Characterization of Vibrio cholerae O1 strains that trace the origin of Haitian-like genetic traits.

Vibrio cholerae O1 is the etiological agent of the severe diarrheal disease cholera. The bacterium has recently been causing outbreaks in Haiti with catastrophic effects. Numerous mutations have been reported in V. cholerae O1 strains associated with the Haitian outbreak. These mutations encompass among other the genes encoding virulence factors such as the pilin subunit of the toxin-co-regulated pilus (tcpA), cholera toxin B subunit (ctxB), repeat in toxins (rtxA), and other genes such as the quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the alteration in the number of repeat sequences at the promoter region of ctxAB. Given the numerous genetic changes in those Haitian isolates, we decided to investigate the possible origins of those variations in the Indian subcontinent. Thus, we determined the genetic traits among V. cholerae O1 strains in Delhi, India. A total of 175 strains isolated from cholera patients during 2004 to 2012 were analysed in the present study. Our results showed that all the tested strains carried Haitian type tcpA (tcpACIRS) and variant gyrA indicating their first appearance before 2004 in Delhi. The Haitian variant rtxA and ctxB7 were first detected in Delhi during 2004 and 2006, respectively. Interestingly, not a single strain with the combination of El Tor rtxA and ctxB7 was detected in this study. The Delhi strains carried four heptad repeats (TTTTGAT) in the CT promoter region whereas Haitian strains carried 5 such repeats. Delhi strains did not have any deletion mutations in the rstB like Haitian strains. Overall, our study demonstrates the sequential accumulation of Haitian-like genetic traits among V. cholerae O1 strains in Delhi at different time points prior to the Haitian cholera outbreak.

Isolation and immunohistochemical identification of Flavobacterium psychrophilum from the tissue of catfish, Clarias batrachus.

This study deals with the identification of bacteria in paraffin embedded tissue of Indian catfish, Clarias batrachus experimentally infected with F. psychrophilum to ascertain it's precise role in the occurrence and state of carrier stage of coldwater disease in asymptomatic fishes. Significant (P < 0.05) mortality was recorded in C. batrachus of this region due to persistent occurrence of creamish/yellowish with non smooth edged colonies of rod shaped flexible gram negative bacterium, F. psychrophillum. Serodiagnosis made through polyclonal antiserum dilutions (raised against H antigen of Aqb Fb-6 isolate in rabbit) revealed that staining intensity of this bacterium was dilution dependent as tissue and smears treated with 1:300 and 1:500 antiserum dilutions conferred close relationship at immunofluorescence level with F. psychrophilum and stained deeply in the tissues and smear prepared from liver, kidney and intestine of experimentally infected C. batrachus. Result of this study would be helpful in identifying the fish pathogenic bacteria in carrier fish where mortality occurs without explicit clinical signs of disease.

Lytic bacteriophages specific to Flavobacterium columnare rescue catfish, Clarias batrachus (Linn.) from columnaris disease.

This investigation was aimed to find out appropriate strategy against antibiotic resistant bacterial fish pathogen, F. columnare. This pathogen was found persistently associated with fishes causing columnaris disease and ensuing mass mortality in hatchery and culture system of Sub - Himalayan region. Nine lytic F. columnare phages (FCP1 - FCP9) specific to its fifteen isolates were isolated from the water and bottom sediments of various geo-climatic regions of North India. The F. columnare phage FCP1 (made of hexagonal head and non contractile long tail belonging to family Podovariedae, a member of DNA virus) exhibited broader host range to lyse 9 out of 15 isolates of F. columnare. Therapeutic ability of FCP1 phage was assessed in C. batrachus inoculated intramuscularly (im) with virulent bacterial isolate FC8 and post inoculated (PI) with FCP1 phage (@ 10(8) : 10(6):: cfu : pfu) through intramuscular (im), immersion (bath) and oral (phage impregnated feed) treatment. Significant (p < 0.001) reduction (less than 10(-3) cfu ml(-1)) in host bacterium in the sera, gill, liver and kidney of challenged fishes was noted after 6 hr of phage treatment. Quantum of phage played a significant role in bringing down bacterial population as in the sera of dose 1 (@ 4.55 x 10(6) pfu ml(-1)) and dose 2 (@ 9.15 x 10(6) pfu ml(-1)) treated fishes mean log10 cfu value reduced by 3 logs (58.39%) and 5 logs (73.77%) at 96 hr, respectively. Phage treatment led to disappearance of gross symptoms, negative bacteriological test, detectable phage and 100% survival in experimentally infected C. batrachus. Result of this study provides evidence of profound lytic impact of FCP1 phage and represents its interesting therapeutic importance against antibiotic resistant F. columnare.

