Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN-0001November30HealthcareCURLI FIBRES AND PRION STRUCTURAL REVERSIONS - IMPLICATIONS TO PHYSICAL BIOLOGY STUDIES English0103A. BarboraEnglishNew investigations in the field of protein structures, bacterial exudes and prion research; and related experimental indications derived suggest that when infectious forms of prion plaques placed in or with resistance – providing variant form(s) are irradiated with appropriate laser power; reversions of prion plaques into structures like coils, helices, etc. may hypothetically take place. These structures are less stable than the beta sheets, hence more susceptible to being biologically cleared off the plaque residues. This provides for a means to effectively cure or prevent prion – like amyloid plaque related diseases; with further implications for physical biology. EnglishCurli fibres, Prions, Amyloids, Protein structure - coils, Helices, Biofilms, etcINTRODUCTION Curli fibrils are proteins in the extracellular matrix secreted by Enterobacteriaceae to make biofilms; which aid primarily in adhesion to surfaces and invasion of host cells. They belong to the class of Amyloid fibre proteins which include the prion proteins. They are known to interact with various other proteins some of which lead to pathogenesis as in the case of bovine mastitis (1). Curli fibres are considered functional amyloids as they possess many properties of amyloid proteins, but not as a consequence of their misfolded nature. Curli fibrils possess a cross-β structure which characterises protein amyloids. But solid state NMR studies indicated that the Curli structures are not based on in-register parallel β-sheet architecture, which is common to most human disease-associated amyloids and the yeast prion amyloids. (2). Generally, the histopathological definition of amyloid is an extracellular, proteinaceous deposit with beta sheet structure. When stained with Congo red dye and seen under a polarized light they give an apple – green birefringence; which in general is commonly used to identify cross – beta type structures (3). Biophysically a polypeptide which polymerizes to form a cross-beta structure, in vivo or in vitro, may be called an amyloid. However, of late amyloid species have been observed in distinct intracellular locations as well (4); some of which, although demonstrably show cross-beta sheets in structure, actually do not have the classic histopathological characteristics such as the Congo-red birefringence (5). Prions as transmissible agents causing lethal neurodegenerative diseases and are composed of assemblies of misfolded cellular Prion Protein (PrP) (6). All known prions can induce formation of amyloid folds, wherein proteins polymerise into aggregates consisting of tightly packed beta sheets. Amyloid aggregates are fibre - like growing from their ends and replicating when breakage causes two growing ends to become four growing ends (7).The PrP variant, G127V, under positive evolutionary selection during the epidemic of Kuru, has been reported to have provided strong protection against this disease in the heterozygous state (8). Transgenic mice expressing both variant and wild type human PrP are completely resistant to both Kuru and classical CJD prions (which are closely similar) but can be infected with variant CJD prions. However, mice expressing only PrP V127 are completely resistant to all prion strains and demonstrates a different molecular mechanism to M129V providing for the relative protection against classical CJD and Kuru in the heterozygous state. A single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as a deletion of the protein. Transgenic mice expressing different ratios (either hetero or homo – zygous) of variant and wild type PrP revealed that PrP V127 is completely refractory to prion conversion besides acting as a potent dose-dependent inhibitor of wild type prion propagation (9); although the actual modus operandi of this phenomenon is yet to be found out. Studies involving the N-terminally truncated form of the prion protein, PrP 27-30, and its corresponding recombinant protein, rPrP revealed that solubilising it in SDS caused transitions induced by changing the conditions from 0.2% SDS to physiological conditions; which are characterized by changes in solubility, resistance to proteolysis, secondary structure and multimerization. The alpha-helical structures of PrP can be transformed into beta-sheets within a minute consisting mainly of dimers. Large oligomers found after 20 minutes and larger multimers found several hours later exhibit resistance to proteolysis. Thus, monomeric alphahelical conformations are stable in SDS or when attached to the membrane; but multimeric conformations of beta – sheet structures, forming at the state of lowest free energy, become stabilised at neutral pH in aqueous solution (10). Crystals with beta – pleated sheets are one of the most stable secondary structures of proteins, a plausible reason for their pathogenesis in the formation of plaques in neuropathies like Alzheimer’s disease. It is supposed that the betapleated-sheet crystals in the dry solid state are so stable that they do no melt upon been given heat energy alone. Peggy et al. demonstrated that the beta-pleated-sheet crystals can directly melt, changing from their solid states into random coils, helices, and turns. With fast scanning chip calorimetry performed at 2,000 K/s they reported the first thermally reversible melting of beta-pleated-sheet crystals of proteins (11). Their experiments using beta – pleated silk fibrin and synthetic polymers confirms the similarity of thermal melting behaviour of crystals of lamellar synthetic polymers with that of beta-pleated-sheet polypeptides. Macedo et al. demonstrated that recombinant murine PrP and its C-terminal domain (90–231) can attain amyloid conformations inside bacteria. And the inclusion bodies formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity (12). Such indications can form the basis of new investigations; wherein infectious forms of the prion plaques placed in or with the resistance – providing variant forms get irradiated with appropriate laser power and are then checked for reversions into structures like coils, helices, etc. which are less stable than the beta sheets and therefore more susceptible to being biologically cleared off the plaque residues; hence providing for means to effectively cure or prevent prion – like amyloid plaque related diseases? One must however take into consideration that “Energy loss due to the skin barrier for continuous HeNe (632nm) laser is 90%, for continuous GaAlAs (820 nm) and Nd:YAG (1064 nm) IR lasers, 80% and for GaAs (904 nm) infrared pulse laser, 50%” (13); these being the lasers commonly used on biological tissues, most of the time. In this scenario; there arises the problem of living tissue getting burnt with increasing laser powers; potentially restricting such advancements till the development of special techniques or systems only. DISCUSSION The implications of these advancements in studies of protein structure and their associated manifestations indicate the exciting possibilities of developing novel techniques to bioremediation. However, such techniques even if they were to be established in non – living systems, would require still higher efficiency for the focusing of high power lasers, etc. into microscopic cellular niches inside living bodies. This in turn calls for the development of better precision instruments for able accessorising of such technology. These technologies would find widespread applications in other varied fields as well; like cleaning of bacterial plaques instead of plasma cleaning and so and so forth. CONCLUSION Protein structures are directly correlated to the medium in which they fold. The chemical environment of protein folding is ultimately determined by the physical properties of the solution like density, specific gravity, etc.; which are in correspondence of the gravitational field and interplay of other fundamental forces and their manifestation(s) in the medium. Applications at the interface of protein structure and high energy irradiations can lead to the development of useful techniques whereby translated proteins may be effectively dealt with for misfoldings and its related repercussions without genome editing; thus, providing for effective biomedical supplementary methods to cure and prevent till – date incurable diseases. These indications encourage further interesting experiments wherein such biophysical interactions of protein – high power lasers are investigated as to how these work out in the natural environment of the biosphere of the Earth. It would also be interesting to study such experiments and look into their manifestations change over time on extra terrestrial Earth – analogue planetary surfaces like that of Mars where physical properties are variant. Surely, exciting implications relating ultimately to the evolution of molecular conformations into living reactions and life forms shall be found. ACKNOWLEDGEMENT The author thanks Prof. K. Ray, Dept. of Biological Sciences, TIFR, Mumbai, for checking the manuscript and providing advice with scientific insight. The author also thanks all the laboratory members for useful discussions. Author acknowledges the immense help received from the scholars whose articles are cited and included in the references. The author is also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Source of Funding The author thanks TIFR for funding in support of this work. Conflict of Interest The author declares that there is no conflict of interest. Englishhttp://ijcrr.com/abstract.php?article_id=214http://ijcrr.com/article_html.php?did=2141. Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annu Rev Microbiol. 2006;60:131-47. 2. Shewmaker F,Ryan P, McGlinchey,Thurber KR,McPhie P,Dyda F et al. The Functional Curli Amyloid Is Not Based on In-register Parallel β-Sheet Structure. J Biol Chem. 2009 Sep 11;284(37):25065-76. doi: 10.1074/jbc.M109.007054. Epub 2009 Jul 1. 3. Amyloid. March 25, 2016. InWikipedia. Retrieved April 13, 2016, https://en.wikipedia.org/wiki/Amyloid. 4. Lin CY, Gurlo T, Kayed R. Toxic human islet amyloid polypeptide (h-IAPP) oligomers are intracellular, and vaccination to induce anti-toxic oligomer antibodies does not prevent h-IAPPinduced beta-cell apoptosis in h-IAPP transgenic mice. Diabetes. 2007 May;56(5):1324-32. Epub 2007 Mar 12. 5. Fändrich M. On the structural definition of amyloid fibrils and other polypeptide aggregates. Cell Mol Life Sci. 2007 Aug;64(16):2066-78. 6. Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci. 2001;24:519-50. 7. Masel J, Jansen VA, Nowak MA. Quantifying the kinetic parameters of prion replication.Biophys Chem. 1999 Mar 29;77(2- 3):139-52. 8. Mead S, Whitfield J, Poulter M, Shah P, Uphill J, Campbell T et al. A novel protective prion protein variant that colocalizes with kuru exposure.N Engl J Med 2009; 361:2056-2065November 19, 2009DOI: 10.1056/NEJMoa0809716. 