Identification of Alternatively Spliced Novel Isoforms of Human HSPB8 Gene.

HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics.

Alternative splicing generates a novel ferroportin isoform with a shorter C-terminal and an intact iron- and hepcidin-binding property.

Ferroportin (FPN) is a transmembrane protein and is the only known iron exporter that helps in maintaining iron homeostasis in vertebrates. To maintain stable iron equilibrium in the body, ferroportin works in conjunction with a peptide called hepcidin. In this study, we have identified an alternatively spliced novel isoform of the human SLC40A1 gene, which encodes for the FPN protein and is found to be expressed in different tissues. The novel transcript has an alternate last exon and encodes 31-amino acid long peptide sequence that replaces 104 amino acids at C-terminal in the novel transcript. Molecular modelling and molecular dynamics (MD) simulation studies revealed key structural features of the novel isoform (FPN-N). FPN-N was predicted to have 12 transmembrane domains similar to the reported isoform (FPN), despite being much smaller in size. FPN-N was found to interact with hepcidin, a key regulator of ferroportin activity. Also, the iron-binding sites were retained in the novel isoform as revealed by the MD simulation of FPN-N in bilipid membrane. The novel isoform identified in this study may play important role in iron homeostasis. However, further studies are required to characterize the FPN-N isoform and decipher its role inside the cell.

Molecular characterization of resistance determinants and mobile genetic elements of ESBL producing multidrug-resistant bacteria from freshwater lakes in Kashmir, India.

BACKGROUND: Antibiotic resistance conceded as a global concern is a phenomenon that emerged from the bacterial response to the extensive utilization of antimicrobials. The expansion of resistance determinants through horizontal transfer is linked with mobile genetic elements (MGEs) like transposons, insertion sequences, and integrons. Heavy metals also create consequential health hazards. Metal resistance gene in alliance with antibiotic resistance genes (ARGs) and MGEs is assisting bacteria to attain exalted quantity of resistance. METHODOLOGY: The present work was carried out to study ARGs blaCTX-M, AmpC, qnrS, MGEs like ISecp1, TN3, TN21, and Int I by performing PCR and sequencing from Wular and Dal lakes of Kashmir; India. The genetic environment analysis of blaCTX-M-15 was carried out using PCR amplification, and sequencing approach followed by in-silico docking and mutational studies. Co-occurrence of ARGs and HMRGs was determined. Plasmid typing was done using PCR-based replicon typing (PBRT) and conjugation assay was also performed. RESULTS: Out of 201 isolates attained from 16 locations, 33 were ESBLs producers. 30 ESBL displaying isolates were perceived positive for CTX-M gene, followed by AmpC (17), qnrS (13), ISecp1 (15), TN3 (11), TN21 (11), Int I (18), and SulI (14). The genetic environment of blaCTX-M-15 was observed as (ISEcp1-blaCTX-M-15-orf477), classical promoter-10 TACAAT and -35 TTGAA was found at the 3' region. The 3D structure of CTX-M-15 and ISEcp1 was generated and CTX-M-15-ISEcp1 (R299L) docking and mutation showed a reduction in hydrogen bonds. Co-occurrence of antibiotics and HMRGs (mer, sil, and ars) was found in 18, 14, and 8 isolates. PBRT analysis showed the presence of Inc. groups- B/O, F, I1, HI1, FIA, HI2, N, FIB, L/M. Molecular analysis of transconjugants showed the successful transfer of ARGs, MGEs, and HMRGs in the E. coli J53 AZR strain. CONCLUSION: This study highlights the occurrence of ESBL producing bacteria in the aquatic environment of Kashmir India that can serve as a reservoir of ARGs. It also discussed the molecular mechanisms of MGEs which can help in containing the spread of antibiotic resistance.

Colistin Interaction and Surface Changes Associated with mcr-1 Conferred Plasmid Mediated Resistance in E. coli and A. veronii Strains.

Colistin, a polycationic antimicrobial peptide, is one of the last-resort antibiotics for treating infections caused by carbapenem-resistant Gram-negative bacteria. The antibacterial activity of colistin occurs through electrostatic interaction between the polycationic peptide group of colistin and the negatively charged phosphate groups of lipid A membrane. This study investigated the interaction of colistin with the outer membrane and surface constituents of resistant and susceptible strains of Escherichia coli and Aeromonas veronii harboring mcr-1 resistance gene. Bacterial membrane and lipopolysaccharide used in this study were isolated from susceptible as well as colistin-resistant strains of E. coli and A. veronii. Interaction of colistin with the bacterial surface was studied by deoxycholate and lysozyme sensitivity test, N-phenyl-1-naphthylamine (NPN) uptake assay, Atomic force microscopy (AFM), Zeta potential measurements and 1H NMR. The binding affinity of colistin was found to be lower with outer membrane from resistant strains in comparison with the susceptible strains. Colistin exposure enhances the outer membrane permeability of the susceptible strains to deoxycholate and lysozyme. However, on the other hand, colistin dose of 256 µg/mL did not permeabilize the outer membrane of resistant bacteria. The NPN permeability in resistant strains was greater in comparison with susceptible strains. Atomic force microscopy images depicted smooth, featherless and deformed membranes in treated susceptible cells. Contrary to the above, resistant treated cells displayed surface roughness topography even at 256 µg/mL colistin concentration. Surface charge alterations were confirmed by Zeta potential measurements as a function of the growth phase. Mid-logarithmic phase susceptible strains showed a greater negative charge than resistant strains upon exposure to colistin. However, there was no statistical variation in the Zeta potential measurements between resistant and susceptible strains at the stationary phase. NMR analysis revealed line broadening in susceptible strains with increasing colistin: LPS aggregates mass ratio. Moreover, resistant strains did not show line broadening for the outer membrane, even at the highest mass ratio. The findings of this study suggest that the resistant strains of E. coli and A. veronii can block the electrostatic contact between the cationic peptide and anionic lipid A component that drives the first phase of colistin action, thereby preventing hydrophobically driven second-tier action of colistin on the outer lipopolysaccharide layer.

