Protective efficacy of Alum adjuvanted Amidase protein vaccine against Staphylococcus aureus infection in multiple mouse models.

AIMS: Staphylococcus aureus is an opportunistic pathogen of humans. No commercial vaccine is available to combat S. aureus infections. In this study, we have investigated the protective immune response generated by S. aureus non-covalently associated cell wall surface protein N-acetylmuramoyl-L-alanine amidase (AM) in combination with Alum (Al) and heat-killed S. aureus (hkSA) using murine models. METHODS AND RESULTS: BALB/c mice were immunized with increasing concentrations of AM antigen or hkSA to determine their optimum concentration for vaccination. Fifty micrograms of AM and hkSA each were found to generate maximum anti-AM IgG antibody production. BALB/c mice were immunized next with 50 µg of AM, 50 µg of hKSA and 1 mg Al vaccine formulation. Vaccine efficacy was validated by challenging immunized BALB/c mice with S. aureus Newman and three clinical methicillin-resistant S. aureus strains. AM-hkSA-Al-immunized mice generated high anti-AM IgG antibody response with IgG1 and IgG2b as the predominant immunoglobulin subtypes. Increased survival (60%-90%) with decreased clinical disease symptoms was observed in the vaccinated BALB/c mice group. A significantly lower bacterial load and decreased kidney abscess formation was observed following the challenge with S. aureus in the vaccinated BALB/c mice group. Furthermore, the efficacy of AM-hkSA-Al vaccine was also validated using C57 BL/6 and Swiss albino mice. CONCLUSIONS: Using murine infection models, we have demonstrated that AM-hkSA-Al vaccine would be effective in preventing S. aureus infections. SIGNIFICANCE AND IMPACT OF STUDY: AM-hkSA-Al vaccine elicited strong immune response and may be considered for future vaccine design against S. aureus infections.

COVID-19 related multisystem inflammatory syndrome in children (MIS-C): a hospital-based prospective cohort study from Kerala, India.

Objectives: To study (1) epidemiological factors, clinical profile and outcomes of COVID-19 related multisystem inflammatory syndrome in children (MIS-C), (2) clinical profile across age groups, (3) medium-term outcomes and (4) parameters associated with disease severity. Design: Hospital-based prospective cohort study. Setting: Two tertiary care centres in Kerala, India. Participants: Diagnosed patients of MIS-C using the case definition of Centres for Disease Control and Prevention. Statistical analysis: Pearson χ2 test or Fisher's exact test was used to compare the categorical variables and independent sample t-test or Mann-Whitney test was used to compare the continuous variables between the subgroups categorised by the requirement of mechanical ventilation. Bonferroni's correction was used for multiple comparisons. Results: We report 41 patients with MIS-C, mean age was 6.2 (4.0) years, and 33 (80%) were previously healthy. Echocardiogram was abnormal in 23 (56%), and coronary abnormalities were noted in 15 (37%) patients. Immunomodulatory therapy was administered to 39 (95%), steroids and IVIg both were used in 35 (85%) and only steroids in 3 (7%) patients. Intensive care was required in 36 (88%), mechanical ventilation in 8 (20%), inotropic support in 21 (51%), and 2 (5%) patients died. Mechanical ventilation requirement in MIS-C was associated with hyperferritinaemia (p=0.001). Thirty-seven patients completed 3 months follow-up by April 2021, of whom 6 (16%) patients had some residual echocardiographic changes. Conclusions: Patients with MIS-C in our cohort had varied clinical manifestations ranging from fever with mild gastrointestinal and mucocutaneous involvement to fatal multiorgan dysfunction. Immediate and medium-term outcomes remain largely excellent except for the echocardiographic sequelae in a few patients which are also showing a resolving trend. Hyperferritinaemia was associated with the requirement of mechanical ventilation.

Sub micron aerosol variability and its ageing process at a high altitude site in India: Impact of meteorological conditions.

