Trends in carbapenem resistance in Pre-COVID and COVID times in a tertiary care hospital in North India.

BACKGROUND: Carbapenem resistance is endemic in the Indian sub-continent. In this study, carbapenem resistance rates and the prevalence of different carbapenemases were determined in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa during two periods; Pre-COVID (August to October 2019) and COVID (January to February 2021) in a north-Indian tertiary care hospital. METHODS: Details of patient demographics and clinical condition was collated from the Hospital Information System and detection of carbapenemases NDM, OXA-48, VIM, IMP and KPC was done by Polymerase chain reaction (PCR) in 152 and 138 non-consecutive carbapenem resistant isolates during the two study periods respectively. Conjugation assay and sequencing of NDM and OXA-48 gene was done on a few selected isolates. RESULTS: As compared to Pre-COVID period, co-morbidities and the mortality rates were higher in patients harbouring carbapenem resistant organisms during the COVID period. The overall carbapenem resistance rate for all the four organisms increased from 23 to 41% between the two periods of study; with Pseudomonas aeruginosa and Klebsiella pneumoniae showing significant increase (p < 0.05). OXA-48, NDM and co-expression of NDM and OXA-48 were the most common genotypes detected. NDM-5 and OXA-232 were most common variants of NDM and OXA-48 family respectively during both the study periods. CONCLUSION: Higher rate of carbapenem resistance in COVID times could be attributed to increase in number of patients with co-morbidities. However, genetic elements of carbapenem resistance largely remained the same in the two time periods.

Carbapenem resistance in Escherichia coli and Klebsiella pneumoniae among Indian and international patients in North India.

The aim of the study was to find out the carbapenem resistance rate and prevalence of different carbapenemase genes in Klebsiella pneumoniae and Escherichia coli from a North Indian corporate hospital that receives both Indian and international patients. A total of 528 clinical isolates of E. coli and K. pneumoniae were included in the study. All isolates that were found resistant to carbapenems by MIC testing (Vitek II Compact®) were screened for NDM, OXA-48, VIM, and KPC genes by PCR. Sequencing of NDM gene and transmissibility by conjugation assay were checked on 22 randomly selected NDM-positive isolates. One hundred and fifty-six isolates (29.54%) were carbapenem-resistant. The rate of carbapenem resistance was significantly higher in K. pneumoniae as compared to E. coli (53.9% vs. 15.6%; p < 0.05). The NDM gene was found in 34.6% (54/156), OXA-48 in 31.4% (49/156), co-expression of NDM + OXA-48 in 15.3% (24/156) of the carbapenem-resistant isolates. VIM and KPC were absent in all isolates. NDM gene was significantly more prevalent in E. coli than K. pneumoniae (p < 0.05). All the tested isolates formed transconjugants and NDM-5 was the most common variant in both species (15/22). The presence of plasmid-based NDM calls for stricter surveillance measures in our hospital settings.

Growth arrest of lung carcinoma cells (A549) by polyacrylate-anchored peroxovanadate by activating Rac1-NADPH oxidase signalling axis.

Hydrogen peroxide is often required in sublethal, millimolar concentrations to show its oxidant effects on cells in culture as it is easily destroyed by cellular catalase. Previously, we had shown that diperoxovanadate, a physiologically stable peroxovanadium compound, can substitute H2O2 effectively in peroxidation reactions. We report here that peroxovanadate when anchored to polyacrylic acid (PAPV) becomes a highly potent inhibitor of growth of lung carcinoma cells (A549). The early events associated with PAPV treatment included cytoskeletal modifications, increase in GTPase activity of Rac1, accumulation of the reactive oxygen species, and also increase in phosphorylation of H2AX (γH2AX), a marker of DNA damage. These effects persisted even at 24 h after removal of the compound and culminated in increased levels of p53 and p21 together with growth arrest. The PAPV-mediated growth arrest was significantly abrogated in cells pre-treated with the N-acetylcysteine, Rac1 knocked down by siRNA and DPI an inhibitor of NADPH oxidase. In conclusion, our results show that polyacrylate derivative of peroxovanadate efficiently arrests growth of A549 cancerous cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress and DNA damage.

Acute and chronic effects of endosulfan on the haemato-immunological and histopathological responses of a threatened freshwater fish, spotted murrel, Channa punctatus.

