBP2X, PBP2B and PBP3 with low inhibition constants (50 μM). Against PBP2B and PBP3, higher binding affinities were observed for ketorolac (-6.45 and -6Kcal/mol respectively) and etodolac (-6.36 and -6.55Kcal/mol respectively) than penicillin (-5.95 and -5.85Kcal/mol respectively) and cefotaxime (-5.08 and -5.07Kcal/mol respectively). The binding affinities were contributed by conventional H-bonds and non-canonical interactions with active site residues of PBPs. Structural dynamics simulations further indicated the overall stability of the drug-bound complexes through minimal overall average root-mean square fluctuations (RMSFs) (1.0 Å). The average binding affinities of Ketorolac and Etodolac with PBPs were marginally higher than other Streptococcal targets and comparable to their conventional inflammatory targets.Pharmacological and structural profiles indicated that ketorolac and etodolac can potentially subdue the cause and effects of streptococcal meningitis and hence encourage experimental validations.

Metallo-β-lactamases (MBLs) producing bacteria especially the ones with New Delhi metallo-beta-lactamase-1 (NDM-1) and its variants can potentially hydrolyse all the major β-lactam antibiotics, ultimately escalating anti-microbial resistance world-wide. There is a dearth of approved inhibitors to combat NDM and other MBLs producing bacteria. Hence we focussed to find novel inhibitor(s) in-silico which can potentially suppress the activity of NDM/MBLs. A total of 2400 compounds were virtually screened to identify a promising carboxylic acid-containing compound (CID-53986787) analogous to NDM antagonist Captopril. Our lead compound can bind adjacent to the active site zinc ions (Zn1 and Zn2) in all highly resistant NDM variants. CID-53986787 possesses ~ 5–8% higher binding affinity than Captopril, exhibiting molecular interactions with crucial residues that can destabilize the hydrolytic activity of NDM. CID-53986787 was virtually evaluated to ascertain its safe pharmacological/toxicity profile. Molecular dynamics simulation studies further elucidated its stable interaction with the target protein (NDM-1).

The emergence of multidrug resistance (MDR), extensively drug-resistant, and total drug-resistant Mycobacterium tuberculosis (Mtb) strains have hampered the treatment of tuberculosis (TB). Capreomycin and Bedaquiline are currently used for MDR-TB treatment. To understand the impact of these antibiotics on Mtb genes, we have curated the gene expression data where the Mtb cultures were exposed to the Bedaquiline and Capreomycin. Based on the P value cut off (0.05) and logFC (-0.5 and+0.5) values, we have selected the top differentially expressed genes during the antibiotic exposures. We have observed that the top differentially expressed Mtb genes were related to universal stress genes, two-component regulatory systems, and drug efflux pumps. We have curated the Mtb gene datasets and carried out the functional over-representation analysis using the individual gene expression values. We further, constructed the gene interaction networks of antibiotic resistance genes and virulence genes of Mtb to understand the impact of the antibiotics at the molecular level and thus to understand the antimicrobial resistance and virulence patterns. Our study elucidates the impact of antibiotics on the Mtb genes at the molecular level and the positively enriched pathways, operons, and regulons data are helpful in understanding the resistance patterns in Mtb. The upregulated genes during the exposure of Bedaquiline and Capreomycin can be considered as potent drug targets for the development of new anti-TB drugs.