The effect of relative humidity and temperature on the submicron aerosol variability and its ageing process was studied over a high altitude site, Mahabaleshwar in south-west India. The mass composition of non-refractory particulate matter of 1 µm (NR-PM1) size was obtained using Time of Flight Aerosol Chemical Speciation Monitor (ToF-ACSM) along with the measurements on a few trace gases during winter (December 2017-February 2018) and summer season (20th March - 5th May 2018). Sulfate exhibited strong dependence on the relative humidity (RH) as its mass fraction increased with the increase in RH. The Sulfate oxidation ratio (SOR) calculated during summer season also showed an increasing trend with RH indicating the influence of aqueous phase oxidation on sulfate fraction. On the other hand, OOA showed remarkable enhancement in its mass fraction with the increase in temperature along with the corresponding increase in f44 and tropospheric ozone. OOA, ozone and f44 ratio increased 14-34%, 8-26% and 25-43% respectively with the increase in temperature from 18 to 30 °C. This is indicative of the dominance of photochemical ageing processes during high temperature conditions. The extent of photochemical ageing was found to be higher during summer season (mean temperature ∼25.4 ± 2.6 °C) as compared to winter season (mean temperature ∼20.5 ± 2.6 °C). The nitrate diurnal was majorly governed by gas to particle partitioning process during winter season, whereas the summertime nitrate diurnal was influenced primarily by its formation rate. The non parametric wind regression analysis revealed that the mass concentration during winter was majorly contributed by distant sources from north east direction while during summer the local sources were more dominant.

The cell surface adhesins of Mycobacterium tuberculosis.

Bacterial cell surface adhesins play a major role in facilitating host colonization and subsequent establishment of infection. The surface of Mycobacterium tuberculosis, owing to the complex architecture of its cell envelope, expresses numerous adhesins with varied chemical nature, including proteins, lipids, lipoproteins, glycoproteins and glycopolymers. Studies on mycobacterial adhesins show that they bind with multifarious host receptors and extracellular matrix (ECM) components. In this review we have highlighted the adhesins that are abundantly present on the mycobacterial surface and their interactions with host receptors. M. tuberculosis interacts with various host cell surface receptors such as toll like receptors, C-type lectin receptors, scavenger receptors, and Fc and complement receptors. Apart from these, ECM components like fibronectin, collagen, elastin, laminin, fibrillin and vitronectin also provide binding sites for surface adhesins of the tubercle bacilli. M. tuberculosis adhesins include proteins with and without signal peptide sequence and transmembrane proteins. Other surface adhesin macromolecules of M. tuberculosis comprises of lipids, glycolipids and glycopolymers. The interaction between the mycobacterial adhesins and their host receptors result in adhesion of the microbe to the host cells, induction of immune response and aid in the pathogenesis of the disease. A thorough understanding of the different M. tuberculosis surface adhesins and host receptors will provide a better picture of interaction between them at molecular level. The information gained on adhesins and host receptors will prove beneficial in developing novel therapeutic strategies such as the use of anti-adhesin molecules to hinder the adhesion of bacteria to the host cells, thereby preventing establishment of infection. The surface molecules discussed in this review will also benefit in identification of new drug targets, diagnostic markers or vaccine candidates against the deadly pathogen.

Combination of Repurposed Drug Diosmin with Amoxicillin-Clavulanic acid Causes Synergistic Inhibition of Mycobacterial Growth.

Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.

Antistaphylococcal and Neutrophil Chemotactic Injectable κ-Carrageenan Hydrogel for Infectious Wound Healing.

Staphylococcus aureus wound infection is a major concern due to the resistance of S. aureus to topical antibiotics and capacity to inhibit neutrophil migration at the infection site. To overcome these problems, we have developed 0.01% (v/v) octenidine dihydrochloride (Oct) and 0.5% (w/w) chitosan-treated serum (CTS) containing 1.5% (w/v) κ-carrageenan hydrogel (κC). Oct is an antiseptic agent, against which no resistance is reported so far, and CTS has neutrophilic attractant properties. The prepared Oct-CTS-κC hydrogel is injectable and biocompatible. Using in vitro experiments, we demonstrated CTS can induce the migration of polymorphonuclear neutrophils (PMNs) and fibroblasts that can facilitate tissue regeneration at a wound site. In vitro release studies revealed a sustained release of Oct and serum proteins from the Oct-CTS-κC hydrogel. Antibacterial properties of developed hydrogels were tested against S. aureus and its clinical isolates. Further, the in vivo antibacterial efficacy of the prepared hydrogel was evaluated in an S. aureus-infected Sprague-Dawley (SD) rat wound. Both in vitro and in vivo studies showed that the Oct-CTS-κC hydrogel inhibited S. aureus growth. Thus, the developed Oct-CTS-κC hydrogel can be potentially exploited for S. aureus-infected wound healing.