9. Asante EA, Smidak M,Grimshaw A,Houghton R, Tomlinson A,Jeelani A et al. A naturally occurring variant of the human prion protein completely prevents prion disease. Nature 522, 478–481 (25 June 2015) doi:10.1038/nature14510. 10. Post K, Pitschke M, Schäfer O,Wille H,Appel TR, Kirsch D et al. Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation. Biol Chem. 1998 Nov;379(11):1307- 17. 11. Cebe P, Hu X, Kaplan DL, Zhuravlev E,Wurm A, Arbeiter D et al. Beating the heat--fast scanning melts silk beta sheet crystal. Sci Rep. 2013;3:1130. doi: 10.1038/srep01130. Epub 2013 Jan 24. 12. Macedo B, Sant’Anna R, Navarro S, Cordeiro Y, Ventura S. Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria. Microbial Cell Factories201514:174 DOI: 10.1186/s12934-015-0361-y 13. Bjordal JM, Couppé C, Chow RT, Tunér J, Ljunggren AE. A systematic review of low level laser therapy with locationspecific doses for pain from joint disorders. Aust J Physiother. 2003;49(2):107-16.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN2016August11HealthcareCOMPARATIVE EVALUATION OF PLASMODIUM LACTATE DEHYDROGENASE BASED RAPID IMMUNOCHROMATOGRAPHIC TEST ASSAY AND ROUTINE MICROSCOPIC TEST IN DIAGNOSIS OF MALARIA AMONG PATIENTS ATTENDING IN A RURAL TEACHING HOSPITAL, SANGAREDDY English0410Prudhvi Chand MallepaddiEnglish Nagababu PyadalaEnglish Soumendra Nath MaityEnglish Rajaneesh BorugaddaEnglish Rohit C. P.English Sudhakar PodaEnglish Rathnagiri PolavarapuEnglishObjectives: The present study was aimed to evaluate routine microscopic examination and plasmodium lactate dehydrogenase (pLDH) based immuno chromatographic assay for the rapid detection of malaria. Methods: This study was carried out at the Department of Research, MNR medical collage & Hospital, sangareddy, Telangana from 2012 to 2015. Thick and thin blood smears were stained by giemsa staining followed by microscopy. All the specimens were tested by rapid test kit and confirmed by Western blot method. Results: In our study out of 1870 clinically suspected cases, 295 (15.78%) were positive for P. falciparum by microscopy and 296 (15.82%) were positive by ICT method. 72 (3.85%) cases were positive for P. vivax by both the methods. Conclusion: Although microscopy is gold standard method to detect malaria parasites from blood smear, it requires well experienced person and well established laboratory. On other hand rapid ICT kit is a very simple, inexpensive, user-friendly, point of care and effective diagnostic assay that can be done at the bedside for detecting malarial parasites. EnglishImmunochromatgraphic test (ICT), Plasmodium Lactate Dehydrogenase (pLDH), Plasmodium falciparum (pf), Plasmodium vivax (pv), Positive Predictive Value (PPV), Negative Predictive Value (NPV).INTRODUCTION Globally malaria has become one of the major health problems. According to WHO malaria report, an estimated of 3 to 5 billion cases are annually reported and 90% of the cases are reporting in Africa alone. [1] Worldwide Malaria is present in more than 100 countries. Most of them are developing or under developing countries. So, it has a great impact on their economy. In India, according to National Vector Borne Disease Control Programme (NVBDCP) around 0.85 million confirmed cases are reported annually, of which 40-50% is due to plasmodium falciparum. [2] Malaria is an acute parasitic disease caused by Plasmodium falciparum or Plasmodium vivax in India. It is one of the most endemic diseases, especially countries like India and African tropical countries. In the past malaria is widely spread among mankind. But now it is mostly ignored as a simple disease. The main clinical presentation is fever with chills; however, nausea and headache can also occur. The majority affected being children under 5 years and almost all the deaths are attributed to Plasmodium falciparum (PF). [2,3] In rural areas, clinical diagnosis is widely used for detecting malaria, where laboratory facility does not exist. [4] According to WHO 2011 guidelines, clinical diagnosis of malaria based on signs and symptoms alone is not recommended. It has low specificity and increased the chance of misdiagnosis of the patient and leads to misuse of drugs. [5,6] A laboratory diagnosis of malaria is one of the possibility in the management of a patient presenting with fever. Microscopic examination of the Giemsa-stained blood smears is widely used as a routine method for the detection of malaria parasites and remains the gold standard for diagnosis. [7] But, it is not 100% sensitive and specific. [8] Expert microscopy gives information about parasite stage and parasitemia. However, maintaining a high standard of microscopy requires trained technicians, supervision, quality control, and regular provision of reagents and also it will take 60 minutes to prepare a blood smear slide, which is a time consuming process. [9,10] In India, a majority of malaria cases occur in rural areas. Where there is a little or no access to reference laboratories. So, WHO recommended another diagnostic method called as Rapid diagnostic tests (RDTs) that detect malarial parasitic proteins by Immunochromatography have been used as a complementary detection method for malaria diagnosis. [11] RDTs detect a variety of proteins, including P. falciparum Histidine-rich protein 2 (PfHRP2) and plasmodium lactate dehydrogenase (pLDH), both specific to P. falciparum, and also Plasmodium LDH (pLDH) and aldolase, enzymes shared by the 5 human-pathogenic Plasmodium species. [12] The main goal of this study was to evaluate, routine microscopic examination and pLDH based immunochromatgraphic assay for the rapid diagnosis of malaria. METHODS AND MATERIALS This study was carried out at the Department of Research, MNR medical collage and Hospital, sangareddy, Telangana from 2012 to 2015. 3 ml volume of venous blood was drawn with aseptic precautions and collected into a sterile Ethylene diamine tetra acetic acid (EDTA) collection tubes for microscopy and immunochromatgraphic testing. The thick and thin blood films were made shortly after being drawn to prevent alteration in the morphology of malarial parasites. The total number of subjects in our study was 1870 numbers and at an age group was from 10 years to > 61 years. The subjects were selected from medical ward, who were suspected to have malaria i.e., fever with two or more of following clinical findings splenomegaly, pallor, convulsions and jaundice. Patient with infective Hepatitis and other known causes of anaemia are excluded from the study. For all the clinically suspected cases of rapid malaria plasmodium lactate dehydrogenase (pLDH) based immuno chromatography test (ICT) assay (Genomix Malaria Pf/Pv antigen Rapid Detection kit, Genomix Molecular Diagnostics (P) Ltd. Hyderabad, India), was done at bed side and simultaneously thick and thin smears are prepared and sent for microscopic examination. The study was to compare immuno chromatography test (ICT) method with conventional microscope with respect to the Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Efficiency. RESULTS In our study out of 1870 clinically suspected cases of malaria, 165 (8.82%) patients were belongs to 10 to 20 years, 465 (25.02%) were in 21 -30 years, 512 (27.37%) were in 31- 40 years, 370 (19.78%) were in 41-50 years, 248 (13.26%) were in 51-60 years and 110 (5.88%) were belongs to > 61 years as shown in the table 1. In our study, among malaria suspected cases 1089 (58%) are males and 781 (42%) are females as shown in the figure 1. All clinically suspected cases of malaria had fever, majority of them had splenomegaly, pallor and convulsions. Very few of them presented with jaundice as shown in table 2. In our study out of 1870 clinically suspected cases, 295 (15.78%) were positive for P. falciparum by microscopy and 296 (15.82%) were positive by ICT method, 72 (3.85%) cases were positive for P. vivax by both the methods as shown in table 3. Among 368 positive cases of malaria, 73 (19.83%) were in 10- 20 age group, 112 (30.44%) were in 21-30, 96 (26.09%) were in 31-40 year, 48 (13.04%) were in 41- 50 year, 22 (5.97%) were in 51-60 year and 17 (4.62%) were in >61 age group as shown in table 4. In our study, among malaria positive cases 219 (60%) are males and 149 (40%) are females as shown in the figure 2. All the malaria proved cases had fever 368 (100%), 318 (86.4%) had splenomegaly, 251 (68.20%) had pallor, 159 (43.20%) had convulsions and 69 (18.75%) had jaundice as shown in the table 5. DISCUSSION The resurgence of malaria has renewed interest in developing not only preventive measures, but also rapid diagnostic techniques. Several methods have been developed to supplement and replace the conventional microscopic method. The most promising new malaria diagnostics are the serological rapid immuno chromatography test (ICT) kit. Genomix Malaria (Pf/Pv) Antigen Rapid Detection kit is one amongst them. We employed the test and compared it with conventional smear examination for diagnosis of malaria. In our study, among 1870 clinically suspected cases of malaria, microscopy showed 367 positive cases, in that 295 (15.78 %) cases were shown positive for P. falciparum and 72(3.85 %) for P. vivax. Whereas Genomix Malaria rapid ICT kit showed 368 positive cases in that, 296 (15.82%) cases positive for P. falciparum and 72 (3.85%) cases for P. vivax. One case of P. falciparum was not detected by microscopic method. The probable cause could be misdiagnosis of a specimen; due to the lack of technical experience in handling and observing the slides and also might be the low levels of parasitemia count in the specimen. In general, the highly qualified microscopist can detect up to 200 p/µl of malaria parasites easily, whereas the ICT can detect up to 100 p/µl. [13,14] In continuation to the Microscopy and lateral flow results, the specific sample which showed false negative (pf) in microscopy and positive (pf) in lateral flow was further confirmed by using the western blot analysis. The pLDH anti-sera immunoreactivity signal was clearly formed at near to 37 kd position on nitrocellulose membrane (the size of pLDH in plasmodium falciparum is 33 kd (15) in western blot stating that the specific sample was positive for malarial parasites as shown in figure 3. In our study among the 368 proved malaria cases 219 (60%) were male and 149 (40%) were females. All the patients had fever 368 (100%), splenomegaly 318(86.4%), pallor 251(68.20%), convulsions 159 (43.20%), and jaundice 69(18.75%). The rapid ICT method had excellent sensitivity and specificity (100%) for detecting P. vivax and P. falciparum. Rapid ICT had positive predictive valve of 100%, negative predictive value of 100% and efficiency of 100%.Whereas microscopic method is showing sensitivity (99.72%), specificity (100%), positive predictive valve of 100%, negative predictive value of 99.93% and efficiency of 99.94% shown in table 6 and 7. The results of the present study were similar to the study done by Diarra et al. in 2012 at Burkina Faso [16]. This study revealed that ICT had a high level of sensitivity and specificity compared with microscopy which is considered as the gold standard method for malaria diagnosis. Therefore, the results of this study further substantiated that ICT is an effective and sensitive tool in the diagnosis of malaria. Compared to microscopy the ICT kits are simple, rapid, inexpensive, point of care, easy to use diagnostic test kits for disease diagnosis. Worldwide, from the past few years, the ICT kits played a major role in the diagnosis and became backbone for most of the commercial diagnostic assays. CONCLUSION The present study reveals that ICT is a very simple, inexpensive, user-friendly, point of care and effective diagnostic assay that can be done at the bedside for diagnosing malaria. It has sensitivity, specificity, PPV, NPV, and efficiency were more or less similar to conventional microscopy and do not require highly skilled personnel to perform or interpret the results. Early diagnosis and treatment are imperative in preventing the complications. Microscopy is the gold standard for malaria parasites. But it is laborious and requires experts to interpret the results. Rapid immunochromatgraphic test that detects pLDH produced by malaria parasite in the blood which can be performed at bedside in 10-15 minutes. ACKNOWLEDGEMENT We thankful to The Vice-Chairman, MNR Medical Collage and Hospitals. Ethical Clearance: The study was approved by the Institutional Human Ethical Committee. Source of Funding: None  Conflict of interest: None Englishhttp://ijcrr.com/abstract.php?article_id=215http://ijcrr.com/article_html.php?did=2151. WHO, World Malaria Report 2013, World Health Organization, Geneva, Switzerland, 2013. 