In the era where issue of antibiotic resistance is increasing at an alarming rate and resistance to most antibiotics has occurred, carbapenems have been the choice of drug against most infection because of its unique structure. However due to lack of antibiotic policy carbapenems too have achieved resistance and led to the emergence of carbapenem resistant Enterobacteriaceae (CRE). Members of Enterobacteriaceae family produce New Delhi metallo-beta-lactamase (NDM), a type of carbapenemase that can cleave carbapenems. Therefore, new anti-microbial targets need to be identified and for this purpose a holistic model is designed in this approach in order to find out the influential genes that are liable in imparting carbapenem resistance owing to the presence of NDM. In this research approach, firstly genes associated with resistance owing to NDM are collected from NCBI database. A total of 419 genes from E. coli that have been obtained from NCBI were subjected to STRING for protein-protein interaction analysis. Out of 419 genes, only 59 genes showed interaction. STRING gave 1679 functional partners at different confidence levels. In the next stage, clustering analysis of the 1679 functional partners was done using MCODE app of Cytoscape software which generated 71 clusters. Topological properties of the 804 functional partners obtained from the 71 clusters were done using Network Analyser plugin of Cytoscape software. 797 functional partners were streamlined and they were subjected to functional enrichment analysis by DAVID Database. This database finally gave 21 genes from 15 clusters, thus indicating that these 15 genes might have a crucial role in the development of carbapenem resistance owing to NDM. These genes can be targeted for designing new anti-microbial drugs in future by the way of gene silencing or altering the genetic expression.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium, which is a leading causal agent for nosocomial infections. Penicillin, cephalosporin and carbapenems along with the inhibitors such as tazobactam, sulbactam and clavulanic acid are prescribed for the treatment of K. pneumoniae infections. Prolonged exposure to β-lactam antibiotics leads to the development of resistance. The major reason for the β-lactam resistance in K. pneumoniae is the secretion of the enzyme K. pneumoniae carbapenemase (KPC). Secretion of KPC-2 and its variant KPC-3 by the K. pneumoniae strains causes resistance to both the substrate imipenem and the β-lactamase inhibitors. Hence, molecular docking and dynamics studies were carried out to analyze the resistance mechanism of KPC-2-imipenem and KPC-3-imipenem at the structural level. It reveals that KPC-3-imipenem has the highest c-score value of 4.03 with greater stability than the KPC-2-imipenem c-score value of 2.36. Greater the interaction between the substrate and the β-lactamase enzyme, higher the chances of hydrolysis of the substrate. Presently available β-lactamase inhibitors are also ineffective against KPC-3-expressing strains. This situation necessitates the need for development of novel and effective inhibitors for KPC-3. We have carried out the virtual screening process to identify more effective inhibitors for KPC-3, and this has resulted in ZINC48682523, ZINC50209041 and ZINC50420049 as the best binding energy compounds, having greater binding affinity and stability than KPC-3-tazobactam interactions. Our study provides a clear understanding of the mechanism of drug resistance and provides valuable inputs for the development of inhibitors against KPC-3 expressing K. pneumoniae. Communicated by Ramaswamy H. Sarma.

Background The incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity. Methods Twenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp. Results The CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors. Conclusion Increasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

BAG3, a co-chaperone protein with a Bcl-2-associated athanogene (BAG) domain, has diverse functionalities in protein-folding, apoptosis, inflammation, and cell cycle regulatory cross-talks. It has been well characterised in cardiac diseases, cancers, and viral pathogenesis. The multiple roles of BAG3 are attributed to its functional regions like BAG, Tryptophan-rich (WW), isoleucine-proline-valine-rich (IPV), and proline-rich (PXXP) domains. However, to study its structural impact on various functions, the experimental 3D structure of BAG3 protein was not available. Hence, the structure was predicted through in silico modelling and validated through computational tools and molecular dynamics simulation studies. To the best of our knowledge, the role of BAG3 in bacterial infections is not explicitly reported. We attempted to study them through an in-silico protein-protein interaction network and host-pathogen interaction analysis. From structure-function relationships, it was identified that the WW and PXXP domains were associated with cellular cytoskeleton rearrangement and adhesion-mediated response, which might be involved in BAG3-related intracellular bacterial proliferation. From functional enrichment analysis, Gene Ontology terms and topological matrices, 18 host proteins and 29 pathogen proteins were identified in the BAG3 interactome pertaining to Legionellosis, Tuberculosis, Salmonellosis, Shigellosis, and Pertussis through differential phosphorylation events associated with serine metabolism. Furthermore, it was evident that direct (MAPK8, MAPK14) and associated (MAPK1, HSPD1, NFKBIA, TLR2, RHOA) interactors of BAG3 could be considered as therapeutic markers to curb down intracellular bacterial propagation in humans.

