Invasive Fungal Infections in Immunocompromised Conditions: Emphasis on COVID-19.

The COVID-19 pandemic caused death of 6 million lives globally, primarily from respiratory failure, but also a significant number from invasive fungal co-infections in these patients, owing to the immune dysfunction in hospitalized patients. Such complications occurred more often in critically ill, hospitalized patients particularly those admitted in intensive care units and were reported as the major reason associated with a high mortality rate worldwide. Fungal pathogens most commonly associated with COVID-19 patients comprise members of the Mucorales (such as Rhizopus, Mucor, and Lichtheimia), as well as genera Aspergillus and Candida. In India, the prevalence rate of mucormycosis is relatively high than aspergillosis and candidiasis, and the predisposing risk factors associated with such infections included uncontrolled diabetes, underlying lung disease, leukopenia, neutropenia, malignancies and prolonged steroid therapy. However, co-infection with other fungi, including Alternaria and Scedosporium was also sporadically reported. These devastating invasive fungal infections are associated with differential mortality (high-low) and morbidity rates even after active management. The diagnosis of such infections is often challenging due to lack of sensitivity in contemporary diagnostic methods and poses an enormous challenge to healthcare experts. Thus, the role of early and accurate diagnosis, and management of such fungal infections, is vital in preventing life-threatening situations. Hence, this review focusses primarily on the epidemiology, predisposing risk factors, host environment, diagnosis and treatment of the most common medically important invasive fungal infections in immunocompromised conditions associated with COVID-19.

Sporosarcina hypophthalmichthys sp. nov. Isolated From Gastrointestinal Tract of Fish Hypophthalmichthys molitrix (Valenciennes, 1844).

A rod-shaped, motile, Gram-stain-positive bacterial strain RKN2T, was isolated from gut of silver carp (Hypophthalmichthys molitrix) residing in Gobindsagar reservoir, Himachal Pradesh, India. Having the greatest sequence similarity to Sporosarcina koreensis F73T (98.51%), Sporosarcina luteola Y1T (98.4%) and Sporosarcina aquimarina SW28T (98.36%), the 16S rRNA gene phylogeny confirmed the belonging of strain RKN2T to genus Sporosarcina. Digital DNA-DNA hybridization values were 21.7%, 20.6%, and 19.2%, and average nucleotide identity values were 76.42%, 80.16%, 76.51%, of strain RKN2T with Sporosarcina koreensis F73T, Sporosarcina luteola Y1T, and Sporosarcina aquimarina SW28T, respectively. The genomic analysis of strain RKN2T showed various biological properties including nitrate reduction, genes responsible for carbohydrate-active enzymes production, antimicrobial compounds, as well as potential metabolism of aromatic compounds and heavy metals. G+C composition of RKN2T genome was 52.7%. This strain can grow in temperatures between 10°C and 40°C (optimum, 28°C-30°C), NaCl concentrations up to 6.0% (w/v), and 6.0-8.0 (optimum, 6.5-7.5) pH range. MK-7 was the dominant respiratory quinone, A-4 type cell wall peptidoglycan was present with anteiso-C15:0, iso-C15: 0, and anteiso-C17:0 being the major fatty acids and Lys-Glu being main amino acids. Diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were the strain RKN2T's three main polar lipids. The strain is a novel species under genus Sporosarcina based on polyphasic approach and the name Sporosarcina hypophthalmichthys sp. nov. is given for strain RKN2T. RKN2T is a type strain (= MCC 4365T = JCM34522T = CCM9112T).

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Characterization and Antimicrobial Studies of Iturin-Like and Bogorol-Like Lipopeptides From Brevibacillus spp. Strains GI9 and SKDU10.

