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आयतन 5, मुद्दा 2 (2021)

खुला एक्सेस

Stable isotope probing coupled Raman microscopy: An efficient way to study single cell biochemistry

Hemanth Nag Noothalapati Venkata

Lipid droplets have been hypothesized to be intimately associated with intracellular proteins. However, there is little direct evidence for both spatiotemporal and functional relations between lipid droplets and proteins provided by molecular -level studies on intact cells. To elucidate the interplay between them at the single cell level, Raman microscopy was coupled with a very powerful strategy, namely, stable isotope labeling. Here, I present in vivo time lapse Raman imaging, coupled with stableisotope (13C) labeling, of single living Schizosaccharomyces pombe cells. Our results show that the proteins newly synthesized from incorporated 13C-substrate are localized specifically to lipid droplets as the lipid concentration within the cell increases. Lipids, which help to store energy in a compact form, have variety of roles in biological systems and their metabolism is central to life. Here, we show that combination of stable isotope probing (SIP), Raman micro-spectroscopy and multivariate curve resolution analysis can serve as a valuable approach in metabolomics research. We studied ergosterol biosynthesis in single living fission yeast cells, grown in mixtures of normal (12C) and 13C-glucose as the sole carbon source.

Microbial communities are essential to many ecosystems and interact in complex ways with almost all eukaryotes. Therefore, a detailed understanding of the functioning of such communities is a basic requirement for biologists, including microbiologists and microbiome researchers. Using a single Raman microspectroscopy cell, the biochemical fingerprints of cells by other microbials can be obtained without the label and without injury. When combined with stable isotope testing (SIP), Raman spectroscopy can directly detect the activities of microorganisms in their natural environment. This review provides an overview of the various SIP methods that should be integrated with the various Raman distribution processes and shows how a single Raman SIP cell can be directly linked to omics-centric analysis pipelines investigating viral communities. By carefully looking into the biosynthetic pathways and by comparing the observed peak positions with calculation results on isotope-substituted ergosterol, it is possible to understand how 13C is incorporated in the conjugated C=C moiety of the molecule. The multivariate spectral data analysis revealed intrinsic spectra and their relative abundances of all isotopomers.

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Chlorflavonin, a flavone-type fungal metabolite with potent and selective antitubercular activity

Georgios Daletos

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is one of the leading causes of mortality and morbidity caused by pathogenic microorganisms. Treatment of TB is currently facing serious problems due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains. The latter is practically untreatable with the currently available anti-TB drugs on the market and thus that there is an urgent need for novel antibiotics against TB. During our ongoing search for new potential anti-TB drug leads, we investigated the endophytic fungus Mucor irregularis, which was isolated from the Cameroonian medicinal plant Moringa stenopetala. The ethyl acetate extract of M. irregularis yielded two flavonoid-type derivatives, chlorflavonin and dechlorflavonin, the latter only differing by the absence of a chlorine atom in the B ring. The design of the mediation modification using complete genome sequencing, chemical supplementation tests, and cell suspension tests and enzymatic descriptions revealed that chlorflavonin specifically inhibits acetohydroxyacid synthase catalytic subunit IlvB1, which produces auxotic chain amxotrophies and pantothenic acid. While showing the bacteriostatic effect on monotreatment treatment, chlorflavonin has shown the effects of first-line anti-antibiotic isoniazid interaction and especially with delamanid, which has led to the complete closure of the fluid civilization in combination therapy.

Using a fluorescent reporter type, the intracellular activity of chlorflavonin against Mycobacterium tuberculosis within infected macrophages was demonstrated and was superior to streptomycin treatment.

In contrast to dechlorflavonin, chlorflavonin exhibited strong growth inhibitory activity against M. tuberculosis, indicating that chlorination plays an important role for anti-TB activity. Importantly, chlorflavonin showed no cytotoxicity against the human fibroblast (MRC-5) and macrophage-like human acute monocytic leukemia (THP-1) cell lines up to concentrations of 100 μM. Mapping of resistance-mediating mutations revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase IIvB1, which mediates the first step in branched chain amino acids and pantothenic acid biosynthesis. Chlorflavonin displayed synergistic effects in combination with the first-line antibiotic isoniazid leading to a complete sterilization and no resistance in liquid culture during combination treatment. Moreover, chlorflavonin exhibited potent activity against XDR M. tuberculosis strains, which highlights the potential of this compound as a promising anti-TB agent.

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Elimination of micro-pollutants and pathogenic (antibiotic resistant) bacteria by advanced waste water treatment technologies

Claudia Gallert

Elimination of bacteria by ozonation in combination with adsorption onto activated carbon or slow sand filtration is a possibility for advanced sewage treatment in order to improve the quality of treated sewage and to reduce the potential risk for human health and of receiving surface waters. To determine the elimination of sewage bacteria, inflowing and leaving waste water of different treatment processes was analyzed in a culture-based and qPCR approach for its content of Escherichia coli, Enterococci and staphylococci and their resistance against selected antibiotics over a period of 17 months. For Enterococci, single species and their antibiotic resistances were identified. In comparison to the standard waste water treatment process, ozonation plus adsorption onto activated carbon and/or sand filtration reduced the concentrations of total and antibiotic resistant E. coli, Enterococci and staphylococci. The discovery of new chemical pollutants and biodiversity in the aquatic environment has become a growing problem of environmental concern. Therefore, wastewater treatment plants (WWTPs) play an important role in the spread of so-called emerging viruses and antimicrobials.