Background The issue of carbapenem resistance in E.coli is very concerning and it is speculated that cumulative effect of both primary resistance genes and secondary resistance genes that act as helper to the primary resistance genes are the reason behind their aggravation. Therefore, here we attempted to find the role of two secondary resistance genes (SRG) ccdB and repA2 in carbapenem resistance in E. coli (CRE). In this context influential genes belonging to secondary resistome that act as helper to the primary resistance genes like blaNDM and blaCTX-M in aggravating β-lactam resistance were selected from an earlier reported in silico study. Transcriptional expression of the selected genes in clinical isolates of E.coli that were discretely harboring blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7 and blaCTX-M-15 with and without carbapenem and cephalosporin stress (2 μg/ml) was determined by real time PCR. Cured mutants sets that were lacking (i) primary resistance genes, (ii) secondary resistance genes and (iii) both primary and secondary resistance genes were prepared by SDS treatment. These sets were then subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method. Results Out of the 21 genes reported in the in silico study, 2 genes viz.repA2 and ccdB were selected for transcriptional expression analysis. repA2, coding replication regulatory protein, was downregulated in response to carbapenems and cephalosporins. ccdB, coding for plasmid maintenance protein, was also downregulated in response to carbapenems except imipenem and cephalosporins. Following plasmid elimination assay increase in diameter of zone of inhibition under stress of both antibiotics was observed as compared to uncured control hinting at the reversion of antibiotic susceptibility by the-then resistant bacteria. Conclusion SRGs repA2 and ccdB help sustenance of blaNDM and blaCTX-M under carbapenem and cephalosporin stress.

Background Cardiovascular Disease (CVD) is one of the most common causes of mortality in humans. Presently, the role of pathogens in the initiation and progression of the CVDs is not clearly understood. Hence, it is essential to understand the molecular-level interactions between the human proteins and the microbial proteins to deduce their functional roles in the CVDs. Method The host-pathogen interactions (HPI) related to CVDs in the case of E. coli str. O157:H7 colonization were curated, and also the protein-protein interactions (PPI) between humans and E. coli were collected. Gene interaction network (GIN) and functional enrichment analyses (FEA) were utilized for this. Results The GIN revealed dense interactions between the functional partners. The FEA indicated that the essential pathways played a significant role in humans as well as in E. coli. The primary responses against most of the bacterial pathogens in humans are different from that of E. coli; Terpenoid biosynthesis and production of secondary metabolite pathways aid the survival of the E. coli inside the host. Interestingly, network analysis divulged that the E. coli genes ksgA, rpsT, ispE, rpsI, ispH, and the human genes TP53, CASP3, CYCS, EP300, RHOA communicated by significant numbers in direct interactions. Conclusions The results obtained from the present study will help researchers understand the molecular-level interactions in the CVDs between the human and the E. coli genes. The important genes with vital interactions can be considered as hub molecules and can be exploited for new drug discovery.

No commercially available drug candidate has yet been devised which is unique to and not repurposed against SARS-CoV-2 and has high efficacy or safe toxicity profile or both. Taking curcumin as a reference compound, we identified a new commercially available cyclohexanone compound, ZINC07333416 with binding energy (−8.72 kcal/mol) better than that of popularly devised anti-Covid-19 drugs like viral protease inhibitor Lopinavir, nucleoside analogue Remdesivir and the repurposed drug hydroxychloroquine when targeted to the active-site of SARS-CoV-2 Main protease (Mpro) through docking studies. The ligand ZINC07333416 exhibits crucial interactions with major active site residues of SARS-CoV-2 Mpro viz. Cys145 and His41 involving in the protease activity; as well as GLU-166 and ASN-142 which plays the pivotal role in the protein-dimerization. The protein-ligand stable interaction was further confirmed with molecular dynamics simulation (MDS) studies. Based on virtual assessment, ZINC07333416 also have significant values in terms of medicinal chemistry, pharmacokinetics, synthetic accessibility and anti-viral activity that encourage its experimental applications against COVID-19., Highlights •We screened commercially available compounds with curcumin as a reference to specifically pertain to the anti-viral potential and safe toxicity profile of the later. •We checked the binding properties of lead compound and previously reported compounds against SARS-CoV-2 Mpro, besides comparing their pharmacological and toxicity profile. A novel cyclohexanone compound was identified with favourable drug attributes and eliciting stable molecular interactions with SARS-CoV-2 Mpro.