Accession numbers for whole-genome sequence of Brevibacillus sp. strain GI9 and SKDU10 are CAGD01000001 to CAGD01000061 and LSSO00000000, respectively. Members of the genus Brevibacillus have been demonstrated to produce a variety of bioactive compounds including polyketides, lipopeptides and bacteriocins. Lipopeptides are non-ribosomally synthesized surface-active compounds with antimicrobial, antitumor, and immune-stimulatory activities. They usually exhibit strong antifungal and antibacterial activities and are considered as promising compounds in controlling fungal diseases. In this study, we have characterized two lipopeptides from Brevibacillus sp. strains GI9 and SKDU10. The corresponding lipopeptides were purified by reverse-phase high-performance liquid chromatography. Mass analysis and characterization by MALDI-TOF-MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analysis revealed production of an iturin-like lipopeptide by strain GI9 and bogorol-like lipopeptide by strain SKDU10. Both lipopeptides exhibited broad spectrum antibacterial activity and inhibited the growth of various fungi. They showed minimum inhibitory concentration (MIC) values between 90 and 300 µg/ml against indicator strains of bacteria and drug-resistant Candida indicator strains. The lipopeptides did not show phytotoxic effect in seed germination experiments but caused hemolysis. Further, both lipopeptides inhibited the growth of fungi on fruits and vegetables in in vitro experiments, thereby exhibited potential use in biotechnological industry as effective biocontrol agents.

Xanthomonas sontii sp. nov., a non-pathogenic bacterium isolated from healthy basmati rice (Oryza sativa) seeds from India.

We report three yellow-pigmented, Gram-negative, aerobic, rod-shaped, motile bacterial isolates designated as PPL1T, PPL2, and PPL3 from healthy basmati rice seeds. Phenotypic and 16S rRNA gene sequence analysis assigned these isolates to the genus Xanthomonas. The 16S rRNA showed a 99.59% similarity with X. sacchari CFBP 4641T, a sugarcane pathogen. Further, biochemical and fatty acid analysis revealed it to be closer to X. sacchari. Still, it differed from other species in general and known rice associated species such as X. oryzae (pathogenic) and X. maliensis (non-pathogenic) in particular. Interestingly, the isolatess in this study were isolated from healthy rice plants but are closely related to species that is pathogenic and isolated from diseased sugarcane. Accordingly, in planta studies revealed that PPL1T, PPL2, and PPL3 are non-pathogenic to rice plants upon leaf inoculation. Taxonogenomic studies based on orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) values with type strains of Xanthomonas species were below the recommended threshold values for species delineation. Whole genome-based phylogenomic analysis revealed that these isolates formed a distinct monophyletic clade with X. sacchari CFBP 4641T as their closest neighbour. Further, pangenome analysis revealed PPL1T, PPL2, and PPL3 isolates to comprise NRPS cluster along with a large number of unique genes associated with the novel species. Based on polyphasic and genomic approaches, a novel lineage and species associated with healthy rice seeds for which the name Xanthomonas sontii sp. nov. is proposed. The type strain for the X. sontii sp. nov. is PPL1T (JCM 33631T = CFBP 8688T = ICMP 23426T = MTCC 12491T) and PPL2 (JCM 33632 = CFBP 8689 = ICMP 23427 = MTCC 12492) and PPL3 (JCM 33633 = CFBP 8690 = ICMP 23428 = MTCC 12493) as other strains of the species.

Hymenobacter terrestris sp. nov. and Hymenobacter lapidiphilus sp. nov., isolated from regoliths in Antarctica.

A group of four psychrotrophic bacterial strains was isolated on James Ross Island (Antarctica) in 2013. All isolates, originating from different soil samples, were collected from the ice-free northern part of the island. They were rod-shaped, Gram-stain-negative, and produced moderately slimy red-pink pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, MALDI-TOF MS, rep-PCR analyses, chemotaxonomic methods and extensive biotyping was used to clarify the taxonomic position of these isolates. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to the genus Hymenobacter. The closest relative was Hymenobacter humicola CCM 8763T, exhibiting 98.3 and 98.9% 16S rRNA pairwise similarity with the reference isolates P5342T and P5252T, respectively. Average nucleotide identity, digital DNA-DNA hybridization and core gene distances calculated from the whole-genome sequencing data confirmed that P5252T and P5342T represent two distinct Hymenobacter species. The menaquinone systems of both strains contained MK-7 as the major respiratory quinone. The predominant polar lipids for both strains were phosphatidylethanolamine and one unidentified glycolipid. The major components in the cellular fatty acid composition were summed feature 3 (C16:1 ω7c/C16:1ω6c), C16:1ω5c, summed feature 4 (anteiso-C17:1 B/iso-C17:1 I), anteiso-C15:0 and iso-C15 : 0 for all isolates. Based on the obtained results, two novel species are proposed, for which the names Hymenobacter terrestris sp. nov. (type strain P5252T=CCM 8765T=LMG 31495T) and Hymenobacter lapidiphilus sp. nov. (type strain P5342T=CCM 8764T=LMG 30613T) are suggested.