2. National vector borne disease control programme, Annual report 2014-15. 3. Ashok KK. Malaria in children. IAP text Book of Paediatrics, New Delhi: Jaypee Bro pub; 2010, 423- 439. 4. Federal Republic of Ethiopia Ministry of Health, National Malaria Guide Lines, FMOH, Addis Ababa, Ethiopia, 3rd edition, 2012. 5. WHO, World Malaria Report, 2011, http://www.who.int/malaria/world malaria report 2011/WMR2011 factsheet.pdf. 6. T. Leslie, A. Mikhail, I. Mayan et al., “Over diagnosis and mistreatment of malaria among febrile patients at primary health care level in Afghanistan,” British Medical Journal, vol. 345, Article ID e4389, 2012. 7. Cooke AH, Doherty T, Moody AH. Comparison of pLDH based immunochromatographic antigen detection assay with microscopy for the detection of malaria parasites in human blood samples. Am.J.Microbiol 8. B. S. C. Uzochukwu, L. O. Chiegboka, C. Enwereuzo et al., “Examining appropriate diagnosis and treatment of malaria: availability and use of rapid diagnostic tests and artemisinin based combination therapy in public and private health facilities in south east Nigeria,” BMC Public Health, vol. 10, pp. 486– 495, 2010. 9. P. J. Rosenthal, “How do we best diagnose malaria in Africa?”The American Journal of Tropical Medicine and Hygiene, vol. 86, no. 2, pp. 192–193, 2012. 10. Davidson HH, Mickey N, Deza et al., Improved diagnostic testing and malaria treatment practices in Zambia. JAMA, May 2007; 297 (20): 2227-2231. 11. D. Bell, C. Wongsrichanalai, and J.W. Barnwell, “Ensuring quality and access for malaria diagnosis: how can it be achieved?” Nature Reviews Microbiology, vol. 4, no. 9, pp. S7–S20, 2006. 12. A. Moody, “Rapid diagnostic tests for malaria parasites,” Clinical Microbiology Reviews, vol. 15, no. 1, pp. 66–78, 2002. 13. Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 1993; 58: 283-292. 14. McManus DP, Bowles J. Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics. Int J Parasitol 1996; 26: 687-704. 15. David J. Bzik, Barbara A. Fox, Kenneth Gonyer. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli Molecular and Biochemical Parasitology Volume 59, Issue 1, May 1993, Pages 155-166 16. Amidou Diarra , Issa Nébié , Alfred Tiono , Souleymane Sanon , Issiaka Soulama , Alphonse Ouédraogo , Adama Gansané , Jean B Yaro , Espérance Ouédraogo , Alfred S Traoré and Sodiomon B Sirima. Seasonal performance of a malaria rapid diagnosis test at community health clinics in a malaria-hyper endemic region of Burkina Faso. Parasites and Vectors 2012, 5:103.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN2016August11HealthcarePRODUCTION, IMMOBILIZATION AND INDUSTRIAL USES OF PENICILLIN G ACYLASE English1122Mohamed E. HassanEnglishPenicillin Gacylase in one of the most important enzymes, it belonging to β-lactam antibiotics, first report on the enzyme penicillin acylase was in 1950 when they found in the mycelium of a Penicillium sp. The enzyme appeared to be a periplasmaticheterodimeric N-terminal serinehydrolase with a molecular mass of 86,183 Da, with a 23,817 Da (209 amino acids)α-subunit and a 62,366 Da (566 amino acids) β-subunit. This enzyme is capable of hydrolyzing penicillin G into phenyl acetic acid and 6-aminopenicillanic acid (6-APA) so this enzyme isthe starting material for the manufacture of penicillin derivatives, which are the most widely used β-lactam antibiotics. Both natural and semi-synthetic penicillins contain 6-aminopenicillanic acid. English?-lactamantibiotics, Penicillin gacylase, Classification, Industrial uses.INTRODUCTION The β-lactam antibiotics One of the most important groups of antibiotics, both historically and medically, is the β-lactam group. The β-lactam antibiotics include penicillins, cephalosporins, and cephamycins, all medically useful antibiotics. These antibiotics are called β- lactams because they contain the β- lactam ring system, a complex heterocyclic ring system. The β-lactam antibiotics act by inhibiting peptidoglycan synthesis in eubacterial cell walls. The target of these antibiotics is the transpeptidation reaction involved in the cross-linking step of peptidoglycan biosynthesis. Because this reaction is unique to bacteria, the β- lactam antibiotics have high specificity and relatively low toxicity. Penicillin G, The first β-lactam antibiotic discovered, is active primarily against Gram-positive bacteria. Its action is restricted to Gram-positive bacteria primarily because Gramnegative bacteria are impermeable to the antibiotic. A vast number of new penicillins have been discovered, some of which are quite effective against Gram-negative bacteria. One of the most significant developments in the antibiotic field over the past several decades has been the discovery and development of these new penicillins. The basic structure of the penicillins is 6-aminopenicillanic acid (6-APA)(Fig.1), which consists of a thiazolidine ring with a condensed β-lactam ring. The 6-APA carries a variable acyl moiety (side chain) in position 6. If the penicillin fermentation is carried out without addition of side-chain precursors, the natural penicillins are produced. The fermentation can be better controlled by adding to the liquid nutrient medium a side-chain precursor, so that only one disk penicillin is produced. Over 100 such biosynthetic penicillins have been produced in this way. In commercial processes, however, only penicillin G, penicillin V, and very limited amounts of penicillin O are produced (Fig.2)1 . In order to produce the most useful penicillins, those with activity against Gram-negative bacteria, a combined fermentation/chemical approach is used which leads to the production of semi synthetic penicillins. The starting material for the production of such semi synthetic penicillins is penicillin G, which serves as the source of the 6-APA nucleus. Penicillin G is split either chemically or enzymatically (using penicillin acylase) and the 6-APA obtained is then coupled chemically to another side chain. The production of penicillin and cephalosporin antibiotics is a multi-thousand tons industrial operation. Benzyl and phenoxymethylpenicillins (penicillin G and penicillin V respectively) are fungal fermentation products and the precursors to a wide range of semi-synthetic antibiotics (amoxicillin, ampicillin etc.). The chemical modification of the fermentation product is initiated by removal of the natural acyl group leaving the 6-aminopenicillanic acid (6-APA) as a penicillin nucleus. Alternative synthetic acyl groups can then be added to confer novel properties to the antibiotic such as resistance to stomach acid, a certain degree of penicillinase resistance or an extended range of antibiotic activity. The method of choice for the conversion to 6-APA at the industrial scale is the use of penicillin acylase. Penicillin acylases or amidases (EC 3.5.1.11) are a group of enzymes which can cleave the acyl chain of penicillins to yield 6-amino penicillinic acid (6-APA) and the corresponding organic acid, and in a number of cases the same enzyme can be used to direct the synthesis of the new antibiotic by the addition of the novel acyl group (Fig. 3)2 . Penicillin G Acylase Discovery and Occurrence The first report on the enzyme penicillin acylase was in 1950 by Sakaguchi and Murao when they found in the mycelium of a Penicillium sp. the enzyme capable of hydrolyzing penicillin G into phenyl acetic acid and the unknown 6-APA (named “penicin”)3 . In the early years it was thought that penicillin G acylases were mainly produced by bacteria and penicillin V acylases mainly by molds. It is now well established that they are ubiquitous in bacteria, actinomycetes, fungi, and yeasts4 . In Escherichia coli the gene encoding for penicillin acylase is located in a cluster of genes involving the metabolism of 4-hydroxyphenylacetic acid, where it is thought to have a function in the degradation of aromatics 5 . Classification The penicillin acylasesare divided into three classes according to their substrate specificity. The penicillin V acylases (Type I) have a high affinity for phenoxyacetic acid derivatives whereas the penicillin G acylases (E.C. 3.5.1.11) (Type II) have a high affinity for phenyl acetic acid derivatives. The α-aminoacyl hydrolases (Type III) specifically hydrolyse α-aminoacyl β-lactam antibiotics. The penicillin acylases are classified as a new enzyme super family called the N-terminal nucleophile hydrolases or Ntn-hydrolases6 . Structure and Catalytic Machinery The crystal structure of E. coli penicillin G acylase was resolved by Brannigan et al., in19956 . The enzyme appeared to be a periplasmaticheterodimeric N-terminal serinehydrolase with a molecular mass of 86,183 Da, with a 23,817 Da (209 amino acids)α-subunit and a 62,366 Da (566 amino acids) β-subunit. The enzyme is kidney-shaped (Fig. 4), approximate dimensions are 70×50×55 Å, with a deep cupshaped depression leading to the active site. It has a singleamino-acid catalytic center, the β-chain N-terminal serine γ-hydroxyl. Penicillin acylase is believed to be activated by its own Ser β1 free α-amino group using a bridging water molecule, though more recently it was found that the reaction proceeds by a direct nucleophilic attack by the Ser β1O γ without the help of a bridging water molecule7 . Structural insights in the catalytic machinery of E. coli penicillinacylase have been gained by resolving the crystal structures of several native and mutant enzyme-substrate complexes, e.g. with phenylacetic acid, phenylmethanesulfonyl fluoride, penicillin G sulfoxide, penicillin G, and Dα-methylphenylacetic acid 8 . The catalytically important amino acids can be divided into several