Potentiation of antibiotic against Pseudomonas aeruginosa biofilm: a study with plumbagin and gentamicin.

AIMS: Pseudomonas aeruginosa is one of the fatal biofilm-forming pathogens which pose to be a problem in clinical infections, contamination of food and marine ecosystems. In this report, a naphthoquinone-plumbagin has been explored for its antimicrobial (antibacterial and antibiofilm) activity against P. aeruginosa biofilm. The ability of plumbagin to enhance the bioactivity of a known broad-spectrum antibiotic was further assayed by combining the sub-MIC doses of plumbagin with sub-MIC doses of gentamicin against P. aeruginosa biofilm. METHODS AND RESULTS: This combinatorial approach was used for a series of experiments for understanding the mechanism of action for antibiofilm activity against P. aeruginosa (MTCC 424, MTCC 2488). Antibiofilm activity was studied by safranin staining, estimating total protein, visualization of biofilms and extra polymeric substances quantification. Antivirulent activity of these doses was studied by azocasein degradation, expression of virulent factors and molecular docking. Expression of quorum sensing (QS) phenotypes was studied by motility assessment and mRNA expression pattern of virulence genes. It was observed that plumbagin alone and the combinatorial doses of plumbagin and gentamicin exhibit significant antibiofilm and antivirulent activity coupled with the reduction in the expression of QS phenotypes and virulence genes. Molecular docking study revealed that plumbagin had variable affinity for different QS proteins. CONCLUSION: Low doses of plumbagin and gentamicin exhibit synergistic activity against P. aeruginosa biofilm while maintaining their effectiveness. SIGNIFICANCE AND IMPACT OF THE STUDY: As the P. aeruginosa biofilms are reservoir of persister bacteria, thus, the increasing concern of antibiotic tolerance has to be dealt with combinatorial approaches. In this report, plumbagin has been explored in potentiating the antibiofilm effect of a broad-spectrum antibiotic gentamicin for better therapeutic efficacy.

Fractional flow reserve: intracoronary versus intravenous adenosine induced maximal coronary hyperemia.

BACKGROUND: Fractional Flow Reserve (FFR), a measure of coronary stenosis severity is based on the achievement of maximal hyperemia of coronary microcirculation. The most widely used pharmacological agent is adenosine which is administered either by intra coronary or intra venous routes. IV route is time consuming, has more side effects and expensive. This study is undertaken to compare the two routes of administration. METHODS: FFR was assessed in 50 patients with 56 intermediate focal lesions using both IV and intracoronary (IC) adenosine. FFR was calculated as the ratio of the distal coronary pressure to the aortic pressure at maximal hyperemia. RESULTS: A total of 25 left anterior descending, 8 right, 21 circumflex, and 2 left main coronary arteries were evaluated. The mean percent stenosis was 63.91 ± 13.13 SD and, the mean FFR was 0.831 ± 0.0738 SD for IV and 0.832 ± 0.0707 SD for IC adenosine. There was a strong and linear correlation between 2 sets of observations with IV dose and IC adenosine dose (R = 0.964, y = 0.065 + 0.923x; p < 0.001) (y = IV dose, x = IC dose). The agreement between the two sets of measurements was also high, with a mean difference of: 0.001 ± 0.0197. The changes in heart rate and blood pressure were significantly higher in IV adenosine group. Different incremental doses were well tolerated, with fewer systemic adverse events with IC adenosine. Transient AV blocks were observed with both IV and IC adenosine. CONCLUSIONS: This study suggests that IC adenosine is equivalent to IV infusion for the determination of FFR. The administration of IC adenosine is easy to use, cost effective, safe and associated with fewer systemic events.

In-vitro metabolism of celecoxib, a cyclooxygenase-2 inhibitor, by allelic variant forms of human liver microsomal cytochrome P450 2C9: correlation with CYP2C9 genotype and in-vivo pharmacokinetics.

In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.

Expression of human cytochrome P450 enzymes in yeast and bacteria and relevance to studies on catalytic specificity.

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences.

Helicobacter pylori infection and upper gastrointestinal patholgy in a British immigrant Indian community.

