Prevalence and molecular characterization of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli isolated from dogs suffering from diarrhea in and around Kolkata.

Background: Dogs are the favorite companion animals among humans. The close interaction between dogs and people increases the risk of antibiotic resistance spreading. Surveillance for antimicrobial resistance and the identification of ESBL-producing Escherichia coli as an indicator bacterium is an important tool for managing antimicrobial drug therapy. Aims: The present study targeted to identify and characterize ESBL-producing E. coli among dogs suffering from diarrhea in and around Kolkata. Methods: Isolation and identification of E. coli from dogs suffering from diarrhea (n=70) along with screening for the production of both ESBL and AmpC. The isolates were further characterized through antimicrobial resistance profiling, resistance genes (bla CTX-M, bla TEM, and bla SHV) screening, and phylogenetic group study. Results: Among the 70 isolates, 21 (30%) were confirmed ESBL producers. An antibiogram typing of ESBL-producing E. coli revealed that the majority of them were resistant to norfloxacin (85.7%) followed by tetracycline (61.90%), doxycycline (57.14%), piperacillin/tazobactam (52.38%), cotrimoxazole (47.62%), gentamicin (42.62%), amikacin (23.81%), and chloramphenicol (19.05%). Major resistance genes included bla CTX-M (100%), bla TEM (28.57%), and bla SHV (9.50%). The predominant phylogenetic groups were phylogroup A (76%) followed by phylogroup D (24%). Conclusion: The current investigation reported a high prevalence of both ESBL and AmpC ß-lactamase (AmpC) producing E. coli, co-resistance to a distinct group of antibiotics, and co-existence of different ESBL genes in dogs. Our findings highlight the importance of diagnostic antimicrobial susceptibility testing for proper antimicrobial therapy and to prevent antimicrobial resistance from spreading to humans from dogs in Kolkata and the surrounding area.

Mutation in Cytochrome B gene causes debility and adverse effects on health of sheep.

Cytochrome B is the mitochondrial protein, which functions as part of the electron transport chain and is the main subunit of transmembrane cytochrome bc1 and b6f complexes affecting energy metabolism through oxidative phosphorylation. The present study was conducted to study the effect of mutation of Cytochrome B gene on the health condition of sheep, which the first report of association of mitochondrial gene with disease traits in livestock species. Non-synonymous substitutions (F33 L and D171N) and Indel mutations were observed for Cytochrome B gene, leading to a truncated protein, where anemia, malfunctioning of most of the vital organs as liver, kidney and mineral status was observed and debility with exercise intolerance and cardiomyopathy in extreme cases were depicted. These findings were confirmed by bioinformatics analysis, haematological and biochemical data analysis, and other phenotypical physiological data pertaining to different vital organs. The molecular mechanism of cytochrome B mutation was that the mutant variant interferes with the site of heme binding (iron containing) domain and calcium binding essential for electron transport chain. Mutation at amino acid site 33 is located within transmembrane helix A, a hydrophobic environment at the Qi site and close to heme binding domain, and mutation effects these domain and diseases occur. Thermodynamic stability was also observed to decrease in mutant variant. Sheep Cytochrome B being genetically more similar to the human, it may be used as a model for studying human diseases related to cytochrome B defects. Future prospect of the study includes the therapeutic application of recombinant protein, gene therapy and marker-assisted selection of disease-resistant livestock.

Isolation, molecular characterization and antibiotic resistance of Shiga Toxin-Producing Escherichia coli (STEC) from buffalo in India.

In total, 363 Escherichia coli were isolated from 165 faecal samples of healthy buffaloes in West Bengal, India. Twenty-four of these isolates (6·61%) were found to carry at least one gene characteristic for Shiga toxin-producing Escherichia coli (STEC). These STEC strains belonged to 13 different O-serogroups. The stx1 gene was present in 23 (95·8%) of total STEC isolates, whereas 20 (83·3%) STEC isolates carried the gene stx2. Twelve strains of E. coli (50% of total STEC isolates) possessed enterohaemolysin (ehxA) gene in combination with others. Fourteen (58·33%) isolates found to possess saa gene. However, no E. coli was detected harbouring gene for intimin protein (eaeA). Of 23 stx1 -positive isolates, seven (30·43%) were positive for genes of the stx1C subtype. Of the 20 isolates with the stx2 gene, 25% (5/20) possessed stx2C and 10% (2/20) possessed stx2d gene. The phylogenetic analysis after RAPD of STEC strains revealed six major clusters. The isolated STEC strains were resistant most frequently to erythromycin (95·83%), cephalothin (62·5%), amikacin (54·17%), kanamycin (45·83%) and gentamicin (41·67%) group of antibiotics. No ESBL-producing (blaCTXM , blaTEM , blaSHV ) or quinolone resistance gene (qnrA) was detected in the STEC isolates.