classes. First one is the catalytically active nucleophile Ser β1. Second are the oxyanion hole formers Ala β69 and Asn β241 that stabilize the oxyanion transition-state intermediate. Third are the residues that play a role in enhancing the nucleophilicity of the Ser β1. These are Gln β23 (at 2.9 Å and 3.2 Å from Ser β1) and Asn β241 (3.0 Å from Ser β1). Fourth are the residues that are important for substrate binding. Where penicillin acylase has two substrate binding pockets, the most specific one, S1, is made up by mainly hydrophobic residues. The enclosed structure and largely hydrophobic character of the S1 pocket makes the enzyme very selective to the benzyl structure with some room for substitutions on the bridging C α and aromatic ring, e.g., -OH, -NH2, -CH3, -OCH3, -CN, -F, -Cl, -Br. Ring structures other than phenyl are also accepted, e.g., pyridyl, thiophene, thiazole, 1H-tetrazole, and furanyl. The principle residues that enclose the S1 pocket are Met α142, Phe α146, Phe β24, Phe β57, Trp β154, Ile β177 with Ser β67 at the closed end. The residues that complete the enclosed structure are Pro β22, Gln β23, Val β56, Thr β68, Phe β71, Leu β253, and Phe β256. The S2 pocket, or (β-lactam) nucleophile-binding pocket, is in reality the bottom of the cup-shaped depression mentioned before and therefore makes for a very broadsubstrate specificity of this pocket. In contrast to the S1 pocket, the S2 pocket is enantioselective and can therefore be used for e.g. amine resolution 9 . It is formed by Arg α145, Phe α146, Phe β71, and Arg β263. The catalytic mechanism of E. coli penicillin G acylase catalyzed amide hydrolysis isshown in figure 5. The first step in penicillin acylase catalysis is the nucleophilic attack of the active Ser β1: O γ hydroxyl on the electrophilic carbonyl carbon of the amide substrate. The tetrahedral oxyanion transition-state intermediate is stabilized by hydrogen bonding with two amino acids in the oxyanion hole (Ala β69: N and Asn β241: N δ2). Next, the covalent acyl-enzyme intermediate is formed when the carbonyl group is restored under release of the product P (e.g., 6-APA, NH3, CH3OH in the case of penicillin G, D-phenylglycine amide (D-PGA) or methyl ester (D-PGM), resp.). In the following step the acyl moiety is transferred to either a water molecule (hydrolysis) or to an amine nucleophile (e.g., a β-lactam nucleus or any other amine) in which case an amide bond is formed (synthesis). Penicillin acylases (PGAs) family is divided into classes based on their substrate preference7 . It has been suggested that PGA in Escherichia coli may function during the freeliving mode of the bacterium to degrade phenylacetylated compounds generating phenyl acetic acid (PAA), which may be utilized by the organism as a carbon source 10. Penicillin acylases are valuable pharmaceutical enzymes and are used in the synthesis of many semi-synthetic penicillin derivatives and in hydrolysis of β-lactam antibiotics11. Penicillin G acylase catalyzes the conversion of benzyl penicillin, via hydrolysis of the amide bond in the benzyl penicillin side chain, to release phenyl acetic acid and 6-aminopenicillanic acid (6-APA), the latter being the important precursor utilized in industrial synthesis of semi-synthetic penicillins10. In nature, PGA is initially produced as a single-chain precursor in the cytoplasm of Escherichia coli, and after removal of several polypeptides, the enzyme reaches a mature state in the periplasm10. The mature enzyme is a heterodimer of a small α-unit (209 residues) and a large β-unit (557 residues. PGA is characterized as an N-terminal-nucleophile (Ntn)hydrolase; the Ntn super family is comprised of enzymes that share a common fold around the active site and that contain a catalytic serine, cysteine, or threonine at the N-terminal position 8 . The two chains form a pyramidal shaped structure, and in the middle of the pyramid resides the hydrophobic active site. The active site of PGA is comprised of several hydrophobic amino acid residues, making it very specific for the phenyl acetyl group of PG 11. Since the discovery of penicillin acylases (PGAs) in the 1950’s, they have been incorporated throughout the pharmaceutical industry and exploited for industrial purposes. Penicillin acylases are widely distributed among many microorganisms, including bacteria, actinomycetes, yeasts, and fungi12. Penicillin G acylase (EC 3.5.1.11) is commonly isolated from Escherichia coli strain W (ATCC 1105) for its use in pharmaceuticals 13. An immobilized form is used commercially to cleave benzyl penicillin (PG) via hydrolysis of the side-chain amide bond, to yield phenyl acetic acid (PAA) and 6-aminopenicillanic acid (6-APA), the latter of which is an important precursor for the synthesis of several semi-synthetic, β-lactam antibiotics (Fig. 6)14,15. The combined industrial uses of penicillin G acylase and penicillin V acylase results in the annual production of 9000 tons of 6-APA16. Though the mechanism and function of PGA for commercial use is widely understood, much uncertainty surrounds the In Vivo function and mechanism of PGA. The In Vivo expression of PGA has been observed to be regulated by both temperature and phenyl acetic acid, leading to the putative role that it is employed during the free-living mode of the organism to generate a carbon source, by degrading phenyl acetylated compounds to generate phenyl acetic acid10. Penicillin acylases belong to a super family of enzymes, Nterminal nucleophile hydrolases (Ntn-hydrolases), which are generally synthesized as precursor proteins and undergo a post-translational autocatalytic process to generate the mature protein with an active catalytic center at the new N-terminus 17.The mature, active enzyme is formed by removing a linker peptide (30-50 amino acids) in the proenzyme, resulting in a heterodimer (A and B Chains) with a free N-terminal nucleophile, either a serine, cysteine, or threonine18. In the case of mature PGA, the N-terminal nucleophile is a serine residue (Ser1) on the carboxyl terminal of the B-chain. This super family of enzymes shares not only a similar generation process, but also a similar tertiary structure18. The characteristic fold of Ntn-hydrolases consists of a four layered catalytically active αββα-core that is comprised of two antiparallel β-sheets packed against one another and covered by a layer of antiparallel α-helices on one side (Fig.7)19, 20. Penicillin G acylase(PGA) is synthesized in the cytoplasm of E. coli as a single chain precursor. The precursor contains a signal sequence of 26 amino acids and a spacer peptide composed of 54 amino acids (Fig.8a)17. The signal sequence is used to direct the translocation of the proenzyme to the periplasm, while the spacer sequence, between chains α and β blocks the active site and is also thought to influence the final folding of the protein 17.The signal sequence and the linker peptide are removed via an autocatalytic process, resulting in the mature form of the enzyme in the periplasm of E. coli10. The mature, active E. coli PGA enzyme is an 86 KD heterodimer, with chains α (209 amino acids) and β (557 amino acids) closely intertwined and held together by non-covalent forces (Fig.8b)10. The crystalline structure of PGA isolated from E. coli W and grown in presence of ethylene glycol has been determined to a resolution of 1.3 Å. The enzyme forms a pyramidal structure with a centrally located deep depression, at the bottom of which is the active site17.The binding pocket is lined with hydrophobic residues from the α and β subunits, defining its specificity for the phenyl acetyl group of penicillin G 11. Conditions for production, extraction and immobilization of PGA The most widely used organism is E. coli, containing either the endogenous gene or a heterologous gene. Fermentations are performed in up to 250m3 fed batch, stirred fermenters. Seed cultures are grown at 37 ο C for a prescribed time; usually a pH change indicates optimal time for transfer, then up to 10% seed can be used to inoculate the production fermenter. After an initial growth period, the temperature is reduced and carbon feeding is initiated. This is the trigger for PGA production. The fermentation is continued until maximum activity is achieved and then the fermentation is harvested2 . The level of dissolved oxygen is required to be maintained at 15% or higher to avoid repression of PGA. In addition, since glucose is a known repressor of PGA activity when used as carbon source its rate of supply has to be limited. Alternative carbon sources such as sucrose or glycerol are commonly used. The pH of the culture needs to be maintained in the range 6.8 to 7.1 for optimal PGA production. Likewise, the production temperature has to be maintained below 30 ο C. Since the expression of the pac gene can be differentially modulated depending on the exact nature of the plasmid constructed, diverse expression inducers can be used to switch on the pac gene fused to a particular upstream sequence. Examples include galactose21, and isopropyl-betaD-thiogalactopyranoside (IPTG) to induce the lac promoter, and rhamnose to switch on the rhaBAD promoter. Since the active PGA enzyme is localized in the periplasmic space in E. coli this immediately affords a purification step as less than 5% of cellular proteins are found in this compartment2 . The periplasmic fraction can be separated from the cytoplasmic fraction using selective permeabilization. Cell permeabilization by osmotic shock in combination with EDTA has been reported to yield 70% protein without affecting cell viability. Polar organic solvents and aqueous solutions of ionic and non-ionic detergents have been used to permeabilize cells. For example, on a laboratory scale, treatment with guanidine/EDTA has been reported to give a 93% yield with a 25-fold purification. Solvents with dielectric constants below five and having high hydrophobicity released the most active PGA protein. The production of reverse micelles using AOT in water/hexane resulted in the selective release of the enzyme without cell breakage22.More specific means of purification of PGA have been demonstrated. It is achieved that greater than 65–fold purification by immobilized metal affinity chromatography (IMAC). The advantage of this technology is that it can be scaled up and the matrices are suitable for sanitization. Whilst the permeabilization methods outlined above can be applied to small scale cultures, the release of PGA from industrial fermentations poses greater problems. To this end the large scale release of PGA usually involves physical disruption followed by partial purification. After fermentation cells are harvested by centrifugation or settled with flocculants. The concentrated cells are then homogenized and cell debris removed. The extract is then purified by the method of choice. The most general means of purification is chromatography and/or ammonium sulphate precipitation. A typical method may involve a pH shift and heating to selectively denature sensitive proteins leaving PGA active in the mix. The use of two-phase affinity partitioning as a means of