Virgicin, a novel lanthipeptide from Virgibacillus sp. strain AK90 exhibits inhibitory activity against Gram-positive bacteria.

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.

Bacteriocins: perspective for the development of novel anticancer drugs.

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

Cysteine-rich low molecular weight antimicrobial peptides from Brevibacillus and related genera for biotechnological applications.

The production of natural antimicrobial peptides (AMPs) is an innate immunity trait of all life forms including eukaryotes and prokaryotes. While these AMPs are usually called as defensins in eukaryotes, they are known as bacteriocins in prokaryotes. Bacteriocins are more diverse AMPs considering their varied composition and posttranslational modifications. Accordingly, this review is focused on cysteine-rich AMPs resembling eukaryotic defensins such as laterosporulin from Brevibacillus spp. and associated peptides secreted by the members of related genera. In fact, structural studies of laterosporulin showed the pattern typically observed in human defensins and therefore, should be considered as bacterial defensin. Although the biosynthesis mechanism of bacterial defensins displayed high similarities, variations in amino acid composition and structure provided the molecular basis for a better understanding of their properties. They are reported to inhibit Gram-positive, Gram-negative, non-multiplying and human pathogenic bacteria. The extreme stability is due to the presence of intra-molecular disulfide bonds in prokaryotic defensins and reveals their potential clinical and food preservation applications. Notably, they are also reported to have potential anticancer properties. Therefore, this review is focused on multitude of diverse applications of bacterial defensins, exploring the possible correlations between their structural, functional and possible biotechnological applications.

Anticancer properties of a defensin like class IId bacteriocin Laterosporulin10.

Laterosporulin10 (LS10) is a defensin like peptide from Brevibacillus sp. strain SKDU10 that inhibited microbial pathogens. However, in this study, anticancer activity of LS10 was examined against different cancer cell lines and compared with normal cells. LS10 displayed cytotoxicity against cancer cells like MCF-7, HEK293T, HT1080, HeLa and H1299 at below 10 µM concentration, but not against prostate epithelium cells RWPE-1. Additionally, no hemolysis was observed at significantly higher concentration compared to IC50 values observed for different cancer cell lines. Release of lactate dehydrogenase from cancer cell lines at 15 µM concentration upon 120 min treatment indicated the lytic ability of LS10. Accordingly, electron microscopy experiments also confirmed the necrotic effect of LS10 at 15 µM concentration against cancer cells. Furthermore, flow cytometry analysis of treated cancer cell lines revealed that LS10 induce apoptosis even at 2.5 µM concentration. Nevertheless, RWPE-1 cells remained viable even at 20 µM concentration. These results provide evidence that LS10 is an anticancer bacteriocin, which causes apoptotic and necrotic death of cancer cells at lower and higher concentrations, respectively. Taken all results together, the present study signifies that LS10 is an anticancer peptide that could be further developed for therapeutic applications.

Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC-MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis.

The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module.

The growing emergence of antibiotic-resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram-positive and Gram-negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all-ß conformation of this peptide, with four ß-strands forming a twisted ß-sheet. All six intrinsic cysteines are intramolecularly disulfide-bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in-solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [C(I)-C(V), C(II)-C(IV), and C(III)-C(VI)] like that of ß-defensins and a striking architectural similarity with α-defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

Screening of serine protease inhibitors with antimicrobial activity using iron oxide nanoparticles functionalized with dextran conjugated trypsin and in silico analyses of bacterial serine protease inhibition.

Plants produce a variety of proteins and peptides which are involved in their defense against pathogens. Serine protease inhibitors are a well-established class of inhibitors correlated with plant defense. Increased levels of protease inhibitors delay cell damage and expand the cell's life-span. Recently, the rapid emergence of antibiotic-resistant microbial pathogens has prompted immense interest in purifying novel antimicrobial proteins or peptides from plant sources. Usually, the purification of protease inhibitors is accomplished by salt-extraction, ultrafiltration and affinity chromatography. Here, we developed a novel approach based on iron oxide nanoparticles conjugated to dextran functionalized with trypsin beads that accelerate the quick screening and purification of antimicrobial peptides with serine protease inhibitor activity. The method described here also works for screening other inhibitors using particular protein kinases, and it is therefore a novel tool for use as the leading method in the development of novel antimicrobial agents with protease inhibitory activity. Finally, and no less important, molecular modelling and dynamics studies of a homologous inhibitor studied here with Escherichia coli trypsin and chymotrypsin are provided in order to shed some light on inhibitor-enzyme interactions.