Clinical utility of the interaction between lectin and serum prostate specific antigen in prostate cancer.

The present investigation was a lectin-based diagnosis of malignant prostate cancer (PC) by the interaction of phytohemagglutinin (PHA lectin) from Phaseolus vulgaris with the glycan part of serum prostate specific antigen (PSA) of patients with prostatic disorder. This was confirmed by the interaction between PHA and purified PSA obtained from serum by electrophoretic separation and finally by HPLC chromatography. The precipitate of carbohydrate content after binding of PHA with purified PSA of PC was significantly higher than that of benign prostate hyperplasia (BPH) and/or normal serum PSA. The results suggest that there may be a striking difference in glycosylation pattern of PSA between BPH and PC. The cut off value > or = 10 microg/ml of the carbohydrate content of PHA-PSA precipitate indicates strong suspicion for PC irrespective of total serum PSA cut off level > or = 4.0 ng/ml by conventional immunoassay method and this may be taken as a guideline in differentiating PC and BPH. Key words: prostate cancer, BPH, PSA, lectin.

Cerebrospinal fluid and serum carcinoembryonic antigen in brain tumors.

Carcinoembryonic antigen (CEA) has been indicated to be a marker for brain tumors. In this study CEA was measured in serum and cerebrospinal fluid (CSF) of 14 patients with benign brain lesions, 16 with primary brain tumors and 8 with metastatic brain tumors by radioimmuno assay. Tumor cyst fluid CEA of 6 patients having intracranial tumors was also measured. The control group (n=20) had no neurological disease. The mean CEA levels in CSF for the control group, patients with benign tumors, primary tumors and metastatic tumors were 0.22 ng/ml, 0.31 ng/ml, 0.92 ng/ml, and 6.3 ng/ml respectively. Corresponding serum CEA levels were 2.5, 2.7, 3.0 and 5.2 ng/ml. Results showed that CEA level in CSF may play an important role in differential diagnosis of primary and metastatic brain tumors and consequently management of the treatment. To our knowledge this is the first such study on brain tumors from India.

Separation and identification of thyroid autoantibodies in patients with thyroid disorders by hydrophobic column.

A method has been developed to separate and identify thyroglobulin autoantibody (TgAb) and thyroid peroxidase autoantibody (TPOAb) in serum obtained from normal and autoimmune thyroid diseases using phenyl Sepharose CL-4B hydrophobic column. The protein peaks obtained from hydrophobic column were identified as TgAb and TPOAb by comparing the elution profile of commercially purified standard thyroid autoantibodies. The similarity of the inhibitory effects of eluted proteins and of standard thyroid autoantibodies on lectin concanavalin A-RBC interaction confirmed the separation of TPO-Ab and TgAb by the hydrophobic, column. The eluted fractions from the hydrophobic column were estimated by the radio immunoassay (RIA) to confirm the presence of both auto-antibodies. This hydrophobic column method offers an advantage of visual inspection of this autoantibodies by graphic representation of peak height along with their estimation in autoimmune thyroid disorders.

Hypothalamic-pituitary-thyroid axis status of humans during development of ageing process.

We have investigated hypothalamic-pituitary-thyroid function in four groups of healthy elderly male humans. Group A (n=18, age range 20-45 years) served as healthy younger controls, group B (n=10, age range 50-60 years), group C (n=15, age range 60-70 years) and group D (n=16, age range 70-85 years) are the subjects of this study. Groups C and D showed significantly lower T3 and thyroid-stimulating hormone (TSH), and higher T4 levels with respect to controls. Evidence for TSH circadian modulation was found in group A (control) and group B subjects. The TRH-stimulated TSH peak was reduced among all elderly subjects with respect to controls and appeared to be pronounced with the ageing process. The maximal prolactin response was also inhibited with increasing age. Our study suggest that a resetting of the pituitary threshold for the TSH feed-back suppression along with complex alterations in peripheral thyroid hormone concentrations may, in turn, develop in older people and that appeared to manifest prominently among the oldest population. Additionally, the TSH nocturnal response appeared to be impaired with increasing age indicating an alteration of hypothalamic function.

Targets of oxidative stress in cardiovascular system.