protein purification is well documented. A polyethylene glycol (PEG) derivative and salt system has been shown to be effective in the purification of PGA23. The PEG derivative is selected on the basis of its expected interaction through hydrophobic, electrostatic and biospecific effects. In most cases the ligand has a structure analogous to the penicillin G substrate of the enzyme. Thus benzoateand phenylacetamide derivatives are useful ligands. The salt phase usually contains sodium sulphate or sodium citrate. Thermostability can be an important feature for enzymes used in industrial processes as higher temperatures can be used to enhance reaction rates, shift thermodynamic equilibria, increase reactant solubility and decrease the reaction viscosity. The catalytic performance of PGA increases at temperatures between 25ο C and 50ο C. However, the enzyme shows poor stability at temperatures above 35ο C and therefore an effective means of providing thermalstability is highly desirable. Many methods of PGA thermo stabilization have been studied. Thermal unfolding of penicillin acylases has been linked to their conformational mobility in water. This mobility can be reduced by diminishing the amount of free water which can be achieved by the addition of stabilizers such as polyolcompounds24, bisimidoesters25, neutral salts or proteins. The stability of PGA was improved by up to 180% by the addition of trehalose after thirty minutes incubation at 60 ο C26. In order to be useful as a biocatalyst the PGA enzyme preparation has to be active, robust and re-usable. One of the most effective ways to enhance stability for many enzymes is to immobilize the enzyme onto a solid support. In addition, immobilization may allow re-use of the catalyst and thus increase cost effectiveness. A number of immobilization methods have been used in this context27. Each method shows superiority to the more traditional use of free cells, extracts or even immobilized whole cells. Immobilized enzyme preparations attain higher activity and specificity and show better control of contamination28. The most common immobilization methods include crosslinking and covalent attachment. Glutaraldehyde is the usual cross-linking agent used. A 15-foldimprovement in thermostability was apparent after cross-linking with dimethyladipimate. The use of different physical forms of chitosan (powder, particles or beads) to immobilize PGA either by adsorption followed by reticulation with glutaraldehyde or by direct cross-linking to the matrix pretreated with glutaraldehyde has been reported29. Also it is used double entrapment methodology for the immobilization of PGA on agar-polyacrylamide resins. A highest specific activity of 322U/g wasobtained by covalent binding of PGA onto vinyl copolymers. At present, commercial manufacturers such as Resindion supply epoxy group resinssuch as Sepabeadsfor use as immobilization matrices for enzymes including PGA. Sepabeads are porous spherical beads with outstanding mechanical stabilityand extensive cross-linking. Multipoint covalent immobilization of PGA fromK. citrophila stabilized the enzyme 10,000-fold compared to the soluble enzyme from E. coli. A further means of PGA enzyme stabilization is afforded by the production of cross-linked enzyme crystals or CLECs. This approach is unique in that it results in both stabilization and immobilization without activity dilution. The protein matrix is both the catalyst and the support. The crystals are produced by stepwise crystallization of the purified enzyme followed by molecular cross-linking to preserve the structure, resulting in a biocatalyst which is extremely stable to both temperature and organic solvents. The stabilization is a consequence of both polar and hydrophobic interactions. The PGA CLEC has been commercialized for both hydrolytic and synthetic activity use, and retains activity over more than 1000 batches30. Another novel preparation called a cross-linked enzyme aggregate (CLEA) has also been reported31. CLEAs are prepared by slowly adding a precipitant such as ammonium sulphate to the enzyme at low temperature. The aggregated enzyme is subsequently linked with glutaraldehyde and is then available for use as a biocatalyst. The CLEA canbe used in aqueous media in both forward and reverse reactions. Unlike the CLEC preparation, the CLEA enzyme need not be purified to near homogeneity. Industrial uses of PGA The most widespread use of PGAs is in the production of 6-APA from bothPen G and Pen V. Immobilized PGA enzymes mainly from E. coli, B. megaterium and A. faecalis are available from a number of commercial suppliers. Reactionsare carried out at >5,000L scale under controlled conditions, the pH being eithercontrolled at approximately 8.0, or slowly ramped from 7.0 to 8.5, depending upon thecatalyst, as high as 8.5. Exposure to high temperature (>30 ο C) and pH (>8.0) isminimized to reduce inactivation of the enzyme and retain high product yield of the otherwise relatively unstable 6-APA. The use of PGA in large scale production of semi synthetic penicillins and cephalosporins is also widespread. These processes are focused on the condensation of an appropriate D-amino acid derivative with a β-lactam nucleus in a PGA catalyzed reaction. This involves the direct acylation of nucleophiles such as 6-APA or 7-ADCA with free acids at low (Englishhttp://ijcrr.com/abstract.php?article_id=216http://ijcrr.com/article_html.php?did=2161. Elnashar, M. M. M.; Hassan, M. E.; Awad, G. E. A. Grafted Carrageenan Gel Disks and Beads with Polyethylenimine and Glutaraldehyde for Covalent Immobilization of Penicillin G Acylase. J Colloid SciBiotechnol (2013); 2, 1–7. 2. David W and Martin R. Penicillin acylase. Industrial enzymes. j.Polaina and A.P. Mac Cabe (eds.), (2007); 583–597. 3. Sakaguchi, K. and Murao, S. A new enzyme, penicillin-amidase. Preliminaryreport J. Agr. Chem. Soc. Japan (1950); (23), 411. 4. Shewale, J.G., Deshpande, B.S., Sudhakaran, V.K. and Ambedkar, S.S. Penicillin acylases: applications and potentials. Process Biochem. (1995); 25, 97–103. 5. Hesham A. El Enshasy · Mohamed S. Mohy El Din · Mohamed El Sayed · Salah El Sayed · Bakry M. Haroun and Elsayed A. Hassan.Optimization and Scaling up of Penicillin Acylase Production Process by Escherichia coli. World Applied Sciences Journal (2009); 6 (10), 1348-1358 6. Brannigan, J.A., Dodson, G., Duggleby, and Murzin, A.G. Penicillin acylase has a single –amino-acid catalytic centre. Nature (1995); 373, 264–268. 7. McVey, C.E., Walsch, M.A., Dodson, G.G., Wilson, K.S., and Brannigan, J.A. Crystal structure of penicillin acylase enzymesubstrate complexes: Structural insights into the catalytic mechanism. J. Mol. Biol, (2001); 313, 139-150. 8. Alkema, W. B., Dijkhuis, A. J., de Vries, E., and Janssen, D. B. The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylaseEur.J.Biochem. (2002); 269, 2093-2100. 9. Van Langen, L.M., Oosthoek, N.H.P., Guranda, D.T., Van Rantwijk, F., Švedas, V.K., and Sheldon, R.A. 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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN2016August11HealthcareA STUDY OF FRESHWATER POND TAXA MARSILEA QUADRIFOLIA & SALVINIA NATANS IN KOLKHETI LOWLAND BLACK SEA COASTLINE English2326Bulbuli BolqvadzeEnglish Izolda MatchutadzeEnglishIn recent years, degradation of freshwater ponds, an important habitat of biodiversity, caused by the anthropogenic factors, has prompted scientists from the Mediterranean countries to evaluate freshwater ponds by the IUCN Red List as threatened ecosystem [1,2]. In 2015 IUCN Red List officially gave the freshwater ponds of the Mediterranean Sea coastal area global status [1,2]. The aim of the paper is to study freshwater species: Marsilea quadrifolia and Salvinia natans. The subject of the present study was freshwater ponds of Nature 2000 and Emerald Network coastal zone with predominance of Salvinia natans and protected by the EUNIS system and Bern Convention from Sarfi to Anaklia. Methodology: During the study a transect method was used; special attention was paid to the habitat type, its ecological state, plant covering density (in %), composition of species and their quantity. Results: Freshwater ponds of the Kolkheti costal area are valuable for being an important habitat for IUCN Red List species: Salvinia natans (LC) and Marsilea quadrifolia (LC). But these habitats and, respectively, plant species are affected by the greatest anthropogenic factors such as implementation infrastructural projects (seaports, terminals) causing their degradation and disappearance. Discussion and Conclusion: In the article spreading of the habitats of Salvinia natans and Marsilea quadrifolia and threats are determined; recommendations for in-situ &ex-situ conservation of habitats and plant species are given. EnglishKolkheti, Freshwater ponds, Marsilea quadrifolia, Salvinia natans, ConservationINTRODUCTION Natural freshwater ponds cover only 1% of the planet; they are distinguished with biodiversity and present one of the important ecosystems being under threat. In recent years, degradation of freshwater ponds, an important habitat of biodiversity, caused by the anthropogenic factors, has prompted scientists from the Mediterranean countries to evaluate freshwater ponds by the IUCN Red List as threatened ecosystems. In 2015 IUCN Red List officially gave the freshwater ponds of the Mediterranean Sea coastal area global status [1,2]. After the natural freshwater ponds were officially recognized by IUCN, donors and private companies took upon themselves an obligation and responsibility to help preservation of the habitats and protect it from any negative influences. This will provide a possibility to create protected territories of a new type. It would be reasonable to create a database and spreading maps [1,2,3]. Freshwater ponds of the Kolkheti coastal area are valuable for being an important habitat for IUCN Red List species: Salvinia natans, Marsilea quadrifolia. Both species are evaluated as of Least Concern (LC) category. This category includes widespread and large taxa and notes that they are not qualified as threatened groups. Subject of the Study Materials and Methods The subject of the study was protected by Nature 2000, Emerald Network and Bern Convention freshwater ponds and living there indicator species Salvinia natans, Marsilea quadrifolia. By the definition, a freshwater pond is a depression in the ground with greenish and brownish clear water, where pH=5-6. Habitats and density of plant cover were studied by the method DAFOR, where D means dominant species, A - Abundant, F – Frequent, O – Occasional, R – Rare. (www. Halcrow, www.TACIS, www.ICWS ). For plant description a transect method was used. Transect is a straight line running across the given habitat for the purpose of studying its plant communities. During phytocenosis study of the given habitat a square method was used on every 50 m space along the transect line. The squares were 4x4 m2 in parameters. For obtaining more detailed data the Domin-Krajina method was also used [1-4]. During the study special attention was paid to the habitat type, its ecological state, plant covering density (in %), composition of species and their quantity, and vitality of single species. The main guideline for field investigations was ecology of dry land plants [1-9]. RESULTS Marsilea quadrifolia is a subject protected by Bern Convention and EUNIS agreement of European Union. The pond with Marsilea quadrifolia was destroyed during the construction of a new boulevard and infrastructure systems. At present, the only habitat of Marsilea quadrifolia in Kolkheti, is a pond near the Tskaltsminda river and village Kvavilnari, close to a costal dune.   