Two experiments, one short-term and one long-term, were conducted to elucidate the acute and chronic effects, respectively, of endosulfan exposure on the haemato-immunological and histopathological responses of Channa puncatatus. In the short-term study, fish were exposed to sublethal endosulfan (8.1 µg l(-1)) for 12, 24, 36, 48, 72 and 96 h. In the long-term study, fish were fed with normal diet and simultaneously either exposed to endosulfan (1.2 µg l(-1)) for 90 days or not. Results showed that the ascorbic acid levels in both the liver and the muscle decreased significantly (P < 0.05) by acute and chronic endosulfan exposure. The haemoglobin (Hb) level reduced significantly (P < 0.05) by 15.5% within 12 h of acute endosulfan exposure, further decreased by 25.8% after 24 h of exposure, however, thereafter the values increased and at the end of 72 h returned to normal levels. Almost similar trend was observed for the erythrocyte (RBC) count. The WBC count and the nitroblue tetrazolium (NBT) value showed a general increasing trend with increase in the duration of acute endosulfan exposure. The chronic exposure of C. punctatus to endosulfan significantly (P < 0.05) lowered the Hb level, RBC and WBC counts, NBT reduction value and the plasma parameters such as plasma protein, albumin (A) and globulin (G) compared with that of the control (except for A/G ratio). Endosulfan exposure also severely altered the liver histological structure. Overall results indicated that both short-term acute and long-term chronic endosulfan exposure had a significant impact on the haemato-immunological parameters and tissue histopathology of C. punctatus.

Mutations in two component system (PhoPQ and PmrAB) in colistin resistant Klebsiella pneumoniae from North Indian tertiary care hospital.

Colistin resistance in Gram negative bacteria is mainly attributed to chromosomal mutations in Two Component Systems(TCS) PhoPQ and PmrAB and plasmid-borne genes(mcr and its variants). The aim of this study was to understand the molecular basis of colistin resistance in Klebsiella pneumoniae and determine clonal transmission, in a North Indian tertiary care hospital over a 2.5 year period. Antimicrobial susceptibility was determined by Vitek and colistin resistance was confirmed by broth microdilution. Carbapenemases(blaKPC, blaVIM, blaIMP, blaNDM, blaOXA-48) and mcr-1 screening was done by PCR. Mutations in chromosomal genes mgrB, phoP, phoQ, pmrA, pmrB were analysed. Sequence typing was performed by Multilocus sequence typing(MLST). OXA-48 was detected in thirteen isolates while three isolates co-expressed OXA-48 and NDM. The mcr-1 gene was absent in all 16 isolates. Deleterious mutations in mgrB included insertion sequences IS903 and ISkpn26 and a premature stop codon. A total of 18 point mutations were identified in PhoPQ and PmrAB TCS; of which, novel mutations were reported in phoQ (K46E, L322V, D152N, F373L, R249G), pmrB (P159R) and pmrA (D149L). Six different sequence types ST231, ST147, ST395, ST42, ST14 and ST101 were identified. Phylogenetic analysis showed that sequence types ST14, ST395 and ST147 are closely related to ST101 and all identified sequence types had a common ancestor ST231. Colistin resistance in K. pneumoniae was attributed to mutations in PhoPQ and PmrAB TCS, while location specific distribution of strains indicates clonal transmission. The results of this study will help in formulation of effective infection prevention and antimicrobial development strategies.

Bayesian estimates of the prevalence of ß-thalassemia trait in voluntary blood donors of central India: a survey.

Early detection of ß-thalassemia (ß-thal) trait is important. Voluntary blood donors represent an important group who are accessible and cooperative for this purpose. However, the usefulness of this population in ß-thal trait detection programs has not been studied in India. We conducted a hematological survey of 5,045 blood donors who visited the Bhopal Memorial Hospital & Research Centre, Bhopal in central India. Using robust Bayesian methods, we estimated the prevalence of ß-thal trait. The overall prevalence of ß-thal trait in the study population was 9.59% [95% confidence interval (95% CI) 8.78-10.4%]. The prevalence of ß-thal trait varied across the states of origin and within the state of Madhya Pradesh. We observed a cline effect for ß-thal trait prevalence in relation to the latitude (p = 0.024). We conclude that blood donors offer an attractive adjunct to ß-thal trait detection in national programs. Our study also offers insights into the ß-thal trait gene flow and migration in India.

Cellular environment controls the dynamics of histone H3 lysine 56 acetylation in response to DNA damage in mammalian cells.

Epigenetic changes play a crucial role in sensing signals and responding to fluctuations in the extracellular environment. How the cellular micro-environment affects DNA damage response signalling in chromatin context is not extensively studied. Histone acetylation is dynamic and very sensitive to changes in the extracellular environment. Existing literature on H3 lysine 56 acetylation (H3K56ac) levels upon DNA damage in mammals presents a conflicting picture. The occurrence of both increased and decreased H3K56ac upon DNA damage in our experiments led us to investigate the role of the micro-environment on H3K56ac. Here, we show that the global levels of H3K56ac increase as cells grow from low density to high density while SIRT1 and SIRT6 expression decrease. Additionally, rising lactic acid levels increase H3K56ac. Our results show that cell density and accumulation of metabolites affect dynamics of H3K56ac in response to DNA damage. Upon DNA damage, H3K56ac increases in low density cells with low initial acetylation, while acetylation decreases in high cell density cells. These results highlight that H3K56ac levels upon DNA damage are dependent on the metabolites in the extracellular milieu which impact chromatin structure by regulating chromatin modifying enzymes. Accumulation of lactic acid at high cell density reflects conditions similar to the tumour micro-environment. As H3K56ac increases in tumours, lactic acid and low pH might alter H3K56ac in tumours, leading to deregulated gene expression, contributing to tumour progression.