Although oxidants such as superoxide (O2.) and hydrogen peroxide (H2O2) play a role in host-mediated destruction of foreign pathogens yet excessive generation of oxidants may lead to a variety of pathological complications in the cardiovascular system. An important mechanism by which oxidants cause dysfunction of the cardiovascular system appears to be due to the increase in intracellular free Ca2+ concentration. Oxidants cause cellular Ca2+ mobilization by modulating activities of a variety of regulators such as Na+/H+ and Na+/Ca2+ exchangers, Na+/K+ ATPase and Ca2+ ATPase and Ca2+ channels that are associated with Ca2+ transport in the plasma membrane and the sarco(endo)plasmic reticular membrane of myocardial cells. Recent research have suggested that the increase in Ca2+ level by oxidants plays a pivotal role in inducing several protein kinases such as protein kinase C, tyrosine kinase and mitogen activated protein kinases. Oxidant-mediated alteration of different signal transduction systems and their interations eventually regulate a variety of pathological conditions such as atherosclerosis, apoptosis and necrosis in the myocardium.

Role of hydroxyl radical in the stimulation of arachidonic acid release caused by H2O2 in pulmonary smooth muscle cells: protective effect of anion channel blocker.

We sought to investigate role of hydroxyl radical (OH.) in H2O2 caused stimulation of arachidonic acid (AA) release from rabbit pulmonary arterial smooth muscle cells, and to ascertain protective effect of the anion channel blocker DIDS in this phenomenon. Exposure of the smooth muscle cells to the oxidant H2O2 (1mM) stimulates iron release and enhances AA liberation from the cells. Pretreatment of the cells with either deferoxamine (DFO) or dimethyl thiourea (DMTU) markedly reduces AA release and prevents OH. production without causing any appreciable reduction of iron release caused by H2O2. Simultaneous treatment of either DFO or DMTU with H2O2 significantly reduces AA release, and also prevents OH. production without causing any significant reduction of iron release. In contrast, addition of either DFO or DMTU even 2 min after exposure of the cells to H2O2 does not cause any significant reduction of AA release, OH. production and iron release. Pretreatment of the cells with DIDS markedly reduces AA release caused by H2O2 without producing any discernible reduction of iron release, and OH. production.

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 microM of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A2 activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfluoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A2 activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A2 activities and arachidonic acid release in cells pretreated with nominal Ca2+ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A2 activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A2 activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated A2 activity (ii) in addition to the presence of extracellular Ca2+, release of Ca2+ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A2 activities does not appear to require new RNA or protein synthesis.

Role of serine esterase in hydrogen peroxide-mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to hydrogen peroxide cause dose-dependent stimulation of [14C] arachidonic acid (AA) release and enhancement of the cell membrane-associated phospholipase A2 activity as well as of the cell membrane-bound serine esterase activity tested against synthetic substrate p-tosyl-L-arginine methyl ester. While pretreatment of cells with serine protease inhibitors, viz. phenyl methyl sulphonyl fluoride, diisopropyl fluorophosphate and alpha-1-proteinase inhibitor, and antioxidant vitamin E prevents H2O2 stimulation of AA release and the cell membrane-bound serine esterase and PLA2 activities, that with actinomycin D and cycloheximide is devoid of any effect on H2O2 caused stimulation of AA release and the smooth muscle cell membrane associated serine esterase and PLA2 activities. Treatment of the smooth muscle cell membrane suspension with the serine protease trypsin markedly stimulates PLA2 activity. These results suggest that on exposure to H2O2 the smooth muscle cell membrane-bound serine esterase plays an important role in stimulating the cell membrane associated PLA2 activity thereby resulting in an increase in AA release.

Myocardial changes in neonates dying of asphyxia neonatorum.

Ten neonates, asphyxiated at birth, were studied by Apgar score, ECG ischaemic score grading (ECGisg), Cardiothoracic (CT) ratio, biochemical parameters like CPK, CPK-MB fraction during life; and they were subjected to postmortem study with particular attention to the changes in the heart. The study revealed that 7 out of 10 asphyxiated neonates showed variable evidences of myocardial damage; but the extent of damage though well correlated with biochemical parameters, did not correspond well with the extent of asphyxia and the survival period. In rest 3 cases, myocardial damage was not overt though there was evidence of asphyxia and evidence of myocardial damage in the form of elevated CPK-MB level. These patients probably had died of "Biochemical Lesion" as described by Rudolf Peter.

Prolactin and alpha-1,4-glucosidase activity in normal and poorly coagulated human semen.

Prolactin and alpha-1,4-glucosidase levels in seminal plasma were measured in poorly coagulated (I), deficiently coagulated (II) and normally coagulated (III and IV) human ejaculates having 0-20%, 21-50% and 51-100% coagulum respectively 4 min after emission. The prolactin concentration (ng ml-1, mean +/- SEM) in poorly coagulated (5.2 +/- 0.48) and deficiently coagulated (7.6 +/- 0.72) samples was significantly lower than in the normally coagulated groups III (51-75% coagulum, 8.2 +/- 0.43) and IV (76-100% coagulum, 9.9 +/- 0.59) as well as the presumably fertile samples (9.2 +/- 0.74). A highly significant positive correlation was observed between the prolactin level and the percentage coagulum of the ejaculates (r = 0.686, n = 58, P less than 0.001). In contrast, the epididymal marker, alpha-glucosidase showed no relationship to seminal coagulation.