Point prevalence surveys of antimicrobial use among eight neonatal intensive care units in India: 2016.

BACKGROUND: Information about antimicrobial use is scarce and poorly understood among neonatal intensive care units (NICUs) in India. In this study, we describe antimicrobial use in eight NICUs using four point prevalence surveys (PPSs). METHODS: As part of the Global Antimicrobial Resistance, Prescribing, and Efficacy in Neonates and Children (GARPEC) study, one-day, cross-sectional, hospital-based PPSs were conducted four times between 1 February 2016 and 28 February 2017 in eight NICUs. Using a standardized web-based electronic data entry form, detailed data were collected for babies on antimicrobials. RESULTS: A total of 403 babies were admitted to NICUs across all survey days, and 208 (51.6%) were prescribed one or more antimicrobials. Among 208 babies, 155 (74.5%) were prescribed antimicrobials for treatment of an active infection. Among 155 babies with an active infection, treatment was empiric in 109 (70.3%). Sepsis (108, 49.1%) was the most common reason for prescribing antimicrobials. Amikacin (17%) followed by meropenem (12%) were the two most commonly prescribed antimicrobials. For community-acquired sepsis, piperacillin-tazobactam (17.5%) was the most commonly prescribed drug. A combination of ampicillin and gentamicin was prescribed in only two babies (5%). CONCLUSIONS: The recommended first-line antimicrobial agents, ampicillin and gentamicin, were rarely prescribed in Indian NICUs for community acquired neonatal sepsis.

Understanding the adhesion mechanism of a mucin binding domain from Lactobacillus fermentum and its role in enteropathogen exclusion.

Lactobacillus species possesses surface exposed Mucin Binding Protein (MucBP) which plays a role in adhesion to gastrointestinal mucin. MucBP contains one or more mucin binding domain (MBD), the functionality of which has yet not been characterized thoroughly. Here, we have characterized a 93-amino acid MBD (MBD93) of MucBP (LAF_0673) from Lactobacillus fermentum. Multiple sequence alignment of L. fermentum MBD93 exhibited ∼60% sequence homology with MBDs from other Lactobacillus species. Further, we cloned, expressed and purified MBD93 from Escherichia coli as N-terminal histidine-tagged protein (6X His-MBD93). The purified MBD93 was able to bind to mucin and showed strong affinity towards the terminally expressed mucin glycans viz. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), Galactose (Gal), and Sialic acid (N-acetylneuraminic acid; Neu5Ac). In silico experiments further confirmed the interaction between homology modeled MBD93 to mucin glycans through hydrogen-bonding with its surface amino acid residues Ser57, Pro58, Ile60, Tyr63 and Ala65. We also have demonstrated that MBD93 was able to inhibit the adhesion of enteric pathogens, including E. coli, Salmonella Paratyphi A, Shigella sonnei and Proteus vulgaris to mucin. Our results suggested that L. fermentum MBD93 is a functionally sufficient unit to act as an adhesin and to protect from invading enteric pathogens.

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C4-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

Point Prevalence Surveys of Antimicrobial Use among Hospitalized Children in Six Hospitals in India in 2016.

The prevalence of antimicrobial resistance in India is among the highest in the world. Antimicrobial use in inpatient settings is an important driver of resistance, but is poorly characterized, particularly in hospitalized children. In this study, conducted as part of the Global Antimicrobial Resistance, Prescribing, and Efficacy in Neonates and Children (GARPEC) project, we examined the prevalence of and indications of antimicrobial use, as well as antimicrobial agents used among hospitalized children by conducting four point prevalence surveys in six hospitals between February 2016 and February 2017. A total of 681 children were hospitalized in six hospitals across all survey days, and 419 (61.5%) were prescribed one or more antimicrobials (antibacterials, antivirals, antifungals). Antibacterial agents accounted for 90.8% (547/602) of the total antimicrobial prescriptions, of which third-generation cephalosporins (3GCs) accounted for 38.9% (213/547) and penicillin plus enzyme inhibitor combinations accounted for 14.4% (79/547). Lower respiratory tract infection (LRTI) was the most common indication for prescribing antimicrobials (149 prescriptions; 24.8%). Although national guidelines recommend the use of penicillin and combinations as first-line agents for LRTI, 3GCs were the most commonly prescribed antibacterial agents (55/149 LRTI prescriptions; 36.9%). In conclusion, 61.5% of hospitalized children were on at least one antimicrobial agent, with excessive use of 3GCs. Hence there is an opportunity to limit their inappropriate use.