Description of a novel actinobacterium Microbacterium assamensis sp. nov., isolated from water sample collected from the river Brahmaputra, Assam, India.

A Gram-positive, yellow pigmented actinobacterium, strain S2-48(T) was isolated from water sample collected from the river Brahmaputra, Assam, India and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(15:0) Anteiso, iso C(16:0) and C(17:0) Anteiso), estimated DNA G+C content (70.2 mol%) and 16S rRNA gene sequence analysis showed that strain S2-48(T) belonged to the genus Microbacterium. Strain S2-48(T) exhibited highest 16S rRNA gene sequence similarity with Microbacterium testaceum (97.0%); however, the DNA-DNA relatedness value between strain S2-48(T) and M. testaceum was 9.1%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain S2-48(T) should be classified within the genus Microbacterium as a novel species, for which the name Microbacterium assamensis is proposed. The type strain is S2-48(T) (=MTCC 10486(T) = DSM 23998(T)).

Chryseomicrobium imtechense gen. nov., sp. nov., a new member of the family Planococcaceae.

A Gram-stain-positive, rod-shaped, yellow, non-motile, non-spore-forming, strictly aerobic bacterial strain, designated MW 10(T), was isolated from seawater of the Bay of Bengal, India, and was subjected to a polyphasic taxonomic study. Analysis of the 16S rRNA gene sequence revealed that strain MW 10(T) showed highest similarity to the type strains of Psychrobacillus psychrodurans (96.15 %) and Psychrobacillus psychrotolerans (96.01 %) and showed less than 96 % similarity to members of the genera Paenisporosarcina, Planococcus, Sporosarcina and Planomicrobium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain MW 10(T) formed a clade separate from members of closely related genera. The morphological, physiological and chemotaxonomic characteristics of strain MW 10(T) differed from those of members of closely related genera. The major fatty acid in strain MW 10(T) was iso-C(15 : 0) and the menaquinones were MK-7 (48.4 %), MK-8 (32.3 %), MK-7(H(2)) (13.7 %) and MK-6 (5.6 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown phospholipid, an unknown lipid and an unknown glycolipid. The cell-wall peptidoglycan type was l-Lys-d-Asp. The genomic DNA G+C content (53.4 mol%) of strain MW 10(T) was significantly different from those of members of closely related genera. On the basis of its morphological, physiological and chemotaxonomic characteristics as well as our phylogenetic analysis, we conclude that strain MW 10(T) is a member of a novel genus and species, for which the name Chryseomicrobium imtechense gen. nov., sp. nov., is proposed. The type strain of Chryseomicrobium imtechense is MW 10(T) ( = MTCC 10098(T)  = JCM 16573(T)).

Agrococcus carbonis sp. nov., isolated from soil of a coal mine.

An actinobacterial strain, designated G4(T), isolated from a coal mine was subjected to polyphasic taxonomic characterization. Cells were Gram-stain-positive, yellow-pigmented, non-motile and non-spore-forming cocci. This organism possessed a type B peptidoglycan with diaminobutyric acid as diagnostic diamino acid. The major respiratory quinones were MK-9, MK-10 and MK-11. The major fatty acids were anteiso-C(15 : 0) (41.6 %) and anteiso-C(17 : 0) (32.8 %). The predominant cellular polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Cell wall sugars comprised galactose, glucose, ribose and rhamnose. 16S rRNA gene sequence analysis of strain G4(T) showed high similarity with Agrococcus baldri (98.9 %), Agrococcus citreus (97.8 %), Agrococcus jenensis (97.3 %) and Agrococcus terreus (97.0 %). Sequence similarity with the type strains of the other species of the genus Agrococcus was less than 97.0 %. The DNA-DNA relatedness of strain G4(T) with the type strains of Agrococcus baldri, Agrococcus citreus, Agrococcus jenensis and Agrococcus terreus was less than 70 %. On the basis of the physiological, biochemical and chemotaxonomic characteristics, strain G4(T) should be classified as the type strain of a novel species of the genus Agrococcus, for which the name Agrococcus carbonis sp. nov. is proposed. The type strain is G4(T) ( = MTCC 10213(T)  = DSM 22965(T)).