Mapping ACE2 and TMPRSS2 co-expression in human brain tissue: implications for SARS-CoV-2 neurological manifestations.

The Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets respiratory cells, but emerging evidence shows neurological involvement, with the virus directly affecting neurons and glia. SARS-CoV-2 entry into a target cell requires co-expression of ACE2 (Angiotensin-converting enzyme-2) and TMPRSS2 (Trans membrane serine protease-2). Relevant literature on human neurological tissue is sparse and mostly focused on the olfactory areas. This prompted our study to map brain-wide expression of these entry proteins and assess age-related changes. The normal brain tissue samples were collected from cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, brain stem and cerebellum; and were divided into two groups - up to 40 years (n = 10) and above 40 years (n = 10). ACE2 and TMPRSS2 gene expression analysis was done using qRT-PCR and protein co-expression was seen by immunofluorescence. The ACE2 and TMPRSS2 gene expression was observed to be highest in hypothalamus and thalamus regions, respectively. Immunoreactivity for both ACE-2 and TMPRSS2 was observed in all examined brain regions, confirming the presence of these viral entry receptors. Co-localisation was maximum in hypothalamus. Our study did not find any trend related to different age groups. The expression of both these viral entry receptors suggests that normal human brain is susceptibility to SARS-CoV-2, perhaps which could be related to the cognitive and neurological impairment that occur in patients.

Heavy metal-induced selection and proliferation of antibiotic resistance: A review.

Antibiotic resistance is recognized as a global threat to public health. The selection and evolution of antibiotic resistance in clinical pathogens were believed to be majorly driven by the imprudent use of antibiotics. However, concerns regarding the same, through selection pressure by a multitude of other antimicrobial agents, such as heavy metals, are also growing. Heavy metal contamination co-selects antibiotic and metal resistance through numerous mechanisms, such as co-resistance and cross-resistance. Here, we have reviewed the role of heavy metals as antimicrobial resistance driving agents and the underlying concept and mechanisms of co-selection, while also highlighting the scarcity of studies explicitly inspecting the process of co-selection in clinical settings. Prospective strategies to manage heavy metal-induced antibiotic resistance have also been deliberated, underlining the need to find specific inhibitors so that alternate medicinal combinations can be added to the existing therapeutic armamentarium.

Potential of 1-(1-napthylmethyl)-piperazine, an efflux pump inhibitor against cadmium-induced multidrug resistance in Salmonella enterica serovar Typhi as an adjunct to antibiotics.

This study was focused on elucidating inhibition of antibiotic efflux mechanism of cadmium adapted (CdA) Salmonella Typhi Ty2 cells. Herein, upregulated expression of efflux genes (acrB, tolC) and their regulators (soxS, marA) was observed in CdA Ty2 cells by qRT-PCR. The pathogen further elevated the expression of these genes even in the presence of three efflux pump inhibitors (EPIs), i.e., Phe-Arg-ß-naphthylamide, 1-(1-naphthyl-methyl)piperazine, and 5-hydroxy-2-methyl-1,4-naphthoquinone, perhaps by sensing the pressure of the latter in addition to cadmium stress. Interaction of different EPIs with efflux pumps of CdA Ty2 cells was confirmed using ethidium bromide (EtBr) accumulation and efflux assay. All the EPIs could cause retention of EtBr which was indicated by increased fluorescence units. Considering this potential of EPIs, retention of antibiotics was evaluated in CdA Ty2 cells wherein EPIs were used in combination with selected antibiotics (instead of EtBr). A decrease in the effective concentration of antibiotics was observed. This was further validated using the clinical isolates. The data revealed the efficiency of EPIs as they could inhibit the efflux potential of even the overexpressed efflux pumps. Thus, combination of EPI(s)-antibiotics may be exploited in future as one of the strategies for combating metal induced antibiotic resistance.

Peptides as adjuvants for ampicillin and oxacillin against methicillin-resistant Staphylococcus aureus (MRSA).

Fast emerging antibiotic resistance in pathogens requires special attention for strengthening the reservoir of antimicrobial compounds. In view of this, several peptides with known antimicrobial activities have been reported to enhance the efficacy of antibiotics against multidrug resistant (MDR) pathogens. In the present study, potential of peptides having distinct mechanism of action, if any, was evaluated to improve the efficacy of conventional antibiotics against methicillin-resistant S. aureus (MRSA). After primary screening of six peptides, two peptides namely T3 and T4 showing very high minimum inhibitory concentrations (MICs) were selected to assess their role in altering the MICs of antibiotics to which the pathogen was resistant. In the presence of the peptides, the MICs of the antibiotics were found to be reduced as per the fractional inhibitory concentration indices (FICI) and time kill assay. These observations prompted us to look for their mechanism of action. The effect of peptides on the morphology of pathogen by field emission scanning electron microscopy (FE-SEM) revealed no damage to the cells at the sub-inhibitory concentrations of the peptide which correlated well with the higher MIC of the peptide, indicating no direct impact on the pathogen. However, dielectric spectroscopy, confocal microscopy and flow cytometry confirmed the interaction and localization of peptides with the bacterial membrane. The peptides were also found to inhibit efflux of ethidium bromide which is the substrate for many proteins involved in efflux system. Therefore, it is speculated that the peptides after interacting with the membrane of the pathogen might have resulted in the inhibition of the efflux of antibiotics thereby reducing their effective concentrations. The study thus suggests that peptides with no antimicrobial activity of their own, can also enhance the efficacy of the antibiotics by interacting with the pathogen thereby, acting as adjuvants for the antibiotics.

