Combination of alpha-melanocyte stimulating hormone with conventional antibiotics against methicillin resistant Staphylococcus aureus.

Our previous studies revealed that alpha-melanocyte stimulating hormone (α-MSH) is strongly active against Staphylococcus aureus (S. aureus) including methicillin resistant S. aureus (MRSA). Killing due to α-MSH occurred by perturbation of the bacterial membrane. In the present study, we investigated the in vitro synergistic potential of α-MSH with five selected conventional antibiotics viz., oxacillin (OX), ciprofloxacin (CF), tetracycline (TC), gentamicin (GM) and rifampicin (RF) against a clinical MRSA strain which carried a type III staphylococcal cassette chromosome mec (SCCmec) element and belonged to the sequence type (ST) 239. The strain was found to be highly resistant to OX (minimum inhibitory concentration (MIC) = 1024 µg/ml) as well as to other selected antimicrobial agents including α-MSH. The possibility of the existence of intracellular target sites of α-MSH was evaluated by examining the DNA, RNA and protein synthesis pathways. We observed a synergistic potential of α-MSH with GM, CF and TC. Remarkably, the supplementation of α-MSH with GM, CF and TC resulted in ≥ 64-, 8- and 4-fold reductions in their minimum bactericidal concentrations (MBCs), respectively. Apart from membrane perturbation, in this study we found that α-MSH inhibited ≈ 53% and ≈ 47% DNA and protein synthesis, respectively, but not RNA synthesis. Thus, the mechanistic analogy between α-MSH and CF or GM or TC appears to be the reason for the observed synergy between them. In contrast, α-MSH did not act synergistically with RF which may be due to its inability to inhibit RNA synthesis (<10%). Nevertheless, the combination of α-MSH with RF and OX showed an enhanced killing by ≈ 45% and ≈ 70%, respectively, perhaps due to the membrane disrupting properties of α-MSH. The synergistic activity of α-MSH with antibiotics is encouraging, and promises to restore the lost potency of discarded antibiotics

Determining bactericidal concentrations of each tested antibacterial agent.

<p>(a) Killing curves showing bactericidal activity of five different tested antibiotics against clinical MRSA isolate. Concentrations of each antibiotic were selected from 0 to 2048 µg/ml. Symbols; Oxacillin (red diamond), Gentamicin (green square), Rifampicin (yellow triangle), Ciprofloxacin (pink square) and Tetracycline (orange circle). (b) Bactericidal activity of α-MSH (2–160 µg/ml) against clinical MRSA isolate. The killing assay was done in triplicate and repeated on three different occasions. *p value ≤0.001, **p value ≤0.01, ***p value ≤0.05.</p

Minimum bactericidal concentration (MBC₅₀ and MBC₉₀) values of selected antimicrobial agents when used alone and in the presence of 8 µg/ml of α-MSH against clinical MRSA strain.

<p>Note: Fold reduction = (MBC₅₀ of antibiotic alone/MBC₅₀ in presence of α-MSH).</p><p>NA*means MBC₉₀ not achieved.</p

Minimum Inhibitory concentration (MIC) values of selected antibiotics against <i>S. aureus</i> strains as determined by broth microdilution assay following CLSI guidelines [25].

<p>Note: (S) Strain susceptible to tested antibiotic.</p><p>(R) Strain resistant to tested antibiotic.</p

Killing curves of each of antibiotic alone and in presence of α-MSH against clinical MRSA isolate.

<p>(a) Gentamicin, (b) Tetracycline, (c) Ciprofloxacin, (d) Oxacillin, and (e) Rifampicin. Symbols; antibiotic alone (diamond) and antibiotic+α-MSH (8 µg/ml) (square). Experiments were repeated on three independent days. p value ≤0.05 (when multiple comparisons were done among % survival data-sets of different concentrations of same antibiotic with and without α-MSH).</p

Impact of sub-lethal doses of α-MSH on DNA, RNA and protein synthesis of <i>S. aureus</i> ATCC 29213.