The ponds with predominance of Salvinia natans are protected by Bern Convention and EUNIS (European Union Nature Information System) agreement together with Emerald Network and Nature 2000. The development of the Anaklia free zone has destroyed freshwater ponds with predominance of Salvinia and caltrop. The area of Salvinia spreading gradually diminishes. Today the habitats of Salvinia in the Kolkheti lowland are the following: Anaklia – small freshwater ponds. Their number was significantly higher but it has been decreased and only two ponds of 5x5 m size remained; The river Tsivi - there is small number of population in chanals, but only individual species can be found; Churia – along the road to the south of Kulevi terminal. DISCUSSION Historically, in the coastal area of Kolkheti, habitats have been continuously degraded and disappeared as a result of human activity. This process has significantly increased from the beginning of the 20th century, namely: irrigation activities, dams over rivers that accompanied urban projects. In many places freshwater ponds were turned into dumpsites. A dump site at the right side of the Chorokhi River is a good example of it. Understanding of the importance of habitats, their role in the ecosystems is very low among the population. Significant anthropogenic factors are water pollution, uncontrolled tourism, the sea coastal line contaminated by household trash during the warm season. Excessive pasturage is one of the worst dangers as well as uncontrolled hunting and fishing. As a result, favorable conditions are created for proliferation propagation of invasive species. Another problem is that the territories of special value beyond the protected area are not given protected status that allows uncontrolled hunting and fishing. CONCLUSION In order to save the biodiversity of per-humid ecosystems of the Kolkheti coastal area, to implement protection and management of threatened species, it is necessary to activate and toughen the measures for environmental protection as well as create local protected areas. The role in protecting ecosystems cannot be overestimated. They form homeostasis that allows communities of species to function normally. Disappearance of even one species leads to misbalance between species interaction. Therefore all these habitats must be fully protected [10-12]. It is necessary to conduct ex-situ conservation of the species in the Batumi Botanical Garden as well as in the Kolkheti protected areas (the Kolkheti National park and Kobuleti protected areas). For this purpose, it is of utmost importance to create artificial freshwater ponds. ACKNOWLEDGMENT Our thanks are to the scholars whose articles are cited and included in references of this manuscript. We are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. We thank the Editors of IJCRR for their helpful comments and editorial support and for reviews that helped improve the paper. We would also like to pass our thanks to the driver of the expedition, Mr Niko Tavdgiridze, for his assistance to identify Marsilea and Salvinia places as well as his help in providing fuel. Englishhttp://ijcrr.com/abstract.php?article_id=217http://ijcrr.com/article_html.php?did=2171. K. Smith, V. Barrios, W. Darwall, C. Numa (Editors) (2015) The Status and distribution of freshwater biodiversity in the eastern mediterranean, IUCN Red List., 129 p; 2. W. Darwall, S. Carrizo, C. Numa, V. Barrios, J. Freyhot, K. Smith (2015) Freshwater key biodiversity areas in the Mediterranean Basin Hotspot, IUCN Red List, 86 p.; 3. I. Matchutadze, B.Bolqvadze, J.jakeli, M. Tsinaridze (2014) Kolkheti refugee- habitat and biodiversity conservation, wise use, World Biodiversity Congress, Sri-Lanka, abstracts book, 78-79. 4. I. Matchutadze, B.Bolqvadze, T.Bakuradze, M.Gvilava, D.Baratashvili (2013) Coastal Sand Dunes and Freshwater Ponds in Kolkheti – Threats and Needs for Conservation, Nova Publisher, ISBN: 978-1-62808-092-6, Chapter 8. 5. Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora, O.J. L206, 22.07.92. 6. CORINE Biotopes - Technical Handbook, volume 1, p. 73-109, Corine/Biotopes/89-2.2, 19 May 1988. 7. CORINE Biotopes manual, Habitats of the European Community. EUR 12587/3, Office for Official Publications of the European Communities, 1991. 8. EUR27. 2007 The Interpretation Manual of European Union Habitats. European Commission DG Environment. 9. Relation between the Directive 92/43/EEC Annex I. Habitats and the CORINE habitat list 1991 (EUR 12587/3). 10. G. Nakhutsrishvili (1999) The vegetation of Georgia (Caucasus). - Braun-Blanquetia 15:1-74. 11. M. Barbour, J. Burk, W. Pitts, M. Schwartz (1999) Terrestrial Plant Ecology, Third Edition 373 p. 12. G. Nakhutsrishvili, I. Matchutadze (2014) Floristic assessment and creation of biodiversity monitoring program for flora surrounding of Kulevi terminal, 55 p.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN2016August11HealthcareISOLATION AND CHARACTERIZATION OF PHOSPHOLIPIDS OF ERI-PUPAL OIL FROM PUPAE GROWN ON CASTOR AND TAPIOCA LEAVES English2732Thumu RavinderEnglish Shiva Shanker KakiEnglish Konda Reddy KunduruEnglish Sanjit KanjilalEnglish B.V.S.K. RaoEnglish Sarat Kumar SwainEnglish R.B.N PrasadEnglishObjective: The aim of the present study was isolation and characterization of phospholipids from eri pupal oils. Methods: Silkworm lipids were extracted from pupae fed on castor and tapioca leaves and further separated in to individual class of lipids by column chromatography. Fatty acid composition of each lipid class was studied along with positional distribution of fatty acids in major phospholipids by enzymatic hydrolysis. Results and Conclusion: The eri pupal oils were found to contain 96.72-97.33% of neutral lipids, 0.51-0.21% of glycolipids and 2.65-2.39% phospholipids. Phosphatidylethanolamine (PE) was the predominant classes of phospholipid present (65.65 and 64.29%) in tapioca and castor fed pupal oils respectively. Phosphatidylcholine (PC) was observed in 19.50 and 19.22% respectively in tapioca and castor fed pupal oils. Cardiolipin (CL) and phosphatidylinositol (PI) are observed in minor levels. Positional distribution of fatty acids showed that, α-linolenic acid (ALA) was found to be more in sn-2 position of PE. Thus pupal oil offers a good natural source of PE with enriched ALA. EnglishEri silkworm pupae, Lipid classes, Phospholipids, Positional distribution, Fatty acid compositionINTRODUCTION Eri silk is obtained from non-mulberry silkworms and is known for its elegance and is mostly produced in the Northeastern parts of India. Silkworms are reported to show association with the host plants on which they feed for the necessary nutrients required for their metabolic activities and also for silk production1 . The host plants reported for growing of eri silkworms are mainly castor (Ricinus communis Linn.) and tapioca (Manihot utilissima Phol.) followed by some other species also2 . Besides production of silk, sericulture also produces large quantities of silkworm pupae which are thrown as sericulture waste or used as fertilizer and as a constituent of chick and fish feeds3-4. The desilked silkworm pupae are reported to have high nutritional value because of the presence of high protein and fat and these are considered as a source of nutritive components and protein supplements5-7. The silkworm pupae have been reported to be a good source of alpha linolenic acid (ALA) and the oil is reported to contain high amount of phosphorous3 . The study on nutritional and toxicological evaluation of silkworm oil revealed that eri pupal oil is safe and nutritionally equivalent to common vegetable oils, which increase its potential as source of ALA8 . The neutral lipids of eri pupae were isolated and characterized previously and the phosphorous content was reported to be 3-3.5%9 . In mulberry silkworm oil, phospholipid content of 10% was reported where the major PLs identified were PE and PC10. Lecithins from vegetable oils like rice bran11, corn12, rapeseed13, sunflower14, cottonseed and peanut15 are known to be good sources for phospholipids such as PC, PE and PI. In milk phospholipids, PE is the major component where as PC, sphingomyelin are almost in equal portions16. Studies on isolation and characterization of phospholipids from vegetable oils are abundant, whereas PLs from other sources is very rare. Literature reports are also available on fatty acid composition of total lipids of silkworm pupae Bombyx mori L 17. However, there is no report on the detailed study on phospholipids obtained from eri silkworm oil. Therefore, the present investigation was aimed at the determination of phospholipid composition in lipids extracted from pupae fed on both castor and tapioca leaves. MATERIALS AND METHODS Pupae fed on castor and tapioca leaves collected from Shadnagar, Ranga Reddy district, Telangana and Rampachodavaram, East Godavari district, Andhra Pradesh, India were supplied by Central Silk Board, Bengaluru, India. Fatty acid mixtures, references PE, PC, PI, CL and snake venom (Naja naja Atra) phospholipase A2 source were purchased from M/s Sigma Chemicals, St Louis, USA. Silica gel (60-120 mesh) for column chromatography was purchased from Acme Synthetic Chemicals, Mumbai, India. Pre-coated thin layer chromatography (TLC) plates (silica gel 60F254) were procured from Merck, Darmstadt, Germany. HPLC grade solvents procured from M/s Merck, Mumbai, India. All other analytical reagent-grade chemicals and solvents were purchased from M/s SD Fine Chemicals, Mumbai, India. Total Lipid Extraction The matured pupae grown on castor and tapioca leaves were dried at 80-90°C for 6-8 hours using vacuum oven, followed by the oil extraction using Soxhlet extractor with hexane as reported earlier9 . The crude total lipids of castor and tapioca pupal oils were dried under reduced pressure, stored for further use. Separation of Lipid classes Total lipids (TL) of eri silkworm oils were separated in to neutral lipids (NL), glycolipids (GL) and phospholipids (PL) using column chromatography18 by the elution with chloroform, acetone and methanol respectively. GLs and PLs were qualitatively identified by TLC using chloroform/methanol/ water (65:25:4, v/v/v) with suitable spray reagents19-20. The phospholipid mixtures obtained from column chromatography of castor (0.619 g) and tapioca (0.687 g) fed pupal lipid extracts were further individually re-chromatographed to separate individual phospholipids. The column was eluted with a gradient of 5-10, 20, 40 and 50% of methanol in chloroform to obtain