The coronavirus disease (COVID-19) is resulting in millions of infected individuals with several hundred thousands dead throughout the world. Amidst all the havoc, one interesting observation in the present COVID-19 pandemic is the negligible symptoms in the young; particularly children below 10 years of age. We assume the extensive pediatric vaccination with MMR vaccines followed globally could have resulted in innate immune responses, e.g., induction of interferons (IFNs) and activated natural killer (NK) cells, thereby offering natural immunity against SARS-CoV-2 in the young population. Possible cross-protective innate immunity offered by MMR vaccination prompted us to suggest repurposing MMR vaccination for immuno-prophylaxis against COVID-19.

Proteus mirabilis is one among the most frequently identified pathogen in patients with the urinary tract infection. The multidrug resistance exhibited by P. mirabilis renders the treatment ineffective, and new progressive strategies are needed to overcome the antibiotic resistance (AR). We have analyzed the evolutionary relationship of 29 P. mirabilis strains available in the National Center for Biotechnology Information-Genome database. The antimicrobial resistance genes of P. mirabilis along with the enriched pathways and the Gene Ontology terms are analyzed using gene networks to understand the molecular basis of AR. The genes rpoB, tufB, rpsl, fusA, and rpoA could be exploited as potential drug targets as they are involved in regulating the vital functions within the bacterium. The drug targets reported in the present study will aid researchers in developing new strategies to combat multidrug-resistant P. mirabilis.

The objective of the study was to evaluate the

In the present study, we have explored the anti-malarial potential of epoxyazadiradione, the natural entity extracted from the neem seed oil and its chemical derivatives, against Plasmodium falciparum. The Surflex dock analysis of 41 compounds against an indispensable target, plasmepsin I (PM-I) revealed that around 70% of the compounds are found to have good binding capacity with the consensus score (C-score) of 5 to 4 with few hydrogen bonds. To elucidate the major structural requirements, vital for binding with the plasmepsin enzyme and to develop the predictive models, three-dimentional quantitative structural activity relationship (3D-QSAR) - comparative molecular field analysis (CoMFA) was carried out using Sybyl X.0. Robust and predictive models were obtained with cross-validated correlation coefficient (q2) value of 0.967 and the non-cross-validated correlation coefficient (r2) value of 0.825, which were validated by an external test set with the predictive correlation coefficient r2(pred) values of 0.773. Three zones were identified for substitution with bulky groups and one zone for substitution with non-bulky groups. Three positions favouring the electronegative group substitution and one for the electropositive group substitution were identified. The physicochemical properties of ligands with the highest C-score were studied. Communicated by Ramaswamy H. Sarma.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), imposes a significantly high morbidity threat to humans in both developed and developing nations. The resistances acquired by this intracellular pathogen towards the commonly used drugs have made TB treatment cumbersome. Hence, there is an exigency to explore alternative therapeutic candidates against this bacterial pathogen. In our study, 15 natural compounds were selected and we have explored their anti-TB properties using in-silico approaches. We have compared their activity with the known drugs (standards) used against TB and their corresponding protein targets involved in nucleic acid metabolism, membrane integrity and protein translation mechanism. Interaction profiles with crucial functional protein domains and in-silico structural biology analysis revealed that Glycyrrhizin, Swertiamarin, and Laccaic acid displayed better binding affinity than the classical anti-TB drugs. Furthermore, hydrogen bonding patterns and energy profiles ascertained the stability of these docked complexes. Our results revealed that Glycyrrhizin, Laccaic acid and Swertiamarin could be developed as multi-target specific alternative drug candidates and could be subjected to experimental validations against MDR/XDR Mtb.