<p>(a) % of radioactivity of thymidine in untreated control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of ciprofloxacin (blue); (b) % of radioactivity of uridine in control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of rifampicin (blue); (c) % of radioactivity of leucine in control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of tetracycline (blue); (d) killing kinetics of 2 µg/ml of α-MSH (diamond), and 10 µg/ml of α-MSH (square) against ∼10⁸ CFU/ml of <i>S. aureus</i> ATCC 29213. Experiments were done in duplicate and repeated on three independent days. *p value ≤0.001, **p value ≤0.01, ***p value ≤0.05.</p

[2Fe-2S] cluster transfer in iron-sulfur protein biogenesis

Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein-protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins

Effectiveness of ayurvedic formulation, NAOQ19 along with standard care in the treatment of mild-moderate COVID-19 patients: A double blind, randomized, placebo-controlled, multicentric trial

Background: Medicines in indigenous systems such as Ayurveda have strong antimicrobial activity but double-blind randomized control trials are infrequent in this system of medicine. The efficacy of a new ayurvedic formulation was evaluated during the pandemic. Methods: 150 mild-moderate COVID-19 patients were enrolled and randomized in 1:1 to NAOQ19 and placebo group. RT-PCR was done on Day 3, 5 and 7. CBC, CRP, LFT, and KFT were assessed at baseline and exit. Duration of hospital stay was noted and clinical assessment was also performed. Result: The results demonstrated more people turning RT-PCR negative in the NAOQ19 group compared to the placebo group on day 3 (p-value = 0.033). The mean time duration to turn RT-PCR negative was significantly lower in the NAOQ19 group (4.6 days) compared to placebo group (5.2 days) (p-value = 0.018). There was significant reduction in hospital stay among patients in the NAOQ19 arm who were discharged earlier (5.6 days) compared to placebo group (6.4 days) (p-value = 0.046). Patients in NAOQ19 arm did not show any adverse life-threatening events. Conclusion: The ayurvedic preparation given along with standard of care therapy reduced the duration of hospital stay and there was earlier conversion to RT-PCR negative.The integrated approach can help to reduce patient workload in the hospitals as well as limit the transmission of the virus in the community. Study registration: CTRI/2021/05/033790

Epidemiology of Congenital Rubella Syndrome (CRS) in India, 2016-18, based on data from sentinel surveillance.

BACKGROUND:Government of India is committed to eliminate measles and control rubella/congenital rubella syndrome (CRS) by 2020. In 2016, CRS surveillance was established in five sentinel sites. We analyzed surveillance data to describe the epidemiology of CRS in India. METHODOLOGY/PRINCIPAL FINDINGS:We used case definitions adapted from the WHO-recommended standards for CRS surveillance. Suspected patients underwent complete clinical examination including cardiovascular system, ophthalmic examination and assessment for hearing impairment. Sera were tested for presence of IgM and IgG antibodies against rubella. Of the 645 suspected CRS patients enrolled during two years, 137 (21.2%) were classified as laboratory confirmed CRS and 8 (1.2%) as congenital rubella infection. The median age of laboratory confirmed CRS infants was 3 months. Common clinical features among laboratory confirmed CRS patients included structural heart defects in 108 (78.8%), one or more eye signs (cataract, glaucoma, pigmentary retinopathy) in 82 (59.9%) and hearing impairment in 51. (38.6%) Thirty-three (24.1%) laboratory confirmed CRS patients died over a period of 2 years. Surveillance met the quality indicators in terms of adequacy of investigation, adequacy of sample collection for serological diagnosis as well as virological confirmation. CONCLUSIONS/SIGNIFICANCE:About one fifth suspected CRS patients were laboratory confirmed, indicating significance of rubella as a persistent public health problem in India. Continued surveillance will generate data to monitor the progress made by the rubella control program in the country