Understanding the molecular differential recognition of muramyl peptide ligands by LRR domains of human NOD receptors.

Human nucleotide-binding oligomerization domain proteins, hNOD1 and hNOD2, are host intracellular receptors with C-terminal leucine-rich repeat (LRR) domains, which recognize specific bacterial peptidoglycan (PG) fragments as their ligands. The specificity of this recognition is dependent on the third amino acid of the stem peptide of the PG ligand, which is usually meso-diaminopimelic acid (mesoDAP) or l-lysine (l-Lys). Since the LRR domains of hNOD receptors had been experimentally shown to confer the PG ligand-sensing specificity, we developed three-dimensional structures of hNOD1-LRR and the hNOD2-LRR to understand the mechanism of differential recognition of muramyl peptide ligands by hNOD receptors. The hNOD1-LRR and hNOD2-LRR receptor models exhibited right-handed curved solenoid shape. The hot-spot residues experimentally proved to be critical for ligand recognition were located in the concavity of the NOD-LRR and formed the recognition site. Our molecular docking analyses and molecular electrostatic potential mapping studies explain the activation of hNOD-LRRs, in response to effective molecular interactions of PG ligands at the recognition site; and conversely, the inability of certain PG ligands to activate hNOD-LRRs, by deviations from the recognition site. Based on molecular docking studies using PG ligands, we propose few residues - G825, D826 and N850 in hNOD1-LRR and L904, G905, W931, L932 and S933 in hNOD2-LRR, evolutionarily conserved across different host species, which may play a major role in ligand recognition. Thus, our integrated experimental and computational approach elucidates the molecular basis underlying the differential recognition of PG ligands by hNOD receptors.

Bacterial peptidoglycan with amidated meso-diaminopimelic acid evades NOD1 recognition: an insight into NOD1 structure-recognition.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an intracellular pattern recognition receptor that recognizes bacterial peptidoglycan (PG) containing meso-diaminopimelic acid (mesoDAP) and activates the innate immune system. Interestingly, a few pathogenic and commensal bacteria modify their PG stem peptide by amidation of mesoDAP (mesoDAPNH2). In the present study, NOD1 stimulation assays were performed using bacterial PG containing mesoDAP (PGDAP) and mesoDAPNH2 (PGDAPNH2) to understand the differences in their biomolecular recognition mechanism. PGDAP was effectively recognized, whereas PGDAPNH2 showed reduced recognition by the NOD1 receptor. Restimulation of the NOD1 receptor, which was initially stimulated with PGDAP using PGDAPNH2, did not show any further NOD1 activation levels than with PGDAP alone. But the NOD1 receptor initially stimulated with PGDAPNH2 responded effectively to restimulation with PGDAP The biomolecular structure-recognition relationship of the ligand-sensing leucine-rich repeat (LRR) domain of human NOD1 (NOD1-LRR) with PGDAP and PGDAPNH2 was studied by different computational techniques to further understand the molecular basis of our experimental observations. The d-Glu-mesoDAP motif of GMTPDAP, which is the minimum essential motif for NOD1 activation, was found involved in specific interactions at the recognition site, but the interactions of the corresponding d-Glu-mesoDAP motif of PGDAPNH2 occur away from the recognition site of the NOD1 receptor. Hot-spot residues identified for effective PG recognition by NOD1-LRR include W820, G821, D826 and N850, which are evolutionarily conserved across different host species. These integrated results thus successfully provided the atomic level and biochemical insights on how PGs containing mesoDAPNH2 evade NOD1-LRR receptor recognition.

Antimicrobial activity of plumbagin, a naturally occurring naphthoquinone from Plumbago rosea, against Staphylococcus aureus and Candida albicans.