Background: NFX1-123 is the longer splice variant isoform of the NFX1 gene and is highly expressed in cervical cancer. Cervical cancer is caused by high-risk HPV infections, and NFX1-123 is a protein partner with the HPV oncoprotein E6. Together, NFX1-123 and E6 affect cellular growth, longevity, differentiation, and the immune response. The expression status of NFX1-123 in cancers beyond cervical cancer, and its potential as therapeutic target, has not been investigated. Methods: TSVdb of TCGA was used to quantify NFX1-123 expression in 25 primary cancers tissues compared to adjacent normal tissues. The NFX1-123 protein structure was predicted using I-TASSER: Interactive Threading ASSEmbly Refinement tool. The modeled structure was submitted to the MTi-Openscreen Virtual screening web-server using ZINC-Database to retrieve suitable drug molecules, and further screening was evaluated by PyRx interphase and AutoDock Vina. The top four compounds, found to bind in silico to NFX1-123, were tested experimentally to determine their inhibitory effects on NFX1-123-related cellular growth and survival by MTT assay. Results: 44% of cancers (11 of 25) had significant differences in NFX1-123 expression when compared to adjacent normal solid tissues. Nine of 11 cancers (88%) had greater NFX1-123 expression: breast invasive carcinoma, cholangiocarcinoma, colon adenocarcinoma, renal cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, sarcoma, and stomach adenocarcinoma. Two of 11 cancers (18%) had reduced NFX1-123 expression: lung squamous cell carcinoma, and pheochromocytoma and paraganglioma. Additionally, HPV+ head and neck cancers had greater expression of NFX1-123 compared to HPV- head and neck cancers. Bioinformatics and proteomic predictive analysis revealed the 3-D structure of the NFX1-123; with this structure, drug libraries were screened for high binding affinity compounds. 17 drugs with binding energies range from -11.3 to -10 Kcal/mol. were found. The top four compounds were used to treat HPV- and HPV+ cervical and head and neck cancer cell lines in culture, and two (R428 and Ketoconazole) were found to reduce NFX1-123 protein levels and inhibit cell growth and survival. R428 was also found to inhibit NFX1-123 and regulate autophagy and autophagy-mediated cell death. Conclusion: Nine out of 25 (36%) cancers expressed high levels of NFX1-123, and drug targeting of NFX1-123 can lead to cell growth inhibition. NFX1-123 may be a potential novel therapeutic target in cancers that highly express NFX1-123. Citation Format: Sreenivasulu Chintala, Anand Anbarasu, Rachel A. Katzenellenbogen. NFX1-123: A potential therapeutic target in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1210.

The complex and heterogeneous nature of neuroblastoma poses a great challenge to the scientific community for developing an effective therapeutic drug. Neuroblastoma tumors show relapse and resistance to available drugs and it is proving to be an obstacle for efficient treatment options. The tumor accounts for about 7% of the malignancies worldwide and is seen mostly in children around one year. Our present study aims to understand various mechanisms underlying Neuroblastoma. The gene interaction network of 92 Neuroblastoma related genes along with a total of 339 functional interactions are used for the gene interaction network construction and analysis. The clustering analysis resulted in four densely interconnected gene clusters. We further performed functional enrichment analysis and found important pathways associated with neuroblastoma in PI3K/AKT pathway, MAPK pathway, Ras pathway, Rap1 pathway, Cytokine- cytokine interaction, Neurotrophin signaling, EGFR tyrosine kinase inhibitor resistance, and VEGF signaling pathways. The predominant genes involved in the enriched pathways are ANGPT2, FGF2, NGFR, NTRK1, NTRK2, PDGFC, TGFA, TP53, VEGFB, VEGFC, BDNF, NRAS, and VEGFA. The topological parameter analysis has shown the genes with more direct interactions are considered as the hub genes and are important in the network. The genes TP53, VEGFA, BDNF, MYCN, and FGF2 with the maximum number of functional interactors, can be used as potential therapeutic targets for Neuroblastoma. The results obtained in our study will be helpful for researchers in better understanding the molecular level associations during the Neuroblastoma progression.