P-27/HP endolysin as antibacterial agent for antibiotic resistant Staphylococcus aureus of human infections.

Staphylococcus aureus causes a wide range of suppurative infections in humans and animals. Due to its high virulence, ability to adopt various environmental conditions, and acquired multiple drug resistance, treatment of such infections has become difficult. Therefore, there is an immense need to develop alternate drug modalities to control this pathogen. In past few years, phage-encoded endolysin therapy has emerged as a new hope not only due to its ability to specifically kill the target bacteria irrespective of their antibiotic sensitivity but also because of minimum or no side effects, a problem associated with antibiotic therapy. In this article, we report purification of a broad spectrum anti-staphylococcal endolysin (P-27/HP endolysin) encoded by phage P-27/HP isolated from sewage water. On SDS-PAGE endolysin resolved in three polypeptides of molecular weights 33.5, 48.6, and 62.2 kDa. Endolysin exhibited maximum in vitro lytic activity at temperature between 35 and 40°C and pH 7.0. In vivo experiments revealed considerable (99.9%) elimination of S. aureus 27/HP from spleens of endolysin-treated mice and had saved them from death due to bacteremia caused by S. aureus 27/HP challenge infection. Thus, P-27/HP endolysin offers suitable substitute of antibiotics to control S. aureus infections.

Effect of pH and salinity on pathogenicity of Flavobacterium columnare and Myxobacterium sp. in Indian cat fish, Clarias batrachus (Linn.) and Heteropneustes fossilis (Bloch.).

Flavobacterium columnare (FC) and Myxobacterium sp. recorded persistently associated in fish hatchery and culture system of Himalayan and Sub - Himalayan regions were found to be pathogenic. The pH and salinity played a significant role on the pathogenicity of these potent pathogens in case of Clarias batrachus and Heteropneustes fossilis. LD50 value of FC was 10(4.5) CFU in both the fishes and those of Myxobacterium sp it was 10(6) CFU ml(-1) fish(-1). Fish challenged with F. columnare and Myxobacterium sp. (@ 0.2 ml fish(-1)) individually consisting 10(5-6) cfu ml(-1) exhibited explicit symptoms of columnaris disease and marked with ulceration and saddle back lesion on the dorsal side of body. Maximum reisolation of inoculated bacteria was recorded at pH 7.0 and 7.5 and at 0.0-0.5 (F. columnare) and 0.0-1.0% (Myxobacterium sp.) salinity. Foregoing results elucidated that F. columnare was more sensitive to salinity in comparison to Myxobacterium sp. and their pathogenicity significantly (p<0.05) depends on the salinity and pH that might be one of the physical factors to control their proliferation.

Efficacy of polyvalent bacteriophage P-27/HP to control multidrug resistant Staphylococcus aureus associated with human infections.