Candida albicans and Staphylococcus aureus are opportunistic pathogens. Despite causing a number of independent infections, both pathogens can co-infect to cause urinary tract infections, skin infections, biofilm associated infections, sepsis and pneumonia. Infections of these two pathogens especially their biofilm associated infections are often difficult to treat using currently available anti-bacterial and anti-fungal agents. In order to identify a common anti-microbial agent which could confer a broad range of protection against their infections, we screened several phytochemicals and identified plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a phytochemical from Plumbago species as a potent antimicrobial agent against S. aureus and C. albicans, with a minimum inhibitory concentration of 5µg/ml. Antimicrobial activity of plumbagin was validated using an ex-vivo porcine skin model. For better understanding of the antimicrobial activity of plumbagin, a Drosophila melanogaster infection model was used, where D. melanogaster was infected using S. aureus and C. albicans, or with both organisms. The fly's survival rate was dramatically increased when infected flies were treated using plumbagin. Further, plumbagin was effective in preventing and dispersing catheter associated biofilms formed by these pathogens. The overall results of this work provides evidence that plumbagin, possesses an excellent antimicrobial activity which should be explored further for the treatment of S. aureus and C. albicans infections.

Congenital Cystic Adenomatoid Malformation: A Tertiary Care Hospital Experience.

INTRODUCTION: Congenital Cystic Adenomatoid Malformation (CCAM) is an uncommon developmental deformity affecting the terminal respiratory structures. It is characterized by broncho pulmonary foregut malformations. The reason behind it is an arrest in lung development between 4th and 7th week of fetal life. AIM: The present study was conducted to assess the clinical and radiological profile and also to study the role of surgical intervention in patients with CCAM. MATERIALS AND METHODS: All patients with clinical suspicion or provisional diagnosis of CCAM were included in the study. A clinical questionnaire was prepared to collect data. Computed Tomography (CT) chest with High Resolution Computed Tomography (HRCT) was done for all the patients. Patients were assessed by paediatric surgeon and eligible patients were operated. The procedure conducted was usually open thoracotomy under general anaesthesia. The affected lobes were removed and specimens were sent for histopathological analysis. All included patients were followed up prospectively to find out about their current level of health. Via telephonic interview they were asked about their overall growth, quality of life, activity, rate of respiratory infections and requirement of hospital admission. RESULTS: Total 15 patients with diagnosis of CCAM were included in the study. Of them, 8 (53.3%) were male. The commonest presentation was cough 13(86%), breathing difficulty 11(73%), fever 9(60%), recurrent pneumonia 4(26%), hypoxia requiring oxygen supplementation 6(40%), others 2(12%). Thirteen patients required surgical intervention and underwent lobectomy. There were 2 cases of type I, one each of type II and III, 3 case of type IV while 5 were intermediate type. There was no procedure related mortality. The median duration of hospital stay and all were successfully discharged with median duration of stay 11±16 days. CONCLUSION: The study concludes that if recognized early, surgical removal of affected lung prevents the complications like recurrent pulmonary infections. The surgery is well tolerated without any post-operative mortality or morbidity.

Amidase, a cell wall hydrolase, elicits protective immunity against Staphylococcus aureus and S. epidermidis.

The morbidity and the mortality associated with Staphylococcus aureus and S. epidermidis infections have greatly increased due to the rapid emergence of highly virulent and antibiotic resistant strains. Development of a vaccine-based therapy is greatly desired. However, no staphylococcal vaccine is available till date. In this study, we have identified Major amidase (Atl-AM) as a prime candidate for future vaccine design against these pathogens. Atl-AM is a multi-functional non-covalently cell wall associated protein which is involved in staphylococcal cell separation after cell division, host extracellular matrix adhesion and biofilm formation. Atl-AM is present on the surface of diverse S. aureus and S. epidermidis strains. When used in combination with Freund's adjuvant, Atl-AM generated a mixed Th1 and Th2 mediated immune response which is skewed more toward Th1; and showed increased production of opsonophagocytic IgG2a and IgG2b antibodies. Significant protective immune response was observed when vaccinated mice were challenged with S. aureus or S. epidermidis. Vaccination prevented the systemic dissemination of both organisms. Our results demonstrate the remarkable efficacy of Atl-AM as a vaccine candidate against both of these pathogens.