Hydroxyapatite (HAp) is a major mineral component of bones which has excellent bioactivity and biocompatibility making it suitable for various biomedical applications. However, the chemical composi...

Ten important plant parts routinely used in South Indian ethnic food preparation as spices and condiments were investigated for their potential antidyslipidemic properties. The aim of the study was to characterize the biochemical properties of the polyherbal formulation (nutritional supplement) and evaluate its use to control dyslipidemia in patients. Phytochemical evaluation, in vitro α-amylase inhibitory assay, and high performance thin layer chromatography (HPTLC) fingerprinting were carried out with alcoholic extracts of all 10 individual plants and with the nutritional supplement. Investigation in human volunteers was conducted to evaluate the effect on dyslipidemia as measured by serum lipid biomarkers. Sixty-five volunteers were recruited for this study. Biomarker values at baseline and at 6th visit (end of review, 8/9 months) were compared to assess the usefulness of the nutritional supplement in the normalization of lipid biomarkers. In the qualitative analysis of metabolites, the results revealed the presence of various bioactive primary and secondary metabolites that might be responsible for their medicinal attributes. In human volunteers, after supplement intake along with standard therapy, we observed significant decrease in serum cholesterol, triglyceride, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) levels. High-density lipoprotein (HDL) level did not change in test patient volunteers. Reductions in hemoglobin A1C (HBA1C) and postprandial blood sugar levels were observed; the difference was not statistically significant. We believe that the polyherbal formulation of 10 medicinal plants has potent antidyslipidemic activity. Our results contribute for the first time toward documentation of augmented dyslipidemia control by use of the formulation.

Molecular docking of imipenem with OXA-143 CHDL of Acinetobacter baumannii. OXA-143 CHDL - imipenem complex has higher binding interaction than OXA-231 CHDL - imipenem complex. The increased binding affinity of the imipenem against OXA-143 CHDL will increase the rate of hydrolysis and leads to the resistance.Display Omitted Molecular docking and simulations are done for OXA-143 and OXA-231 CHDLs with imipenem.Imipenem has increased binding affinity with OXA-143 CHDL than OXA-231 CHDL.Molecular dynamics study reveals high stability of OXA-143 CHDL- imipenem complex.Lesser stability indicates the effectiveness of imipenem against OXA-231 CHDL. Acinetobacter baumannii (A. baumannii), is a Gram negative, coccobacilli and is associated with nosocomial infections. The bacterium has developed resistance to all known classes of antibiotics. Multi-drug resistant A. baumannii infections have been treated with the carbapenem group of antibiotics like imipenem and meropenem. Recent reports indicate that A. baumannii has acquired resistance to imipenem due to the secretion of carbapenem hydrolysing class D beta-lactamases (CHDLs). Such CHDLs found in carbapenem resistant A. baumannii belongs to OXA-143 and its variant OXA-231, which has Alanine (A) in place of Aspartic acid (D) at sequence position 224. The mutation of the OXA-231 CHDL alters the catalytic activity of the enzyme. Hence, the present study was carried out to find the probable mechanism of imipenem resistance in OXA-143 and OXA-231 (D224A) CHDLs expressing A. baumannii by employing molecular docking and dynamics. Methods Our study reveals that OXA-143 CHDL-imipenem complex has more binding affinity than OXA-231 (D224A) CHDL-imipenem complex. Our results indicate that there is a strong binding affinity of OXA-143 with imipenem when compared with OXA-243 and this mechanism might be the probable reason for imipenem resistance in OXA-143 expressing A. baumannii strains.