OBJECTIVES: To assess the pattern of upper gastrointestinal pathology and the prevalence of Helicobacter pylori infection in the Southall Indian community. DESIGN: A prospective study of endoscopic findings in 124 Indian and 107 whites from the Southall area. In a separate study blood samples were taken from 100 Indian subjects presenting to a single general practitioner in Southall. METHODS: The presence of gastritis and H. pylori infection was assessed histologically in Indian and white patients undergoing endoscopy. Serum samples were analysed using a specific enzyme-linked immunosorbent assay (ELISA) for anti-H. pylori immunoglobulin G. RESULTS: In the endoscopic study, Indian and white patients had the same rate of H. pylori infection (52% vs. 43%, respectively) (P= NS). The pattern of upper gastrointestinal pathology was similar in whites and Indians. In the general practice based study 41 subjects were H. pylori seropositive. Seropositivity increased with age (P<0.05). CONCLUSION: There is no excess of H. pylori-related pathology in Southall immigrant Indians. The similarity of upper gastrointestinal pathology in UK Indian and white patients presenting for endoscopy suggests that the high rates of duodenal ulceration, gastritis and H. pylori infection in India are environmentally rather than racially determined.

Lack of release from hepatocytes in vitro or excretion in vivo of mutagenic chrysoidine metabolites.

Rat liver postmitochondrial supernatant (S9) converted the azo dyes chrysoidine Y and R to products that were mutagenic towards Salmonella typhimurium strain TA100. No such release of mutagens was demonstrated using intact rat hepatocytes as an activation system despite the fact that chrysoidine dyes cause unscheduled DNA synthesis in these cells. It appears that genotoxic products produced within hepatocytes either react within the cell or are detoxified prior to release. Following intraperitoneal administration of chrysoidine Y to rats (100 mg/kg i.p.) there was also no evidence of mutagenic or por-mutagenic products excreted in bile or urine. The S9-derived mutagens appear to be largely independent of bacterial acetylation since they were active in the acetylation-deficient strain TA98/1,8-DNP6 in addition to strain TA98. The ultimate mutagenic form(s) are therefore unlikely to be acetoxyarylamines.

Bacterial mutagenesis and hepatocyte unscheduled DNA synthesis induced by chrysoidine azo-dye components.

5 azo dye components of Gurr chrysoidine 'Y' have been separated, synthesised and identified. Dyes with a methyl substitution (particularly between the two amino groups) were more mutagenic in Salmonella typhimurium strain TA100 with control rat liver S9 than the non-methylated counterpart (range 66-1992 revertants at 50 micrograms/plate). Mutagenicity was also catalysed by human-liver S9 and pre-treatment of rats with either phenobarbitone or beta-naphthoflavone enhanced the activation ability of S9 by greater than 4-fold. Using the most potent promutagenic component (2,4-diamino-3-methylazobenzene), the use of inhibitors of cytochrome P450 (metyrapone: 1.0 mM; alpha-naphthoflavone: 0.075 mM; DPEA: 0.125 mM) and of the flavin monooxygenase (methimazole: 0.75 mM) suggested a major role for cytochrome P448 in the activation of chrysoidine to mutagens. The ability of chrysoidine components to induce unscheduled DNA synthesis in rat hepatocytes in vitro was demonstrated and ranged between 11.92 and 23.5 net nuclear grains at a dose level of 2.5 micrograms/incubation. Since each dye was equi-potent, methyl substitution had little influence on genetic toxicity in hepatocytes.

Clinical evaluation of cyanoacrylate glue in corneal perforations.

Our experience of the use of Cyanoacrylate glue in 50 cases of perforation or impending perforation of cornea has been presented. The method of application has been described. Quite encouraging and useful results have been obtained.

Sonographic evaluation of renal allograft.

Sonography has become an integral part of the care of renal allograft recipients. It is a simple, inexpensive and readily available non-invasive imaging modality. It is indicated as the initial investigation in patients presenting with decreased urine output, pain, infection and hematuria and for doing a percutaneous allograft biopsy. While sonography confirms the diagnosis of obstructive nephropathy and perinephric fluid collections, Doppler is an effective screening modality for the detection of post-transplant vascular complications.

Giant cavernous hemangiomas of liver mimicking metastasis.

