Antibiotic resistance: a global crisis, problems and solutions.

Healthy state is priority in today's world which can be achieved using effective medicines. But due to overuse and misuse of antibiotics, a menace of resistance has increased in pathogenic microbes. World Health Organization (WHO) has announced ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) as the top priority pathogens as these have developed resistance against certain antibiotics. To combat such a global issue, it is utmost important to identify novel therapeutic strategies/agents as an alternate to such antibiotics. To name certain antibiotic adjuvants including: inhibitors of beta-lactamase, efflux pumps and permeabilizers for outer membrane can potentially solve the antibiotic resistance problems. In this regard, inhibitors of lytic domain of lytic transglycosylases provide a novel way to not only act as an alternate to antibiotics but also capable of restoring the efficiency of previously resistant antibiotics. Further, use of bacteriophages is another promising strategy to deal with antibiotic resistant pathogens. Taking in consideration the alternatives of antibiotics, a green synthesis nanoparticle-based therapy exemplifies a good option to combat microbial resistance. As horizontal gene transfer (HGT) in bacteria facilitates the evolution of new resistance strains, therefore identifying the mechanism of resistance and development of inhibitors against it can be a novel approach to combat such problems. In our perspective, host-directed therapy (HDT) represents another promising strategy in combating antimicrobial resistance (AMR). This approach involves targeting specific factors within host cells that pathogens rely on for their survival, either through replication or persistence. As many new drugs are under clinical trials it is advisable that more clinical data and antimicrobial stewardship programs should be conducted to fully assess the clinical efficacy and safety of new therapeutic agents.

Maize Seedlings Colonization with Serendipita indica and Its Colonization Efficiency Analysis.

Maize is one of the most important crops in the world, and ensuring its successful growth and productivity is crucial for global food security. One way to enhance maize growth and productivity is by improving the colonization of its roots by beneficial microorganisms. In this regard, Serendipita indica, a plant growth-promoting fungus, has gained attention for its ability to enhance plant growth and productivity, especially in cereal crops and medicinal plants. Previous studies have shown that S. indica can colonize various plant species, including maize, but the efficiency of the colonization process in maize seedlings has not been extensively characterized. This protocol outlines a method for efficient colonization of maize seedlings with the beneficial fungus S. indica. The protocol includes the preparation of stock solutions, maintenance and growth of S. indica, surface sterilization and germination of seeds, preparation of S. indica chlamydospores, and colonization of maize plants with S. indica. The advantages of this protocol include the use of surface sterilization techniques that minimize contamination, the production of a large number of viable chlamydospores, and efficient colonization of maize seedlings with S. indica. This protocol may be useful for researchers studying the role of S. indica in promoting plant growth and combating biotic and abiotic stress. Additionally, this protocol may be used in the development of biofertilizers using S. indica as a means of increasing crop yields and reducing dependence on synthetic fertilizers. Overall, this protocol offers a reliable and efficient method for colonizing maize seedlings with S. indica and may have potential applications in the agricultural industry. This study also provides a valuable tool for researchers interested in studying plant-microbe interactions in maize and highlights the potential of S. indica as a biocontrol agent to enhance maize productivity under adverse conditions. Key features • This protocol builds upon the method developed by Narayan et al. (2022), and its application optimized for the root endophytic symbiotic fungus S. indica. • This protocol also allows for histochemical analysis to visualize the colonized fungal spores in the root cells of host plant species. • This protocol helps in mathematical calculation of the percent colonization or efficiency of colonization. • This protocol utilizes readily available laboratory equipment, including a light microscope, autoclave, and laminar flow hood, ensuring ease of reproducibility in other research laboratories.

Sulfur nutrition and its role in plant growth and development.

Sulfur is one of the essential nutrients that is required for the adequate growth and development of plants. Sulfur is a structural component of protein disulfide bonds, amino acids, vitamins, and cofactors. Most of the sulfur in soil is present in organic matter and hence not accessible to the plants. Anionic form of sulfur (SO42-) is the primary source of sulfur for plants that are generally present in minimal amounts in the soil. It is water-soluble, so readily leaches out of the soil. Sulfur and sulfur-containing compounds act as signaling molecules in stress management as well as normal metabolic processes. They also take part in crosstalk of complex signaling network as a mediator molecule. Plants uptake sulfate directly from the soil by using their dedicated sulfate transporters. In addition, plants also use the sulfur transporter of a symbiotically associated organism like bacteria and fungi to uptake sulfur from the soil especially under sulfur depleted conditions. So, sulfur is a very important component of plant metabolism and its analysis with different dimensions is highly required to improve the overall well-being of plants, and dependent animals as well as human beings. The deficiency of sulfur leads to stunted growth of plants and ultimately loss of yield. In this review, we have focused on sulfur nutrition, uptake, transport, and inter-organismic transfer to host plants. Given the strong potential for agricultural use of sulfur sources and their applications, we cover what is known about sulfur impact on the plant health. We identify opportunities to expand our understanding of how the application of soil microbes like AMF or other root endophytic fungi affects plant sulfur uptake and in turn plant growth and development.