Malaria is the major health issue in African, Asian and Mediterranean regions of the world. Due to the emerging resistance by the parasites and mosquitoes for the current medications and insecticides, respectively, the malaria free human world can be attained only by the novel design and development of new anti-malarial drugs. Hence, we attempted to carry out in silico screening of benzene sulfonamide substituted pyrazole-pyrazoline series against Histo aspartic protease. Our results reveal that the 65% of the data set with the free binding energy in the range of -11.58 to -11.21 kcal/mol, which is categorized as 'high scoring'. Ligands are docked with the catalytic residues Asp 215, Ser 75, Thr 33 and Ala 217, respectively. Molecular dynamic simulation study of free enzyme and the enzyme complex with 4-(5-(4-methoxyphenyl)-1'phenyl-3'-(p-tolyl)-3,4-1'H,2H-[3,4'-bipyrazol]-2-yl)benezenesulfonamide indicated structural stability. The trajectory analysis of complex reveals that the HAP-ligand complex is more stable than the free HAP. We are of the opinion that our results will be useful for designing potential anti-malarial compounds. AbbreviationsADTauto dock toolsBSPPbenzene sulfonamide substituted pyrazole-pyrazolineCQchloroquineHAPhisto aspartic proteaseKKelvinMDmolecular dockingMM/PBSAmolecular mechanics/Poisson Boltzmann surface areaNVTnormal volume and temperatureNPTnormal pressure and temperatureNsnanosecondsPDBprotein data bank.pdbprogram data base format

The class A β-lactamase penA gene codes for penicillin-binding protein 2 (PBP2) which plays an important role in assembling the peptidoglycans on the outer side of the plasma membrane. The alteration in the structure of PBP2 protein makes the pathogen to gain resistance against penicillin. Thus, it is important to understand the role of drug-resistant mechanism by penA gene to develop potent drugs against penicillin-resistant pathogenic strains. In our study, we have used gene interaction network analysis of penA gene in various bacteria to understand its role in drug-resistant mechanisms. We have collected a total of 1039 interactions from 28 organisms available from STRING database. The penA gene interaction network was constructed using Cytoscape 3.6.1. The network analysis has shown that, along with penA gene, the genes murG, ftsW, murC, ftsA, and ftsQ are observed to have more number of interactors and they may be considered as the key candidates to understand the penA drug-resistant mechanism. Functional enrichment analysis has shown the important GO terms and pathways where penA gene plays an important role. We have also elucidated the evolutionary relationship of penA gene in various Gram-positive and Gram-negative bacteria. Our study helps in understanding the drug-resistant patterns of penA gene in various bacteria and also their evolutionary relationships.

The antimicrobial resistance (AMR) exhibited against broad spectrum and new generation antibiotics used for Pseudomonas infections is a major threat and renders the treatment ineffective. In our present study, we have used a computational approach to understand various drug resistance mechanisms which contribute to Multi-Drug Resistance (MDR) in P. aeruginosa. The interaction network of 60 AMR genes along with the 337 functional interactions was analyzed. Functional enrichment analysis of AMR genes has shown that the genes in the network are mainly associated with efflux pump mechanisms, alginate biosynthesis, biofilm formation, and ampC beta-lactamase biosynthesis. Interestingly, the genes phoP, phoQ, and cat genes are observed to have roles in more than one drug-resistant mechanism. The genes phoP and phoQ apart from their role in two-component regulatory systems also play major roles in multidrug efflux pumps and alteration in drug target. The gene cat involves in alteration of drug target and enzymatic inactivation. The interaction network analysis has shown that the AMR genes oprJ, oprM, oprN, ampC, gyrA, mexA, oprD, mexB and nfxB have higher number of direct interactors and they are considered as the hub nodes in the network and these genes can be used as potential drug targets for developing new drugs. The results from our study will be helpful in better understanding of the antibiotic resistance mechanisms in P. aeruginosa. The gene targets reported, can be used for new drug discovery against Pseudomonas infections.