P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

BACKGROUND: Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. OBJECTIVE: In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. METHODS: Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1ß, Transforming Growth Factor (TGF ß1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene ß-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. RESULTS: The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. CONCLUSIONS: Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

ABC superfamily transporter Rv1273c of Mycobacterium tuberculosis acts as a multidrug efflux pump.

Efflux pump-mediated drug resistance in bacteria is a common occurrence effective for the general survival of the organism. The Mycobacterium tuberculosis genome has an abundance of adenosine triphosphate (ATP) dependent cassette transporter genes but only a handful of them are documented for their contribution to drug resistance. In this study, we inspected the potential of an ABC transporter Rv1273c from M. tuberculosis as a multidrug efflux pump and a contributor to intrinsic drug resistance. Expression of Rv1273c in Escherichia coli and M. smegmatis conferred tolerance to various structurally unrelated antibiotics. Lower accumulation of fluoroquinolones in intact E. coli and M. smegmatis cells expressing the transporter implied its active efflux activity. Energy-dependent efflux by Rv1273c was observed in real time using the lipophilic dye Nile Red. Expression of Rv1273c also resulted in an increase in biofilm formation by E. coli and M. smegmatis cells. Overall, the results indicate the possibility that Rv1273c might be a multidrug transporter with a wide substrate range and a probable contributor to biofilm formation.

A NiCoT family metal transporter of Mycobacterium tuberculosis (Rv2856/NicT) behaves as a drug efflux pump that facilitates cross-resistance to antibiotics.

Metals often act as a facilitator in the proliferation and persistence of antibiotic resistance. Efflux pumps play key roles in the co-selection of metal and antibiotic resistance. Here, we report the ability of a putative nickel/cobalt transporter (NiCoT family), Rv2856 or NicT of Mycobacterium tuberculosis (Mtb), to transport metal and antibiotics and identified some key amino acid residues that are important for its function. Ectopic expression of NicT in Escherichia coli CS109 resulted in the increase of intracellular nickel uptake. Additionally, enhanced tolerance towards several antibiotics (norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid and isoniazid) was observed with NicT overexpression in E. coli and Mycobacterium smegmatis. A comparatively lower intracellular accumulation of norfloxacin upon NicT overexpression than that of the cells without NicT indicated the involvement of NicT in an active efflux process. Although expression of NicT did not alter the sensitivity towards kanamycin, doxycycline, tetracycline, apramycin, neomycin and ethambutol, the presence of a sub-inhibitory dose of Ni2+ resulted in the manifestation of low-level tolerance towards these drugs. Further, substitution of four residues (H77I, D82I, H83L and D227I) in the conserved regions of NicT by isoleucine and leucine resulted in reduced to nearly complete loss of the transport function for both metals and antimicrobials. Therefore, the study suggests that nickel transporter Rv2856/NicT may actively export different drugs and the presence of nickel might drive the cross-resistance to some of the antibiotics.

Balanced cytokine upregulation by diluted ethanolic extract of Bryonia alba in Delta SARS-CoV-2 Spike protein RBD-induced pathogenesis in Gallus gallus embryo.

Background: Bryonia alba extract is a well-known drug which is being utilized as phytomedicines and homoeopathic preparations since more than two centuries. This medicine is frequently used in clinical practice for flu-like conditions, respiratory tract infections, and gastrointestinal diseases, as evidenced by old literature and historical records. The plant contains Bryonicin, Bryonolic acid, Bryodin, Cucurbitacin, etc. The alkaloids in Bryonia alba have been discovered to be a powerful heme-oxygenase-1 inhibitor, which could help reduce oxidative stress during SARS-CoV-2 pathogenesis. During three waves of SARS-CoV-2, extracts of Bryonia alba were used; however, the actual scientific explanation for its mechanism of action is still unknown. In this experiment, we studied cytokine changes by diluted Bryonia alba extract in Delta SARS-CoV-2 spike protein RBD-induced pathogenesis, in fertilized chick (Gallus gallus domesticus) embryos. Results: The recombinant Delta SARS-CoV-2 spike RBD protein was inoculated in 14-day-old chick (Gallus gallus domesticus) embryos along with control, pre-, and post-treatment sets with diluted Bryonia extract. After 48 h, allantoic fluids were collected and stored at - 20 °C for study of different cytokines. Histological changes of the liver were also studied in each animal. Diluted Bryonia extract upregulated IFN-α and IL-10 markedly. In pre-treatment set, IFN-α, IL-8, IL-10, and IL-1ß were markedly decreased, while in the post-treatment set IL-6, IL-10, IL-8, and TGFß1 were significantly decreased, with a tendency of more anti-inflammatory surge than pro-inflammatory cytokines. Conclusions: This experiment indicated an immunomodulatory role of diluted ethanolic extract of Bryonia particularly in the post-treatment set, decreasing pro-inflammatory cytokines with beneficial effect.

Childhood Brucellosis in Eastern India.

OBJECTIVE: To investigate the presence of childhood brucellosis presenting as PUO (pyrexia of unknown origin) cases in Eastern zone of India. METHODS: Blood samples were collected from PUO patients aged ≤18 y. The main diagnostic tools were STAT, RBPT, ELISA- IgM, IgG and PCR. Although mainly PUO cases were selected for the study, other associated clinical manifestations were also noted. RESULTS: The findings revealed significantly higher percentage of infection in female children (14.3%) than in male children (10.9%). The positive results by different diagnostic tools, STAT, RBPT, ELISA- IgM, ELISA-IgG and brucella genus specific PCR were 10.6%, 7.2%, 7.2%, 0.85% and 1.3% respectively. Main associated clinical symptoms were joint pain, low backache, fatigue and night sweat. CONCLUSIONS: This hospital based study reflects a significant number of childhood brucellosis cases in Eastern zone of India, and thus emphasizes the need for further monitoring of such subjects.

Draft Genome Sequence of Ammonia-Producing Acinetobacter sp. Strain MCC2139 from Dairy Effluent.

We report the draft genome sequence of an ammonia-producing, esculin-hydrolyzing, catalase-positive, gram-negative bacterium, Acinetobacter sp. strain MCC2139. This bacterium, isolated from dairy sludge and with optimum growth at 37°C, has a genome size of 2,967,280 bp with a G+C content of 42.3%.