Functional characterization of a high-affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development.

Iron (Fe) is a micronutrient required for plant growth and development; however, most Fe forms in soil are not readily available to plants, resulting in low Fe contents in plants and, thereby, causing Fe deficiency in humans. Biofortification through plant-fungal co-cultivation might be a sustainable approach to increase crop Fe contents. Therefore, we aimed to examine the role of a Piriformospora indica Fe transporter on rice Fe uptake under low Fe conditions. A high-affinity Fe transporter (PiFTR) from P. indica was identified and functionally characterized. PiFTR fulfilled all criteria expected of a functional Fe transporter under Fe-limited conditions. Additionally, PiFTR expression was induced when P. indica was grown under low Fe conditions, and PiFTR complemented a yeast mutant lacking Fe transport. A knockdown (KD) P. indica strain was created via RNA interference to understand the physiological role of PiFTR. We observed that the KD-PiFTR-P. indica strain transported a significantly lower amount of Fe to colonized rice (Oryza sativa) than the wild type (WT) P. indica. WT P. indica-colonized rice plants were healthier and performed significantly better than KD-PiFTR-P. indica-colonized rice plants. Our study offers potential avenues for an agronomically sound amelioration of plant growth in low Fe environments.

Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT.

A deficiency of the essential macronutrient sulfur leads to stunted plant growth and yield loss; however, an association with a symbiotic fungus can greatly improve nutrient uptake by the host plant. Here, we identified and functionally characterized a high-affinity sulfate transporter from the endophytic fungus Serendipita indica. SiSulT fulfills all the criteria expected of a functional sulfate transporter responding to sulfur limitation: SiSulT expression was induced when S. indica was grown under low-sulfate conditions, and heterologous expression of SiSulT complemented a yeast mutant lacking sulfate transport. We generated a knockdown strain of SiSulT by RNA interference to investigate the consequences of the partial loss of this transporter for the fungus and the host plant (maize, Zea mays) during colonization. Wild-type (WT) S. indica, but not the knockdown strain (kd-SiSulT), largely compensated for low-sulfate availability and supported plant growth. Colonization by WT S. indica also allowed maize roots to allocate precious resources away from sulfate assimilation under low-sulfur conditions, as evidenced by the reduction in expression of most sulfate assimilation genes. Our study illustrates the utility of the endophyte S. indica in sulfur nutrition research and offers potential avenues for agronomically sound amelioration of plant growth in low-sulfate environments.

Advanced strategies for development of vaccines against human bacterial pathogens.

Infectious diseases are one of the main grounds of death and disabilities in human beings globally. Lack of effective treatment and immunization for many deadly infectious diseases and emerging drug resistance in pathogens underlines the need to either develop new vaccines or sufficiently improve the effectiveness of currently available drugs and vaccines. In this review, we discuss the application of advanced tools like bioinformatics, genomics, proteomics and associated techniques for a rational vaccine design.

Metagenomic Analysis of Microbial Diversity in Cotton Rhizosphere Soil in Alwar, India.

Cotton is an important cash crop for both the Indian economy and rural livelihoods. In the present study, metagenomic analysis is used to characterize microbial diversity in cotton rhizosphere soil from the Alwar district, located in the semiarid northeast region of the state of Rajasthan in India.

Metagenomic Analysis of the Microbial Diversity in Solid Waste from Okhla Landfill, New Delhi, India.

The Okhla landfill site is consistently in the news for having pollution levels higher than the city average. Here, we report the taxonomic characterization of the microbial diversity of Okhla landfill solid waste. The metagenome analyses revealed the microbial and metabolic diversity of the site.

Deciphering Streptococcal Biofilms.