Therefore, the daily loads released by WWTP are calculated including the model system for the distribution of these loads within the receiving body of water.

However, antibiotic resistant E. coli and staphylococci apparently survived ozone treatment better than antibiotic sensitive strains. Neither vancomycin resistant Enterococci nor methicillin resistant Staphylococcus aureus (MRSA) were detected by a culture-based approach. The decreased percentage of antibiotic resistant Enterococci after ozonation may be explained by a different ozone sensitivity of species: Enterococcus faecium and Enterococcus faecalis, which determined the resistance-level, seemed to be more sensitive for ozone than other Enterococcus species. Overall, ozonation followed by adsorption onto activated carbon or sand filtration led to 0.8-1.1 log-units less total and antibiotic resistant E. coli, Enterococci and staphylococci. Thus, advanced waste water treatment after common sewage treatment is an effective tool for further elimination of microorganisms from sewage before discharge in surface water.

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Assessment of human viruses in municipal waste water in Calgary, Canada

Yuanyuan Qiu

Water shortage is an increasing problem worldwide. Effective treatment of waste water for reuse has attracted interests for public and scientific community. Currently, most of the studies related to the quality of reclaimed waste water focus on the bacteriological indicators such as E. coli and total coliform. There is limited data of human viruses in waste water. In fact, human enteric viruses are present at high levels in waste water, which poses a potential risk to public health and hinders application of reuse. The objective of this study is to assess the effect of UV treatment on the virus removal at two municipal waste water treatment plants in Calgary, Canada. Ten liters of pre and post-UV treatment waste water samples were collected in duplicate monthly for 12 months. Viruses were concentrated by filtration, elution and flocculation followed by nucleic acid extraction. A real-time quantitative PCR viral panel was used for detection and quantification of eight viruses including Norovirus (NoV), Rotavirus (RV), Sapovirus (SaV), Astrovirus (AsV), Adenovirus (AdV), Enterovirus (EV), JC virus (JCV) and Reovirus (ReoV). Viral infectivity was assessed using both cell culture and integrated cell culture and qPCR (ICC-qPCR). NoV, RV, AsV and AdV were detected in all 24, EV and JCV in 23 and SaV in 22 of pre-UV samples, respectively.

The mean viral load (except ReoV) ranged from 3.06 (JCV) to 5.77 (RV) log10 DNA copies/Lin pre-UV samples and 3.23 (JCV) to 5.68 (RV) log10 DNA copies/Lin post-UV samples. 

The average virus removal by UV treatment ranged from -0.42 (JCV) to 0.52 (NoV) log as assessed by qPCR which may be due to the sample variation and the limitation of qPCR in detection of live viruses. Further studies on developing a qPCR method to differentiate the infectious and non-infectious viruses are of interests. Cell culture and ICC-qPCR detected infectious viruses in all 24 pre-UV and 19 out of 24 post- UV samples. Approximately 1.8 log reductions of infectious viruses could be achieved by current industrial standard UV treatment. The results indicate a moderate effect of UV treatment on virus inactivation in municipal waste water which urges further studies to increase the understanding of the effect of UV treatment on human viruses in waste water to improve the waste water reuse and public safety as treated waste water is discharged into river and could become source water for downstream users.

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Photodynamic inactivation of microorganisms in waste water: An effective approach with low environmental impact

Adelaide Almeida

Waste water (WW) with a high content of pathogens, containing even multidrug resistant microorganisms, particularly when hospital effluents are included, is a current area of concern affecting the quality of natural water. Hospital WW effluents are discharged as conventional urban effluents to the municipal sewage system without prior treatment. Secondary treatment of WW is usually considered sufficient; however, the secondary effluent still contains infective concentrations of microorganisms (MO). In order to reduce the concentration of pathogens in WW to levels comparable to those found in natural water, the tertiary effluent is usually subjected to disinfection with chlorine, ozone or ultraviolet light (UV). Chlorination and UV may lead to the formation of toxic products and also might result to the selection of resistant genes. The antimicrobial photodynamic inactivation (PDI) may represent an alternative to the traditional expensive, unsafe and not always effective disinfection methods. PDI with photosensitizers (PS) and visible light has demonstrated to be effective in the destruction of MO via photogeneration of reactive oxygen species (ROS) able to induce microbial inactivation. As PDI is a multi-target approach, the selection of photo-resistant strains after treatment is unlikely.

The main goal of this work was to assess the efficiency of PDI on non-clinical and on clinical multidrug-resistant (MDR) bacteria in domestic and hospital waste water in order to evaluate its potential use to treat WW effluents. 

The efficiency of PDI was assessed using a cationic porphyrin as photosensitizer (PS), non-clinical bacteria and clinical MDR-bacteria either in phosphate buffered saline or in filtrated domestic and hospital waste water. The synergistic effect of PDI and antibiotics (ampicillin and chloramphenicol) was also evaluated. The results show an efficient inactivation of non-clinical and MDR bacteria in PBS (reduction of 6-8 log after 270 min). In waste water, the inactivation of bacteria was also efficient and the decrease in bacterial survival starts even sooner. A faster decrease in bacterial survival occurred when PDI was combined with the addition of antibiotics. It can be concluded that PDI has potential to be an effective alternative for the inactivation of bacteria, even MDR; in waste waters and that the presence of antibiotics in hospital WW may enhance its effectiveness.

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