Current Update on Intrinsic and Acquired Colistin Resistance Mechanisms in Bacteria.

Colistin regained global interest as a consequence of the rising prevalence of multidrug-resistant Gram-negative Enterobacteriaceae. In parallel, colistin-resistant bacteria emerged in response to the unregulated use of this antibiotic. However, some Gram-negative species are intrinsically resistant to colistin activity, such as Neisseria meningitides, Burkholderia species, and Proteus mirabilis. Most identified colistin resistance usually involves modulation of lipid A that decreases or removes early charge-based interaction with colistin through up-regulation of multistep capsular polysaccharide expression. The membrane modifications occur by the addition of cationic phosphoethanolamine (pEtN) or 4-amino-l-arabinose on lipid A that results in decrease in the negative charge on the bacterial surface. Therefore, electrostatic interaction between polycationic colistin and lipopolysaccharide (LPS) is halted. It has been reported that these modifications on the bacterial surface occur due to overexpression of chromosomally mediated two-component system genes (PmrAB and PhoPQ) and mutation in lipid A biosynthesis genes that result in loss of the ability to produce lipid A and consequently LPS chain, thereafter recently identified variants of plasmid-borne genes (mcr-1 to mcr-10). It was hypothesized that mcr genes derived from intrinsically resistant environmental bacteria that carried chromosomal pmrC gene, a part of the pmrCAB operon, code three proteins viz. pEtN response regulator PmrA, sensor kinase protein PmrAB, and phosphotransferase PmrC. These plasmid-borne mcr genes become a serious concern as they assist in the dissemination of colistin resistance to other pathogenic bacteria. This review presents the progress of multiple strategies of colistin resistance mechanisms in bacteria, mainly focusing on surface changes of the outer membrane LPS structure and other resistance genetic determinants. New handier and versatile methods have been discussed for rapid detection of colistin resistance determinants and the latest approaches to revert colistin resistance that include the use of new drugs, drug combinations and inhibitors. Indeed, more investigations are required to identify the exact role of different colistin resistance determinants that will aid in developing new less toxic and potent drugs to treat bacterial infections. Therefore, colistin resistance should be considered a severe medical issue requiring multisectoral research with proper surveillance and suitable monitoring systems to report the dissemination rate of these resistant genes.

Lentic and effluent water of Delhi-NCR: a reservoir of multidrug-resistant bacteria harbouring blaCTX-M, blaTEM and blaSHV type ESBL genes.

Antimicrobial resistance is not restricted to clinics but also spreading fast in the aquatic environment. This study focused on the prevalence and diversity of extended-spectrum ß-lactamase (ESBL) genes among bacteria from lentic and effluent water in Delhi-NCR, India. Phenotypic screening of 436 morphologically distinct bacterial isolates collected from diverse sites revealed that 106 (∼24%) isolates were ESBL positive. Antibiotic profiling showed that 42, 60, 78 and 59% ESBL producing isolates collected from Ghazipur slaughterhouse, Lodhi garden pond, Hauz Khas lake and Jasola wastewater treatment plant, respectively, were multidrug-resistant (MDR). The multiple antibiotic resistance (MAR) index varied from 0.20 to 0.32 among selected locations. The prevalence of ESBL gene variants blaSHV, blaTEM and blaCTX-M were found to be 17.64, 35.29 and 64%, respectively. Furthermore, the analysis of obtained gene sequences showed three variants of blaCTX-M (15, 152 and 205) and two variants of blaTEM (TEM-1 and TEM-116) among ESBL producers. The co-existence of 2-3 gene variants was recorded among 48% ESBL positive isolates. New reports from this study include the blaCTX-M gene in Acinetobacter lwoffii, Enterobacter ludwigii, Exiguobacterium mexicanum and Aeromonas caviae. Furthermore, the identification of blaTEM and blaSHV in an environmental isolate of A. caviae is a new report from India.

Bacterial isolates harboring antibiotics and heavy-metal resistance genes co-existing with mobile genetic elements in natural aquatic water bodies.