Mutational analysis of thalassemia in transfusion-dependent beta-thalassemia patients from central India.

BACKGROUND: Thalassemia and hemoglobin (Hb) disorders are the most common genetic disorders among humans. These disorders entail huge morbidity, economic, and psychological burden on the families of the affected. Genetic counseling and prenatal diagnosis are the steps, which helps to reduce this burden. At present, there is paucity of data on the mutational spectrum of thalassemia from the central Indian region. METHODS: Blood samples were collected from 62 transfusion-dependent patients, demographic and relevant data were collected and screened for the two rare mutations - 88 (C-T) and CAP + 1 (A-G) using amplification refractory mutation system-polymerase chain reaction (PCR) and GAP PCR technique. PCR was performed for rare Hb disorders such as Hb Lepore and δ ß chain disorder by GAP PCR in addition to five common Indian beta-thalassemia mutations IVS1-5 (G-C), IVS1-1 (G-T), Cd41/42 (-TCTT), Cd8/9 (+G), 619 bp deletion. RESULTS: Overall 93.5% of the mutations could be identified. Among the abnormal Hb, sickle cell and HbE were found at 4% and 3% of all the loci studied. We also reported two loci with Hb δ ß and one locus with Hb Lepore in the present samples. IVS I-5 (G-C) was the common mutation (46%) followed by IVS I-1 (G-T) (12%) and 619 bp (9%). CONCLUSION: The identification of the genotypes helps to define the severity of the phenotype, plan therapy and form the basis of the comprehensive diagnostic database that would be useful not only for genetic counseling but prenatal diagnosis as well, contributing to the current focus of the National Policy to prevent and control hemoglobinopathies.

Intracellular distribution of human SIRT7 and mapping of the nuclear/nucleolar localization signal.

Sirtuins belong to a class of NAD-dependent deacetylases, and include seven distinct isoforms, of which SIRT7 is the least studied member. In the present study, the subcellular expression of SIRT7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of nucleolar SIRT7 in young fibroblast was very prominent, decreased in pre-senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT7 in fibroblasts, and report the existence of a steady-state level of SIRT7 in cytoplasm. Selective localization of the high-molecular-mass (47.5 kDa) SIRT7 in the cytoplasmic fraction and the low-molecular-mass (45 kDa) SIRT7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N-terminal antibodies for detection of cytoplasmic SIRT7 was confirmed by RNAi and peptide competition assays. The two forms of SIRT7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site-directed mutagenesis, we defined the role of two distinct SIRT7 sequences in the N-terminal region (amino acids 61-76, LQGRSRRREGLKRRQE) and the C-terminal region (amino acids 392-400, KRTKRKKVT) for nuclear and nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT7 in addition to its well-known nucleolar form, identify distinct localization signals for its nuclear/nucleolar targeting, and describe an association between loss of nucleolar SIRT7 and replicative senescence.

Role of cellular senescence in hepatic wound healing and carcinogenesis.

A state of permanent growth arrest characterises a senescent cell. Both the beneficial and deleterious effects that have accrued in senescent cells are observed in a complex organ, such as the liver. Injury to liver tissues triggers processes of regeneration and associated wound healing. Persistent injury can also lead to the neoplastic state. Recent evidence linked the senescent characteristics of the cells to the beneficial processes of wound healing and tumour surveillance in the liver. On the other hand, the secretory phenotype of senescent cells can also selectively promote undesirable neoplastic progression. In an evolutionary context, a senescent cell can function primarily as an adaptive response featuring the characteristics of altruism, trade-offs and bystander effects. Using the liver cell as a model system, this review focuses on the current knowledge of the role of senescence in these seemingly contradictory cell phenomena.

Diperoxovanadate can substitute for H(2)O(2) at much lower concentration in inducing features of premature cellular senescence in mouse fibroblasts (NIH3T3).

Stress induced premature senescence (SIPS) in mammalian cells is an accelerated ageing response and experimentally obtained on treatment of cells with high concentrations of H(2)O(2), albeit at sub-lethal doses, because H(2)O(2) gets depleted by abundant cellular catalase. In the present study diperoxovanadate (DPV) was used as it is known to be stable at physiological pH, to be catalase-resistant and to substitute for H(2)O(2) in its activities at concentrations order of magnitudes lower. On treating NIH3T3 cells with DPV, SIPS-like morphology was observed along with an immediate response of rounding of the cells by disruption of actin cytoskeleton and transient G2/M arrest. DPV could bring about growth arrest and senescence associated features at 25 µM dose, which were not seen with similar doses of either H(2)O(2) or vanadate. A minimal dose of 150 µM of H(2)O(2) was required to induce similar affects as 25 µM DPV. Increase in senescent associated markers such as p21, HMGA2 and PAI-1 was more prominent in DPV treated cells compared to similar dose of H(2)O(2). DPV-treated cells showed marked relocalization of Cyclin D1 from nucleus to cytoplasm. These results indicate that DPV, stable inorganic peroxide, is more efficient in inducing SIPS at lower concentrations compared to H(2)O(2).