Most hepatic hemangiomas are small and symptomless. These are now being increasingly diagnosed with the greater use of scanning procedures. Hemangiomas can occasionally grow to a large size and become manifest to the patient and the clinician. Giant hemangiomas can produce symptoms including awareness of abdominal mass, pain due to thrombosis, and very rarely, rupture. Though ultrasound is known to be quite suggestive of the diagnosis, large hemangiomas may be mistaken for liver metastases due to their enormous size and variegated picture on the scanning procedure. Dynamic CT scan and at times MRI may be required for confirmation of the diagnosis. Needle biopsy is contraindicated if the diagnosis is suspected.

A multicentre retrospective study to understand anti-platelet treatment patterns and outcomes of acute coronary syndrome patients in India (TRACE).

BACKGROUND: There is limited available information for treatment of acute coronary syndrome (ACS) with respect to outcomes, therapeutic agents and treatment practices. Our retrospective registry study collected and evaluated varying anti-platelet treatment strategies and outcomes of ACS patients who were admitted to 9 different tertiary care hospitals in India. This study was carried out to provide an insight to anti-platelet treatment patterns and analyze outcomes of ACS patients in India. METHODS: All the relevant data, including anti-platelet treatment strategies, outcomes and patient treatment compliance were collected from 500 ACS (defined as STEMI, NSTEMI and unstable angina [UA]) cases from January 2007 to December 2009. These ACS cases were randomly collected from the hospital records and included in the analysis. The patient follow up data was acquired either from the hospital records or via telephonic contact for a period of one year following the event. RESULTS: Out of 500 ACS patients, 59.8% had UA/NSTEMI and 40.2% had STEMI. On hospital admission, aspirin, clopidogrel, statins, beta-blockers and angiotensin converting enzyme inhibitors (ACE-Is) were used by 83%, 83%, 68%, 43.2% and 31.6% patients, respectively. On discharge, aspirin, clopidogrel, statins and beta-blockers were used by 90.2%, 88%, 80.6%, and 59% patients, respectively. The average patient compliance to statins, clopidogrel and aspirin was recorded as 74.28%, 69.7% and 68.66%, respectively during discharge and follow-up visits. Greater than 50% of ACS patients after discharge were lost to follow-up and as a result there was significant drop in the number of clinical events reported. CONCLUSION: This pilot study conducted in tertiary care centers in India showed that patients with ACS were more often diagnosed with UA/NSTEMI as compared to STEMI and reported maximum compliance to statins, clopidogrel and aspirin after discharge over 1 year follow-up. More ACS patients were lost to follow up that resulted in low reporting of clinical outcomes, following discharge upto 1 year.

The paclitaxel-eluting PTCA-balloon in combination with a cobalt-chromium stent in two different sequences to treat de novo coronary artery lesions: an angiographic follow up study.

INTRODUCTION: The paclitaxel-coated balloon catheter (DCB) based on the PACCOCATH(®) technology has yielded angiographic and clinical results superior to drug-eluting stents (DES) in situations like in-stent restenosis (ISR) and a trend towards superior results in small coronary vessels and side branches of coronary bifurcations. Using the DCB followed by cobalt-chromium stent (CoCr) deployment or with a reverse sequence may yield different outcomes in terms of late loss. METHODS: 97 patients with de-novo coronary stenosis (55.6 ± 10.7 years, 79.4% male, ≥70%, length: ≤25 mm, vessel diameter: 2.5-4.0 mm) were randomly treated with the DCB (3 µg/mm²) followed by a CoCr-stent or stent first and DCB later. Six-month angiographic and one-year clinical follow-up intention-to-treat analyses were performed. RESULTS: Angiographic and demographic baseline data was comparable between the two groups. When comparing balloon first versus stent first technique, the primary outcome variables were not statistically different for mean in-segment (0.51 ± 0.56 mm vs. 0.36 ± 0.55 mm, p = 0.23) and in-stent (0.52 ± 0.55 mm vs. 0.46 ± 0.52 mm, p = 0.65) late lumen loss. The lesion related 12-month MACE rates were 5/49 (10.2%) and 2/48 (4.2%) (p = 0.44). Lesion related thrombotic events occurred in three patients in balloon first and in one patient in stent first group, two of which were associated with early discontinuation of continuous dual anti-platelet therapy, two with suboptimal PCI, and one each were performed in a thrombotic lesion and a bifurcation type 1.1.0. CONCLUSION: Drug-coated balloon first followed by cobalt chromium stent deployment versus a reverse sequence is not associated with statistically significantly different 6-month angiographic or 12-month clinical outcomes.