Ramachandran Sarojini Santhosh

Four clove clones (BRC1, BRC3 MRC5 and MRC7) were collected from different places of ponmudi hills, Kerala, India. DNA was extracted and RAPD analysis was designed to evaluate clove clonal variation. Upon analysis bacterial sequences were also identified among clove genome sequences. So the clove and microbial sequences were separated and performed an analysis to identify clove associated metagenome. The major microbes present were Mycobacterium canettii Xanthomaonas euvesicateria, Klebsiella and Psuedomonas . A total of 88 genus of microbes were present altogether in four clones of Syzygium aromaticum . There were few fungal species were also identified, Fusarium pseudograminearum was the major fungus associated with all four different clones of clove.

Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is Toxin-Antitoxin systems (TAS) which are also implicated in a wide range of stress-related phenomena. In a report (Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140-1150) an interesting link between ppGpp mediated stringent response, TAS and persistence was proposed. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for Lon-dependent degradation of YefM, the antitoxin of YefM/YoeB TAS. The Δ10 strain, an Escherichia coli MG1655 derivative in which the ten TAS are deleted, is more sensitive to Ciprofloxacin and Ampicillin compared to wild-type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the implications based on this strain are void. We conclude that there is no direct and specific link between stringent response and the regulation of TAS. The link between TAS and persistence is inconclusive due to altered fitness of Δ10 strain and hence requires thorough inspection and debate. Importance: A model connecting stringent response, endoribonuclease encoding Type II Toxin-Antitoxin systems (TAS) and persistence is widely propagated. It states that “accumulation of ppGpp results in accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxin rendering toxins free to induce persistence”. This work presents a contradiction to and challenges the model. Experimental evidence, literature survey as well as rationale are provided to show that inorganic polyphosphate is not required for the degradation of YefM, the antitoxin in YefM/YoeB TAS. The Δ10 strain is relatively more sensitive to Ciprofloxacin and Ampicillin as well as has lowered fitness. This is likely because of the polar effects on the adjacent genes caused by the genetic manipulation of multiple TAS loci.

Type II toxin–antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are ‘acquired’ DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.

Drug efflux pumps (EP) like Mmr in Mycobacterium transported drugs out of cell, a main reason for drug resistance developing in Mycobacterium tuberculosis. In this in silico study, mainly analysed EP inhibitory potential of a plant-derived flavonoid, quercetin, through docking analysis. Mmr present in Mycobacterium smegmatis and M. tuberculosis, and its homologue EmrE of Escherichia coli was used. Initially, homology modelling of EP monomers and dimers constructed from M. smegmatis, M. tuberculosis and E. coli; the stabilities of models were analysed from Ramachandran plots prepared in PROCHECK. Docking analysis of quercetin with EP protein showed that in all three organisms, the residues for function and stability are important and quercetin had best interactions comparing to compounds such as, verapamil, reserpine, chlorpromazine, Carbonyl Cyanide m- Chloro Phenylhydrazone. Molecular dynamics and simulation studies showed that during the entire course of simulation quercetin-Mmr complex were stable. It insights quercetin can act as a non-antibiotic adjuvant for treatment of tuberculosis by bring down the efflux of drug from bacteria.

Tuberculosis, also called TB, is currently a major health hazard due to multidrug-resistant forms of bacilli. Global efforts are underway to eradicate TB using new drugs with new modes of action, higher activity, and fewer side effects in combination with vaccines. For this reason, unexplored new sources and previously explored sources were examined and around 353 antimycobacterial compounds (Nat Prod Rep 2007; 24: 278–297) [7] have been previously reported. To develop drugs from these new sources, additional work is required for preclinical and clinical results. Since ancient times, different plant part extracts have been used as traditional medicines against diseases including tuberculosis. This knowledge may be useful in developing future powerful drugs. Plant natural products are again becoming important in this regard. In this review, we report 127 antimycobacterial compounds and their antimycobacterial activities. Of these, 27 compounds had a minimum inhibitory concentration of < 10 µg/mL. In some cases, the mechanism of activity has been determined. We hope that some of these compounds may eventually develop into effective new drugs against tuberculosis.