Augmented antibiotic resistance associated with cadmium induced alterations in Salmonella enterica serovar Typhi.

In view of the reports on co-selection of metal and antibiotic resistance, recently we have reported that increased cadmium accumulation in Salmonella Typhi Ty2 leads to increased antibiotic resistance. In continuation, the present study was carried to substantiate this association in clinical isolates. Interestingly, the levels of cadmium were found to be more in the clinical isolates which co-related with their antibiotic sensitivity/resistance pattern. On cadmium accumulation, antibiotic(s) sensitive isolates were rendered resistant and the resistant isolates were rendered more resistant as per their minimum inhibitory concentration(s). Further, after subjecting the pathogen to cadmium accumulation, alterations occurring in the cells were assessed. Transgenerational cadmium exposure led to changes in growth response, morphology, proteome, elevated antioxidants other than SOD, increased biofilm formation, decreased intracellular macrophage killing coupled with upregulation of genes encoding metallothionein and metal transporters. Thus, these results indicate that cadmium, if acquired from the environment, being non-degradable can exert a long-lasting selective pressure on Salmonella in the host which may display antibiotic resistance later on, as a result of co-selection. Therefore, appropriate strategies need to be developed to inhibit such an enduring pressure of heavy metals, as these represent one of the factors for the emerging antibiotic resistance in pathogens.

Management of Staphylococcus Mediated Systemic Infection by Enhancing the Resurging Activity of Co-trimoxazole in Presence of Cryptdin-2.

Resurgence of sensitivity of the antibiotics, to which the pathogen had developed resistance in the past, requires special attention for strengthening the reservoir of antimicrobial compounds. Reports in the recent past have suggested that co-trimoxazole (COT) has regained its activity against methicillin resistant Staphylococcus aureus (MRSA). The present study exploited the use of COT in the presence of an antimicrobial peptide (AMP), cryptdin-2 (a murine Paneth cell alpha defensin), in order to reduce the selective pressure of the antibiotic on the pathogen. In vitro antibacterial activity and in vivo efficacy of the combination was ascertained against MRSA induced systemic infection using a murine model. Observations of the present study might help in restoring the regained activity of conventional antibiotics, such as COT, when used in combination with novel antimicrobial molecules like AMPs. This might prove as a viable strategy to eliminate the chances of re-occurrence of resistance due to their multi-prong targeting and synergistically combating infections caused by these resistant pathogens.

Potential of 2, 2'-dipyridyl diselane as an adjunct to antibiotics to manage cadmium-induced antibiotic resistance in Salmonella enterica serovar Typhi Ty2 strain.

One of the reasons for increased antibiotic resistance in Salmonella enterica serovar Typhi Ty2 is the influx of heavy metal ions in the sewage, from where the infection is transmitted. Therefore, curbing these selective agents could be one of the strategies to manage the emergence of multidrug resistance in the pathogen. As observed in our earlier study, the present study also confirmed the links between cadmium accumulation and antibiotic resistance in Salmonella. Therefore, the potential of a chemically-synthesised compound 2, 2'-dipyridyl diselane (DPDS) was explored to combat the metal-induced antibiotic resistance. Its metal chelating and antimicrobial properties were evidenced by fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and microbroth dilution method. Owing to these properties of DPDS, further, this compound was evaluated for its potential to be used in combination with conventional antibiotics. The data revealed effective synergism at much lower concentrations of both the agents. Thus, it is indicated from the study that the combination of these two agents at their lower effective doses might reduce the chances of emergence of antibiotic resistance, which can be ascribed to the multi-pronged action of the agents.

Better Management of Alcohol Liver Disease Using a 'Microstructured Synbox' System Comprising L. plantarum and EGCG.

Synergistic combination of probiotics with carbohydrate based prebiotics is widely employed for the treatment of various gut related disorders. However, such carbohydrate based prebiotics encourage the growth of pathogens and probiotics, equally. Aim of the study was (i) to explore the possibility of using epigallocatechin gallate (EGCG) a phenolic compound, as a prebiotic for L.plantarum; (ii) to develop and evaluate a microstructured synbox (microencapsulating both probiotic and EGCG together) in rat model of alcohol liver disease (ALD); and, (iii) to confirm whether the combination can address issues of EGCG bioavailability and probiotic survivability in adverse gut conditions. Growth enhancing effect of EGCG on L. plantarum (12.8±0.5 log 10 units) was significantly (p≤0.05) better than inulin (11.4±0.38 log 10 units), a natural storage carbohydrate. The formulated synbox significantly modulated the levels of alcohol, endotoxin, hepatic enzymes and restored the hepatoarchitecture in comparison to simultaneous administration of free agents. Additionally, using a battery of techniques, levels of various cellular and molecular markers viz. NF-kB/p50, TNF-α, IL12/p40, and signalling molecules TLR4, CD14, MD2, MyD88 and COX-2 were observed to be suppressed. Developed microbead synbox, as a single delivery system for both the agents showed synergism and hence, holds promise as a therapeutic option for ALD management.