Streptococci are a diverse group of bacteria, which are mostly commensals but also cause a considerable proportion of life-threatening infections. They colonize many different host niches such as the oral cavity, the respiratory, gastrointestinal, and urogenital tract. While these host compartments impose different environmental conditions, many streptococci form biofilms on mucosal membranes facilitating their prolonged survival. In response to environmental conditions or stimuli, bacteria experience profound physiologic and metabolic changes during biofilm formation. While investigating bacterial cells under planktonic and biofilm conditions, various genes have been identified that are important for the initial step of biofilm formation. Expression patterns of these genes during the transition from planktonic to biofilm growth suggest a highly regulated and complex process. Biofilms as a bacterial survival strategy allow evasion of host immunity and protection against antibiotic therapy. However, the exact mechanisms by which biofilm-associated bacteria cause disease are poorly understood. Therefore, advanced molecular techniques are employed to identify gene(s) or protein(s) as targets for the development of antibiofilm therapeutic approaches. We review our current understanding of biofilm formation in different streptococci and how biofilm production may alter virulence-associated characteristics of these species. In addition, we have summarized the role of surface proteins especially pili proteins in biofilm formation. This review will provide an overview of strategies which may be exploited for developing novel approaches against biofilm-related streptococcal infections.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191.

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Functional Characterization of a Magnesium Transporter of Root Endophytic Fungus Piriformospora indica.

Magnesium (Mg) is a crucial macronutrient required for the regular growth of plants. Here we report the identification, isolation and functional characterization of Mg-transporter PiMgT1 in root endophytic fungus Piriformospora indica. We also report the role of P. indica in the improvement of the Mg nutrition of the plant particularly under Mg deficiency condition. Protein BLAST (BLASTp) for conserved domains analysis showed that PiMgT1 belong to CorA like protein family of bacteria. We have also observed the presence of conserved 'GMN' signature sequence which suggests that PiMgT1 belongs to Mg transporter family. Phylogenetic analysis revealed that PiMgT1 clustered among fungal CorA family members nearer to basidiomycetes. Functionality of PiMgT1 was confirmed by complementation of a yeast magnesium transporter mutant CM66. We have observed that PiMgT1 restored the growth of mutant and showed comparable growth with that of WT. We found statistically significant (p < 0.05) two fold increase in the total intracellular Mg content of mutant complemented with PiMgT1 as compared to the mutant. These observations suggest that PiMgT1 is actively involved in Mg uptake by the fungus and may be helping in the nutritional status of the host plant.

Antioxidant enzymes in chickpea colonized by Piriformospora indica participate in defense against the pathogen Botrytis cinerea.

Piriformospora indica, a root endophytic fungus, promotes growth of the economically important chickpea plant (Cicer arietinum Linn.) and protects it against the pathogenic fungus Botrytis cinerea. Biomass and root development were found to be significantly improved in chickpea plants colonized with P. indica as compared to the plants grown without P. indica as well as from the plants infected with the B. cinerea. Our PCR analyses showed that gradual increase in the colonization of P. indica in the plants result in the inhibition of the colonization of B. cinerea. P. indica colonized plants showed increased antioxidant enzyme activities. Interestingly, there were pronounced decrease in the antioxidant enzyme activities in shoots infected with B. cinerea and colonized with P. indica in alternate and simultaneous mode as compared to plants infected with B. cinerea alone. We conclude that P. indica helps plants to overcome the disease load by enhancing antioxidant enzyme defense system. Our data suggest that, bio-protective action of P. indica might be mediated via systemic induction of antioxidant defense in the host plants.

A Botrytis cinerea KLP-7 Kinesin acts as a Virulence Determinant during Plant Infection.

Botrytis cinerea is a necrotrophic pathogen that infects many important crops. In an attempt to unravel some novel factors that govern pathogenicity in B. cinerea, Agrobacterium tumefaciens mediated transformation (ATMT) was deployed, and a number of tagged transformants were generated. Among these, a mutant, BCM-29 exhibited slower growth rate, reduced conidia size, conidiation and penetration. The mutant was also defective in secretion of oxalic acid (OA) and exhibited reduced activities of polygalacturonase (PG) and pectin methyl esterases (PME). TAIL-PCR followed by BLAST search identified the tagged gene as KLP-7 that encodes for kinesin. Targeted deletion of KLP-7 resulted in several folds decrease in virulence of mutants as compared to WT, while complementation of the gene helped in rescue of virulence traits. This is the first time when a unique kinesin KLP-7 that is mainly found in the phylum Pezizomycotina has been linked to virulence in B. cinerea.

PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers salinity stress tolerance in rice plants.