Gemfibrozil in dyslipidaemia.

A new lipid regulating agent, Gemfibrozil was evaluated in different types of dyslipidaemias. Out of a total of 34 patients, all completed 12 weeks' treatment and 26 completed 24 weeks' of treatment. A significant reduction in total cholesterol, LDL-c triglyceride and apo-B and an increase in HDL cholesterol and its apoprotein--apo-A were observed. The patients belonged to hyperlipidaemias--types IIa, IIb, and IV. Patients' compliance was good and side effects were minimal.

An echo-cardiographic (M-mode & 2D) analysis of thalassaemia major.

25 cases of thalassaemia major were studied by 2D and M-mode echocardiography. A significantly increased (p less than 0.001) mean value (100.8 +/- 27.37 msec, range 80 to 140 msec) of A2-E (early relaxation period) interval on M-mode was observed in thalassemia in comparison to mean level (82.6 +/- 5.7, range 60 to 100 msec) of control population. No significant differences were noted in FS % (fractional shortening) and EF% (ejection fraction) when compared to corresponding normal values respectively. Mean serum iron concentration (142.2 +/- 29.1 micrograms/dl, range 102 to 192 micrograms/dl) was significantly higher in thalassaemia as compared to normal population (mean 106.3 +/- 11.4 micrograms/dl, range 75 to 120 micrograms/dl). There was also a direct correlation between serum iron concentration and A2-E interval. 11 patients (44%) showed abnormal A2-E interval but only 3 patients (12%) showed abnormal percentage of FS and EF. It is therefore concluded that A2-E interval will help to detect early left ventricular dysfunction much before overt and irreversible heart failure becomes manifest and which will also help to optimise transfusion and chelation therapy.

Serum phospholipid fractions measured by two dimensional thin layer chromatography in normal pregnancy.

Serum phospholipid fractions were quantitated by two dimensional thin layer chromatography in 85 apparently normal pregnant women at different gestational period. Fifteen healthy non-pregnant women matched for age served as controls. Normal chromatogram showed eight spots of serum phospholipid viz., phosphatidic acid (PA, 3.9 +/- 0.5%), cardiolipin (C, 7.2 +/- 0.4%), phosphatidylethanolamine (PE, 10.6 +/- 1.1%), phosphatidylglycerol (PG, 3.2 +/- 0.6%), lecithin (L, 34.5 +/- 1.8%), sphingomyelin (S, 31.0 +/- 1.4%), lysolecithin (LL, 8.6 +/- 1.2%) and phosphalidylionositol (PI, 1.0 +/- 0.2%). Serum total phospholipid which increased with gestational age, being lowest at 6 wk (220.2 +/- 4.8 mg/dl) and highest at 38 wk (290.3 +/- 4.5 mg/dl), started declining at term (275.8 +/- 5.8 mg/dl) and returned to levels in normal nonpregnant women (214.1 +/- 4.7 mg/dl) at 4 wk after delivery (217.2 +/- 3.3 mg/dl). PG was absent in both the first and second trimester and first appeared between 31-32 wk, i.e., in the third trimester of pregnancy when the mean per cent of PG was 0.7 +/- 0.4 per cent. During the first trimester when the L/S ratio was less than 1.2 the PI level was low (0.8 +/- 0.2%). Parallel to the increase in the L/S ratio to 2.0, the content of PI increased to 2.6 +/- 0.4 per cent. PG first appeared (0.7 +/- 0.4%) and PI concomitantly decreased (2.1 +/- 0.3%) when the L/S ratio exceeded 2.0 at 31-32 wk. Sequential analysis of maternal serum phospholipid fractions may be used as an additional tool in predicting gestational age of normal growing pregnancy.

Immunological studies on pulmonary tuberculosis: a preliminary report.

An investigation of 71 patients (61 mantoux positive and 10 mantoux negative) with bacteriologically and radiologically proven primary pulmonary tuberculosis was undertaken for immunological analysis to evaluate cell mediated (E-rosette) and humoral (EAC-rosette and serum immunoglobulins; IgG, IgA & IgM) immune status. 41 age and sex matched normal subjects served as controls. An inverse relationship between cell mediated and humoral immune response was observed in tuberculous patients in general. Defects in cell mediated immune response was pronounced in mantoux negative cases. Fractionation of serum protein revealed a significant diminution in albumin level with a concomitant elevation in globulins particularly gammaglobulin levels. It appeared that there is a definite need to boost up T-cell mediated responses and to control the over activity of B-cell in these cases. Proper handling of the immune system by appropriate therapy might change the course of the disease.