The rise in antibiotic-resistant bacteria and contamination of water bodies is a serious issue that demands immense attention of scientific acumen. Here, we examined the pervasiveness of ESBL producing bacteria in Dal Lake and Wular Lake of Kashmir valley, India. Isolates were screened for antibiotic, heavy metal resistant elements, and their coexistence with mobile genetic elements. Out of two hundred one isolates screened, thirty-eight were found positive for ESBL production. Antibiotic profiling of ESBL positive isolates with 16 different drugs representing ß-lactam or -non-ß-lactam, exhibited multidrug resistance phenotype among 55% isolates. Molecular characterization revealed the occurrence of drug resistance determinants blaTEM, AmpC, qnrS, and heavy metal resistance genes (MRGs) merB, merP, merT, silE, silP, silS, and arsC. Furthermore, mobile genetic elements IntI, SulI, ISecp1, TN3, TN21 were also detected. Conjugation assay confirmed the transfer of different ARGs, HMRGs, and mobile elements in recipient Escherichia coli J53 AZR strain. Plasmid incompatibility studies showed blaTEM to be associated with Inc groups B/O, HI1, HI2, I1, N, FIA, and FIB. Co-occurrence of blaTEM, HMRGs, and mobile elements from the aquatic milieu of Kashmir, India has not been reported so far. From this study, the detection of the blaTEM gene in the bacteria Bacillus simplex and Brevibacterium frigoritolerans are found for the first time. Considering all the facts it becomes crucial to conduct studies in natural aquatic environments that could help depict the epidemiological situations in which the resistance mechanism might have clinical relevance.

Plant-Based Phytochemicals as Possible Alternative to Antibiotics in Combating Bacterial Drug Resistance.

The unprecedented use of antibiotics that led to development of resistance affect human health worldwide. Prescription of antibiotics imprudently and irrationally in different diseases progressed with the acquisition and as such development of antibiotic resistant microbes that led to the resurgence of pathogenic strains harboring enhanced armors against existing therapeutics. Compromised the treatment regime of a broad range of antibiotics, rise in resistance has threatened human health and increased the treatment cost of diseases. Diverse on metabolic, genetic and physiological fronts, rapid progression of resistant microbes and the lack of a strategic management plan have led researchers to consider plant-derived substances (PDS) as alternative or in complementing antibiotics against the diseases. Considering the quantitative characteristics of plant constituents that attribute health beneficial effects, analytical procedures for their isolation, characterization and phytochemical testing for elucidating ethnopharmacological effects has being worked out for employment in the treatment of different diseases. With an immense potential to combat bacterial infections, PDSs such as polyphenols, alkaloids and tannins, present a great potential for use, either as antimicrobials or as antibiotic resistance modifiers. The present study focuses on the mechanisms by which PDSs help overcome the surge in resistance, approaches for screening different phytochemicals, methods employed in the identification of bioactive components and their testing and strategies that could be adopted for counteracting the lethal consequences of multidrug resistance.

Prevalence and diversity of blaTEM, blaSHV and blaCTX-M variants among multidrug resistant Klebsiella spp. from an urban riverine environment in India.

In the present study, we have investigated prevalence and diversity of ESBL genes among Klebsiella isolates obtained from highly polluted stretch of river Yamuna, India. Phenotypic screenings of 116 Klebsiella isolates revealed ~30% were positive for ESBL production. Antibiotic profiling showed multidrug resistance phenotype among 90% isolates. Prevalence of blaTEM, blaSHV and blaCTX-M genes were found to be 57, 54 and 48% respectively. Furthermore, we identified eight variants of blaSHV (SHV-1, SHV-11, SHV-27, SHV-28, SHV-38, SHV-61, SHV-144, SHV-148), three each of blaTEM (TEM-1, TEM-116, TEM-206) and blaCTX-M (CTX-M-15, CTX-M-55, CTX-M-188) among Klebsiella spp. Co-occurrence of blaTEM, blaSHV and blaCTX-M (any two or all three) was observed among 45% Klebsiella isolates. Occurrence of blaCTX-M-188 and blaTEM-206 in environmental isolates of K. pneumoniae has not been reported earlier. Identification of blaTEM-206, blaSHV-27 and blaSHV-144 from Klebsiella spp. and blaTEM-116 from K. quasipneumoniae and K. variicola is the first report from India.

Antibiotics, Resistome and Resistance Mechanisms: A Bacterial Perspective.

History of mankind is regarded as struggle against infectious diseases. Rather than observing the withering away of bacterial diseases, antibiotic resistance has emerged as a serious global health concern. Medium of antibiotic resistance in bacteria varies greatly and comprises of target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Further aggravation to prevailing situation arose on observing bacteria gradually becoming resistant to different classes of antibiotics through acquisition of resistance genes from same and different genera of bacteria. Attributing bacteria with feature of better adaptability, dispersal of antibiotic resistance genes to minimize effects of antibiotics by various means including horizontal gene transfer (conjugation, transformation, and transduction), Mobile genetic elements (plasmids, transposons, insertion sequences, integrons, and integrative-conjugative elements) and bacterial toxin-antitoxin system led to speedy bloom of antibiotic resistance amongst bacteria. Proficiency of bacteria to obtain resistance genes generated an unpleasant situation; a grave, but a lot unacknowledged, feature of resistance gene transfer.