In this study, yeast HOG1 homologue from the root endophyte Piriformospora indica (PiHOG1) was isolated and functionally characterized. Functional expression of PiHOG1 in S. cerevisiae ∆hog1 mutant restored osmotolerance under high osmotic stress. Knockdown (KD) transformants of PiHOG1 generated by RNA interference in P. indica showed that genes for the HOG pathway, osmoresponse and salinity tolerance were less stimulated in KD-PiHOG1 compared to the wild-type under salinity stress. Furthermore, KD lines are impaired in the colonization of rice roots under salinity stress of 200 mM NaCl, and the biomass of the host plants, their shoot and root lengths, root number, photosynthetic pigment and proline contents were reduced as compared to rice plants colonized by WT P. indica. Therefore, PiHOG1 is critical for root colonisation, salinity tolerance and the performance of the host plant under salinity stress. Moreover, downregulation of PiHOG1 resulted not only in reduced and delayed phosphorylation of the remaining PiHOG1 protein in colonized salinity-stressed rice roots, but also in the downregulation of the upstream MAP kinase genes PiPBS2 and PiSSK2 involved in salinity tolerance signalling in the fungus. Our data demonstrate that PiHOG1 is not only involved in the salinity response of P. indica, but also helping host plant to overcome salinity stress.

Functional Characterization of a Hexose Transporter from Root Endophyte Piriformospora indica.

Understanding the mechanism of photosynthate transfer at symbiotic interface by fungal monosaccharide transporter is of substantial importance. The carbohydrate uptake at the apoplast by the fungus is facilitated by PiHXT5 hexose transporter in root endophytic fungus Piriformospora indica. The putative PiHXT5 belongs to MFS superfamily with 12 predicted transmembrane helices. It possess sugar transporter PFAM motif (PF0083) and MFS superfamily domain (PS50850). It contains the signature tags related to glucose transporter GLUT1 of human erythrocyte. PiHXT5 is regulated in response to mutualism as well as glucose concentration. We have functionally characterized PiHXT5 by complementation of hxt-null mutant of Saccharomyces cerevisiae EBY.VW4000. It is involved in transport of multiple sugars ranging from D-glucose, D-fructose, D-xylose, D-mannose, D-galactose with decreasing affinity. The uncoupling experiments indicate that it functions as H(+)/glucose co-transporter. Further, pH dependence analysis suggests that it functions maximum between pH 5 and 6. The expression of PiHXT5 is dependent on glucose concentration and was found to be expressed at low glucose levels (1 mM) which indicate its role as a high affinity glucose transporter. Our study on this sugar transporter will help in better understanding of carbon metabolism and flow in this agro-friendly fungus.

Fungal association and utilization of phosphate by plants: success, limitations, and future prospects.

Phosphorus (P) is a major macronutrient for plant health and development. The available form of P is generally low in the rhizosphere even in fertile soils. A major proportion of applied phosphate (Pi) fertilizers in the soil become fixed into insoluble, unavailable forms, which restricts crop production throughout the world. Roots possess two distinct modes of P uptake from the soil, direct and indirect uptake. The direct uptake of P is facilitated by the plant's own Pi transporters while indirect uptake occurs via mycorrhizal symbiosis, where the host plant obtains P primarily from the fungal partner, while the fungus benefits from plant-derived reduced carbon. So far, only one Pi transporter has been characterized from the mycorrhizal fungus Glomus versiforme. As arbuscular mycorrhizal fungi cannot be cultured axenically, their Pi transporter network is difficult to exploite for large scale sustainable agriculture. Alternatively, the root-colonizing endophytic fungus Piriformospora indica can grow axenically and provides strong growth-promoting activity during its symbiosis with a broad spectrum of plants. P. indica contains a high affinity Pi transporter (PiPT) involved in improving Pi nutrition levels in the host plant under P limiting conditions. As P. indica can be manipulated genetically, it opens new vistas to be used in P deficient fields.

Isolation of genes conferring salt tolerance from Piriformospora indica by random overexpression in Escherichia coli.