Emergence of multidrug resistant strains has created serious problem for safe eradication of Staphylococcus aureus infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages kill bacteria irrespective of their antibiotic sensitivity and thus they can be used as potent prophylactic/therapeutic agent to treat such infections. Here, we report isolation of broad host range anti-staphylococcal lytic bacteriophage P-27/HP from sewage water. This phage was able to inhibit 17 of 28 (60%) human disease associated S. aureus isolates. In vitro studies revealed its strong lytic efficacy to diminish S. aureus 27/HP population (c.f.u.) by more than 5.0 logs (P < 0.0001) (equivalent to 99.99%) in 3 h at 0.01 MOI. In vivo lytic efficacy analysis showed that a single subcutaneous injection of phage P-27/HP (10(7) p.f.u.) was sufficient to protect S. aureus 27/HP infected (5 × 10(8) c.f.u.) mice from bacteremia and subsequent death. A considerable decline of more than 6 logs (99.9%) in splenic S. aureus 27/HP c.f.u. count was noted at the 3 days of phage treatment. In conclusion, our results suggest that phage P-27/HP is polyvalent in nature and has high-lytic potential towards S. aureus, thus, a therapy employing this phage would be efficacious to control S. aureus infections.

Does cardiac SPECT using attenuation and scatter correction accurately predict coronary artery disease in a minority women population?

BACKGROUND: Myocardial perfusion imaging (MPI) is a well-established diagnostic method for evaluation and risk stratification of coronary artery disease (CAD). We undertook this study to validate both the positive predictive value (when compared to cardiac catheterization) and the prognosis afforded by MPI in a group of minority women patients. MATERIAL/METHODS: The database of our Nuclear Imaging and Catheterization Laboratory was retrospectively queried for consecutive minority (African-American, Hispanic and Asian) women patients who underwent MPI and cardiac catheterization within 90 days of each other. Patients with recent revascularization were excluded. Attenuation/scatter correction was utilized in the final interpretation of the study. RESULTS: Of the 54 women patients who underwent MPI, 7 underwent exercise stress testing, 26 had stress testing with adenosine, 18 with dipyridamole and 3 with dobutamine. Eighteen patients (53%) had same number of vessels predicted by MPI and coronary angiography (7 patients with triple vessel disease, 7 with 2-vessel disease and 4 with single vessel disease). Five (3 with intermediate and 2 with high risk scans) out of the 54 patients (9.3%) were dead at 2 years. The sensitivity, specificity and positive predictive value of MPI as compared to angiography were 87.2%, 26.7%, 75.6% and 44.4% respectively. CONCLUSIONS: The sensitivity of MPI in this group of patients is comparable to the general population though the specificity is lower in spite of using attenuation and scatter correction. Low risk perfusion scan signifies favorable prognosis at 2 years with regards to mortality.

Aortic sclerosis--a marker of coronary atherosclerosis.

Aortic valve sclerosis is defined as calcification and thickening of a trileaflet aortic valve in the absence of obstruction of ventricular outflow. Its frequency increases with age, making it a major geriatric problem. Of adults aged > 65 years, 21-29% exhibit aortic valve sclerosis. Incidence of aortic sclerosis increases with age, male gender, smoking, hypertension, high lipoprotein (Lp) (a), high low-density lipoprotein (LDL), and diabetes mellitus. Aortic valves affected by aortic sclerosis contain a higher amount of oxidized LDL cholesterol and show increased expression of metalloproteinases. Clinically, it can be suspected in the presence of soft ejection systolic murmur at the aortic area, normal split of the second heart sound, and normal volume carotid pulse, but it can be best detected by echocardiography. Aortic sclerosis may be accompanied by mitral annulus calcification up to 50% of cases. It is associated with an increase of approximately 50% in the risk of death from cardiovascular causes and the risk of myocardial infarction. The mechanism by which aortic sclerosis contributes to or is associated with increased cardiovascular risk is not known. Aortic sclerosis is associated with systemic endothelial dysfunction, and a small percentage of cases may progress to aortic stenosis. Lowering of LDL cholesterol by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to decrease progression of aortic valve calcification. Aortic sclerosis is not a mere benign finding. Once diagnosis of aortic sclerosis has been made, it should be considered a potential marker of coexisting coronary disease. Aggressive management of modifiable risk factors, especially LDL cholesterol lowering, may slow progression of the disease.