Piriformospora indica, a root endophytic fungus identified in the Indian Thar desert, colonizes the roots of plants and provides resistance towards biotic stress as well as tolerance to abiotic stress in the plants. Despite its positive impact on the host, little is known about the P. indica genes that are involved in salt stress tolerance. Therefore this study was conducted to identify and isolate high salinity-tolerance genes from P. indica. Thirty-six salinity-tolerance genes were obtained by functional screening, based on random over expression of a P. indica cDNA library in Escherichia coli grown on medium supplemented with 0.6 M NaCl. The salinity tolerance conferred by these 36 genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. However when the expression of these 36 genes was analysed in P. indica using quantitative RT-PCR, we found only six genes were up-regulated by salt stress. These six genes are involved in different cellular processes, such as metabolism, energy and biosynthetic processes, DNA repair, regulation of protein turnover, transport and salt stress tolerance. This work presents the basis for further molecular analyses of the mechanisms of salt tolerance in P. indica and for the use of this endophyte to confer salt tolerance to plants.

Molecular epidemiology and virulence characteristics of prevalent group A streptococci recovered from patients in northern India.

INTRODUCTION: In this study, the prevalence of M types of Group A Streptococcus (GAS) in North India, invasive behavior of prevalent M types, and inflammatory immune response by host cells were studied. METHODOLOGY: A total of 1,047 clinical samples were collected between 2004 and 2010. Confirmation of GAS was determined by serotyping and M types were identified by emm gene sequencing. The most prevalent serotypes were selected to study their invasive behavior and inflammatory immune response under different temperatures and salt concentrations in A549 and HEp-2 cells. RESULTS: Ninety-two isolates were identified as GAS of which 17 were M types with 18.5% heterogeneity. The most prevalent M types were M1 (21.73%) and M49 (8.7%), respectively. M1 and M49 were used to study virulence potential and inflammatory immune responses. The efficiency of cell infection decreased with increased temperature for both M types, increasing with lowering temperatures compared to the uninfected control (37°C). As salt concentration was increased, cell infection efficiency was lowered with some exceptions; the infection efficiency of M1 strain in A549 cells with 0.6 M NaCl was 50 fold higher (p ≤ 0.03). Significantly increased production of IL-6 and IL-8 was observed in both cell lines infected with GAS and when grown under different environmental conditions compared to uninfected cell lines. CONCLUSIONS: This study determined the prevalence of different M types in North India and showed that environmental conditions can regulate cell infection by GAS . This information may influence the selection of GAS serotypes used in vaccine development.

Epidemiology of Group B Streptococcus in developing countries.

Group B Streptococcus (GBS) causes pneumonia, meningitis and sepsis in neonates. The current distribution pattern of GBS serotypes in developing countries such as India, China and Brazil is not clear. In order to appropriately plan for vaccination programs to address the burden of this disease in these countries, prospective population based studies are urgently needed. In our discussions, we focused on India, China and Brazil because of the membership of our workgroup, but data on other countries are also presented here. Further studies in developing countries are needed so as to better formulate appropriate public health interventions.

Are women satisfied when using levonorgestrel-releasing intrauterine system for treatment of abnormal uterine bleeding?

AIM: To determine the efficacy of levonorgestrel intrauterine system (LNG IUS) in treatment of abnormal uterine bleeding (AUB) in women over 35 years and to determine satisfaction of users of LNG IUS in case of AUB. MATERIALS AND METHODS: This was a multicentric, retrospective, and observational study. Case records of patients with AUB from the hospitals in Pune, Delhi, and Gurgaon for the last 6 years were examined. Records of 80 women who had an LNG IUS inserted were included. The chief complaints and their duration were recorded. Investigation results, histopathology reports, and date of LNG IUS insertion were noted. The incidence of spotting, heavy menstrual bleeding, pain, expulsion, and amenorrhea were recorded at 3, 6, 12, and 18 months following treatment. Following this a telephonic interview was conducted to determine the efficacy of LNG IUS in treating the symptoms. Patients' satisfaction in percentage was also noted and they were asked if they would recommend the LNG IUS to other women. RESULTS: The mean age of women was 42.3 years. 77.5% of the women had menorrhagia as the chief complaint, and the mean duration was 12 months. Fibroids and adenomyosis were the most common pathology, present in 21.3% and 20% of the patients respectively. At 3 months, spotting seemed to be the predominant symptom (59.4%) and only 15% women had heavy bleeding. 49.3% of women were asymptomatic at 6 months. 27.5% had amenorrhea by the end of 18 months. 14 women in whom the device was expelled or removed due to persistent symptoms, underwent hysterectomy at various stages during the study period. Overall patient satisfaction was high at about 80%. Furthermore, 73.8% patients agreed to recommend it to other women. CONCLUSION: LNG IUS seems to be a viable and effective treatment option for AUB in women after 35 years. There is a high rate of patient satisfaction in appropriately selected patients.