CL, PE, PC and PI respectively21. The separated phospholipids were identified by comparison with individual standards and confirmed by spray reagents20. The isolated yields of phospholipids were found to be in the following amounts. For castor based PLs: CL (0.058g, 9.37%), PE (0.398g, 64.29%), PC (0.119g, 19.22%), PI (0.042g, 6.78%) and for tapioca based PLs: CL (0.060g, 8.73%), PE (0.451g, 65.65%), PC (0.134g, 19.50%), PI (0.041g, 5.96%). Positional Distribution of Fatty Acids Positional distribution of fatty acids in individual phospholipids of both was carried by phospholipase A2 mediated regiospecific hydrolysis as described by Christie22. Briefly, PE (50 mg) was dissolved in 4 ml of diethyl ether and to this 300 µL of snake venom solution (6 mg of snake venom in 1 ml of borate buffer; pH 7.0) was added and shaken vigorously for 1 hour. After complete hydrolysis, the ether solution was evaporated under nitrogen. The mixture containing liberated free fatty acids (FFA) and lysophospholipids were separated by column chromatography with the elution of gradient methanol in chloroform23. PC, PI were also hydrolyzed in a similar manner and the fatty acid composition was determined22-23. Fatty Acid Methyl Esters (FAME) All the lipid fractions isolated were converted to FAME using 2% sulfuric acid in methanol24 and the FFA was treated with diazomethane. All the FAMEs were analyzed by GCFID for fatty acid composition. Gas Chromatography (GC) GC was performed on Agilent 6890N series Gas Chromatograph equipped with a flame ionization detector (FID) on a split injector. A fused silica capillary column (DB-225MS, 30 m x 0.25 mm i.d. J and W Scientific, USA) was used for separation. The oven temperature was programmed at 160°C for 2 min, increased to 230°C at 5°C/min and hold for 20 min at 230°C. The injector and detector temperatures were maintained at 230 and 250°C respectively. Nitrogen used as carrier gas with a flow rate of 1 mL/min. Identification of fatty acids was carried out by comparing with the retention time of respective commercial standards. HPLC analysis of Phospholipids The phospholipid mixtures separated by column chromatography were qualitatively analyzed by Agilent HPLC chromatograph equipped with a quaternary pump and an evaporative light scattering detector (ELSD2000, Alltech, Deerfield, IL, USA). The drift tube temperature was set at 50°C and the nitrogen gas flow set at 1.5 L/min. The castor and tapioca phospholipids of 1 mg/mL concentration was separated on a SunFireTM Prep Silica column (5 μm, 4.6 x 250 mm; Sunfire columns, Waters, Ireland) at a mobile phase flow 0.5 mL/ min. A binary gradient mobile phase composed of eluent A [chloroform/ methanol/ammonium hydroxide (80:19.5:0.5, v/v/v)] and eluent B [chloroform/methanol/ammonium hydroxide/water (60:34:0.5:5.5, v/v/v/v)] was used for elution as follows25: 0-10 min, 95% B; 10-15 min, 100% B and 15-20 min, 95% B. Identification of phospholipids was carried out by comparing the retention times of the respective commercial standards. All the analysis was carried out in duplicate. RESULTS AND DISCUSSION The present study describes the separation and characterization of lipid classes from eri pupae (Samia Cynthia ricini) grown on castor and tapioca leaves. Total lipids were extracted from pupae and were found to be in the range of 18-20 % as reported earlier26. The extracted total lipids were separated into 3 lipid classes by column chromatography and the data is shown in Table 1.   The fatty acid composition of total, neutral, glyco and phospholipids of both varieties were determined by GC and are given in Tables 2 and 3. It was observed that, ALA was the major fatty acid followed by palmitic (16:0) and oleic (18:1) in all tapioca lipids compared to castor fed lipids which could be due to the influence of host plants as reported earlier9 . The ALA content in castor and tapioca fed PLs was observed 30.51% and 38.59% respectively. Previous studies on phospholipids from mulberry silkworm oil reported 40% of ALA10. Among other fatty acids, stearic (18:0), oleic and linolenic (18:2) acids were observed in greater amounts in both PLs and GLs of both varieties compared with neutral lipids. However, ALA content was observed to be low in PLs and GLs compared with NLs in both pupal oils. Long chain saturated fatty acids like arachidic (20:0) and behenic (22:0) acids were found slightly higher amounts in polar lipids compared with NLs of both pupal lipids. The phospholipids from both silkworm oils were identified by HPLC and quantified by column chromatography into CL, PE, PC and PI. The results showed that the castor and tapioca phospholipids are major source for PE (64-65%). Such a high PE content was earlier observed in the phospholipids of an obligate intracellular parasitic bacterium, Rickettsia prowazeki, where the PE reported 60-70%27. It was reported that mammalian and plant tissues have lesser occurrence of PE than PC, where as in bacteria PE is the principal phospholipid present28. In vegetable oil phospholipids, only castor seed oil was reported to contain high amounts of PE29. The fatty acid compositions of individual phospholipid classes and their hydrolysis products were determined by GC and are given in Tables 4 and 5. In cardiolipin isolated from castor and tapioca PLs, palmitic acid was the major fatty acid followed by oleic, stearic, ALA and other fatty acids. ALA content was more in PE of both pupal oils, followed by stearic, oleic, palmitic and other fatty acids compared to other PLs of both varieties.   The results (Table 4 and 5) indicate that, ALA was present in higher amounts at sn-2 position of tapioca PE (51.74%) compared to castor PE (45.56%). Saturated fatty acids were majorly located at sn-1 and unsaturated fatty acids were predominantly located at sn-2 position for all the phospholipid classes. A higher content of ALA and their predominance at sn-2 position increases the nutritional importance of the lipid9 . Hence, the phospholipids of castor and tapioca fed oils are rich source of ALA with predominant distribution at sn-2 position. In addition, phospholipids are a major component of cell membranes and required for signal transduction, metabolic regulation and maintenance of living cells30. The higher content of PE with ALA can be helpful for formation of fluid membrane structures which can have potential applications in signal transduction and other biological applications.   CONCLUSIONS In this study, eri pupal oil is shown as a novel source for phosphatidylethanolamine containing ALA. The extracted oil from eri pupae was separated into different lipid classes and further characterization of phospholipids is reported for first time. With a few exceptions, castor and tapioca leaf fed eri silkworm pupal oils showed similar characteristics in lipid classes, fatty acid composition and positional distribution of fatty acids in phospholipids. The phospholipid fraction of eri pupal lipids with a high amount of PE could be a useful product for pharma and food applications. ACKNOWLEDGEMENT Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Englishhttp://ijcrr.com/abstract.php?article_id=218http://ijcrr.com/article_html.php?did=2181. Unni BG, Kakoty AC, Khanikor D, Bhattacharya PR, Pathak MG, et al. Lipid and fatty acid composition of muga silkworm, Antheraea assama, host plants in relation to silkworm growth. J Lipid Mediat Cell Signal 1996; 13: 295-300. 2. Suryanarayna N, Das PK, Sahu AK, Sarmah MC, Phukan JD. Recent advances in eri culture. Indian Silk 2003; 41: 5-12. 3. Udayasekhara Rao P. Chemical composition and nutritional evaluation of spent silkworm pupae. J Agric Food Chem 1994; 42: 2201-2203. 4. Ichhponani JS, Malik NS. Evaluation of deoiled silkworm pupae meal and corn steep liquor as protein sources in chick rations. Br Poult Sci 1971; 12: 31-234. 5. Sing KC, Suryanarayan N. Eri pupae: A popular cuisine too. Indian Silk 2003; 41: 57-58. 6. Wang J, Wu Fu-An, Liang Y, Wang M. Process optimization for the enrichment of α-linolenic acid from silkworm oil using response surface methodology. Afr J Biotechnol 2010; 9: 2956- 2964. 7. Mishra N, Hazarika NC, Narain K, Mahanta J. Nutritive value of non-mulberry and mulberry silkworm pupae and consumption pattern in Assam, India. Nutrition Research 2003; 23: 1303- 1311. 8. Longvah T, Manghtya K, Qadri SSYH. Eri silkworm: a source of edible oil with a high content of α-linolenic acid and of significant nutritional value. J Sci Food Agr 2012; 92: 1988-1993. 9. Kaki SS, Shireesha K, Kanjilal S, Kumar SVLN, Prasad RBN, et al. Isolation and characterization of neutral lipids of desilked eri silkworm pupae grown on castor and tapioca leaves. J Agric Food Chem 2006; 54: 3305-3309. 10. Kotake NE, Yamamoto K, Nozawa M, Miyashita K, Murakami T. Lipid profiles and oxidative stability of silkworm pupal oil. J Oleo Sci 2002; 51: 681-690. 11. Adhikari S, Adhikari DJ. Indian rice bran lecithin. J Am Oil Chem Soc 1986; 63: 1367-1369. 12. Schneider M. In lecithins: Sources, Manufacture and Uses. American Oil Chemical Society, Champaign-III; 1989. P. 109- 130. 13. Sosulki P, Zadernowski R, Babuchowsk K. Composition of polar lipids in rapeseed. J Am Oil Chem Soc 1981; 58: 561-564. 14. Morrison WH. Sunflower lecithin. J Amer Oil Chem Soc 1981; 58: 902-903. 15. Vijayalaxmi B, Rao SV, Achaya KT. The nature of cottonseed phospholipids. Fette Seifen Anstrichm 1969; 1: 757-761. 16. Morrison WR, Jack EL, Smith LM. Fatty acids of Bovine milk Glycolipids and phospholipids and their specific distribution in diacylglycerophospholipids. J Am Oil Chem Soc 1965; 42: 1142-1147. 17. Sreekantaswamy HS, Siddalingaiah KS. Sterols and fatty acids from the neutral lipids of desilked silkworm pupae. Fette Seifen Anstrichm 1981; 83: 97-99. 18. Rouser G, Kritchevsky G, Yamamata A. Column chromatographic and associated procedures for separation and determination of phosphatides and glycolipids. In: Marinetti GV. Lipid chromatographic analysis, Marcel Dekker; 1967. P. 99-161. 19. Jacin H, Mishkin AR. Separation of carbohydrates on borateimpregnated silica gel G plates. J Chromatogr A 1965; 18: 170- 173. 20. Mangold HK. Thin layer chromatography of lipids. J Am Oil Chem Soc 1961; 38: 708-727. 21. Christie WW. Lipid analysis; isolation, separation, identification and structural analysis of lipids. 2nd ed, Pergamon, Oxford; 1982. P. 109-111. 22. Christie WW. Structural analysis of lipids by means of enzymatic hydrolysis. Lipid Analysis. (2nd ed), Pergamon Press, Oxford, UK; 1982. P. 155-166. 23. Hanahan DJ, Brockerhoff H, Barron EJ. The site of attack of phospholipase A on lecithin. J Biol Chem 1960; 235: 1917-1923. 24. Christie WW. The preparation of derivatives of lipids. Lipid Analysis. (2nd ed.). Oxford, UK, Pergamon Press; 1982. P. 51- 61. 25. Avalli A, Contarini G. Determination of phospholipids in dairy products by SPE/HPLC/ELSD. J Chromatogr A 2005; 1071: 185-190. 26. Ravinder T, Kaki SS, Prabhakar INSS, Rao BVSK, Swain SK, Prasad RBN. Effect of Natural and Synthetic Antioxidants on Oxidation of Eri silkworm oils. IAJPR 2015; 5: 3666-3675. 27. Winkler HH, Miller ET. Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs. J Bacteriol 1978; 136: 175-178. 28. Vance JE. Biochemistry of Lipids, Lipoproteins and Membranes. (4th ed) Elsevier, Amsterdam; 2002. 29. Paulose MM, Venkob Rao, Achaya KT. Nature of castor seed phospholipids. Indian J Chem 1966; 4: 529-532. 30. Yanagita T. Nutritional functions of dietary phosphatidyl inositol. Inform 2003; 14: 64-66.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN2016August11HealthcareANTIMICROBIAL RESISTANCE DEMONSTRATED BY UROPATHOGENIC ESCHERICHIA COLI AT A TERTIARY CARE HOSPITAL English3338Snehal S. AnturlikarEnglish Niyati A. TrivediEnglishBackground: Urinary tract infection (UTI) is one of the most common bacterial infections encountered by clinicians in developing countries. Escherichia coli is the most common causative organism of UTI. Development of resistance by E.coli towards different antimicrobial agents is alarming. Hence, our study was planned to analyze the antimicrobial resistance pattern of E.coli isolates at a tertiary care teaching hospital. Materials and method: Culture sensitivity reports of all urine samples sent to microbiology department of a tertiary care teaching hospital during the period of July 2010-June 2013 were screened. Detailed reports were collected for all the samples in which E.coli was identified as a causative organism. Culture sensitivity testing was done by modified Kirby-Bauer disk diffusion (high media) method. Result: E.coli was isolated in total 1155 urine samples during the period of three years. Majority of patients belonged to pediatric age group (823/1155, 71.25%). 52.21% samples were of female patients. Thirty-one antimicrobial agents were tested for 13048 times for their sensitivity towards E.coli. Antimicrobial resistance ranging from 14.58% to 100% was noted among various antimicrobials. E.coli showed 38.23% resistance towards aminoglycosides, 52.27% resistance towards quinolones, 54.95% resistance towards beta-lactams and 67.33% resistance towards miscellaneous group of antimicrobials. Conclusion: Proper selection and wise use of available antibiotics will help in reducing the rate of increase in resistance. Periodic monitoring of antimicrobial susceptibility pattern of causative agent in a particular setting will be helpful in guiding judicious use of antimicrobial agents limiting the spread of resistant strains. EnglishAminoglycosides, Antimicrobial susceptibility, Escherichia coli, Urinary tract infection.INTRODUCTION Urinary tract infections (UTI) is one of the most common bacterial infections encountered by clinicians in developing countries[1] and constitute a great proportion of prescription of antibiotics [2]. It has been estimated that symptomatic urinary tract infections (UTI) occurs in as many as 7 million visits to emergency units and 100,000 hospitalizations annually. UTI has become the most common hospital-acquired infection, accounting for as many as 35% of nosocomial infections, and it is the second most common cause of bacteraemia in hospitalized patients [3]. Escherichia coli is the most common causative organism of urinary tract infections.[4] The emergence of drug resistance to trimethoprim-sulfamethoxazole, the penicillins, cephalosporins, and fluoroquinolones by Uropathogenic Escherichia coli (UPEC) has limited the options for selecting the appropriate antibiotic for the treatment of urinary tract infections [4]. As resistance to commonly prescribed antimicrobial agents is increasing significantly, there is a need of periodic analysis of the pattern and sensitivity of organisms isolated and the results need to be communicated to doctors [5]. Hence, this study was carried out with an aim to analyze the antimicrobial resistance pattern of E.coli towards commonly prescribed antimicrobial agents. MATERIALS AND METHODS Culture sensitivity reports of all urine samples sent to microbiology department of a tertiary care teaching hospital during the period of July 2010-June 2013 were screened. Detailed clinic- epidemiological data were collected for all the samples in which E.coli was identified as a causative organism and the data was entered into Microsoft excel spreadsheet 2007. Antimicrobial susceptibility testing was done by modified Kirby Bauer disc diffusion method [6]. Statistical analysis Data has been presented as percentage of resistance or Mean (SD). Chi square test was performed as a test of significance whenever necessary using GraphPad InStat software version 3.10 (trial version). P value < 0.05 was considered statistically significant. RESULTS Amongst 33,000 samples of positive urine culture received at the Microbiology Department in a tertiary care teaching hospital during the period of 3 years (July 2010- June 2013), E.coli isolates were obtained from 1155 urine samples. Out of 1155 urine samples, 603 samples (52.21%) were of female patients while 552 samples (47.79%) were of male patients. The age of the patients ranged from 1 day to 87 years with mean age of 16.33± 20.05 years. Maximum numbers of samples (823/1155, 71.25%) were obtained from pediatric age group followed by patients belonging to adult age group (270/1155, 23.38%). Only 62 (5.37%) samples belonged to the patients of geriatric age group. E.coli isolates were tested for their susceptibility towards 31 different antimicrobial agents with each E.coli isolate being tested for its susceptibility towards 5 to 17 different antimicrobial agents (Mean 11± 3.89), out of which it showed resistance towards at least 0 to 10 antimicrobials (Mean 5± 3.32). Out of total 13,048 times for which different antimicrobials were tested for their susceptibility towards E.coli, 6824 (52.30%) times resistance was observed while 6188 (47.42%) times sensitivity was observed and for 36 times (0.28%) intermediate sensitivity was observed. Group wise resistance pattern of E.coli isolates. E.coli isolates were tested for their susceptibility towards four different groups of antimicrobial agents including aminoglycosides, quinolones, beta lactams and miscellaneous (which includes nitrofurantoin, doxycycline, tetracycline etc.). Aminoglycosides were tested for 3071 times for their susceptibility towards E.coli out of which resistance was shown towards them for 1174 times (38.23%). (Table1) Among the four different aminoglycosides tested, amikacin was the most commonly tested antimicrobial agent (1084/1155, 93.85%) followed by gentamicin (950/1155, 82.25%). Percentage of resistance amongst aminoglycosides ranged from 77.45% to 27.21% with amikacin sowing least resistance. Total seven quinolones were tested for 3046 times for their susceptibility towards E.coli out of which resistance was observed for 1592 times (52.27%). (Table2) Marked difference in resistance was observed among quinolones with lomefloxacin and levofloxacin exhibiting 100% resistance while gatifloxacin showed 14.58% resistance towards E.coli. Total 14 beta-lactams were tested for total 4719 times for their susceptibility towards E.coli out of which resistance was observed for 2593 (54.95%) times. (Table3) Amongst beta-lactams, piperacillin (1073/1155, 92.90 %) was the most commonly tested antimicrobial agent while ceftriaxone showed highest resistance (100%, 5/5) towards E.coli. Out of 1249 times for which 3rd generation cephalosporins were tested, 841 times (67.33%) resistance was observed towards them. 86.96% resistance (20/23) was observed towards 4th generation cephalosporin. Among the six miscellaneous antimicrobials tested for their susceptibility towards E.coli for 2176 times, resistance was observed for 1465 times (67.33 %). Chloramphenicol (987/1155, 85.45%) was most commonly tested antimicrobial agent as shown in table 4. Highest resistance was observed towards doxycycline (100%, 4/4) followed by cotrimoxazole (76.71%, 56/73). Out of 1155 samples where E.coli was isolated, 925 (80.1%) samples showed resistance to atleast one agent from three or more antimicrobial families and hence was identified as MDR. The pattern of resistance among samples obtained from male and female patients appeared similar for all the antimicrobial agents tested, except for nitrofurantoin. In case of nitrofurantoin higher resistance (p=0.025) was observed among male patients (69.94%, 328/469) as compared to samples obtained from female patients (63.12%, 315/499). Except for amikacin (p=0.013), piperacillin (pEnglishhttp://ijcrr.com/abstract.php?article_id=219http://ijcrr.com/article_html.php?did=2191. Getenet B, Wondewosen T. Bacterial Uropathogens in Urinary Tract Infection and Antibiotic Susceptibility Pattern in Jimma University Specialized Hospital, Southwest Ethiopia. Ethiop J Health Sci 2011;21:141-46. 2. Khameneh ZR, Afshar AT. Antimicrobial Susceptibility Pattern of Urinary Tract Pathogens. Saudi J Kidney Dis Transpl 2009;20:251-53. 3. Patel S, Taviad PP, Sinha M, Javadekar TB, Chaudhari VP. Urinary Tract Infections Among Patients at G.G. Hospital and Medical College, Jamnagar. Natl J Community Med 2012;3:138- 141. 4. Shariff V A AR, Shenoy MS, Yadav T, Radhakrishna M. The Antibiotic Susceptibility Patterns of Uropathogenic Escherichia coli, with Special Reference to the Fluoroquinolones. J Clin Diagn Res 2013;7:1027-30. 5. Tiwari P, Kaur S. Profile and Sensitivity Pattern of Bacteria Isolated from Various Cultures in a Tertiary Care Hospital in Delhi. Indian J Public Health 2010;54:213-15. 6. Bauer AW, Kirby WMM, Sherris JC, Turck M, et al. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-96. 7. Rani H, Kaistha N. Choice of Antibiotics in Community Acquired UTI due to Escherichia coli in Adult Age Group. J Clin Diagn Res 2011;5:483-85. 8. Razak SK, Gurushantappa V. Bacteriology of Urinary Tract Infection and Antibiotic Susceptibility Pattern in a Tertiary Care Hospital in South India. Int J Med Sci Public Health 2012;1:109- 12. 9. World Health Organization. Regional Strategy on Prevention and Containment of Antimicrobial Resistance 2010-2015; 2010. 10. Holloway K, Mathai E, Sorensen TL, Gray A. CommunityBased Surveillance of Antimicrobial Use and Resistance in Resource-Constrained Settings. Report on five pilot projects. Geneva: World Health Organization 2009. 11. Vila J, Pal T. Update on Antibacterial Resistance in Low-Income Countries: Factors Favoring the Emergence of Resistance. The Open Infectious Diseases Journal 2010;4:38-54. 12. Tawanda G. General Principles of Antimicrobial Therapy. In: Brunton L, editor. Goodman and Gilman’s The Pharmacological Basis of Theraupeutics. 12th ed. The McGraw-Hill Companies, Inc., 2011.p. 1375-76. 13. Yilmaz N, Agus N, Yurtsever SG, Pullukcu H, Gulay Z, Coskuner A, et al. Prevalence and antimicrobial susceptibility of Escherichia coli in outpatient urinary isolates in Izmir, Turkey. Med Sci Monit 2009;15:61-65.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241815EnglishN-0001November30HealthcareA STUDY OF AN ANOMALOUS ORIGIN OF CYSTIC ARTERY AND ITS RELATION WITH CALOT’S TRIANGLE- CADAVERIC STUDY English3943Kankhare Sonali B.English Patil Anjali D.English Kate Sarika P.EnglishIntroduction: The Cystic artery is the main blood supply of cystic duct and gall bladder. If there is damage to the cystic artery during surgery may lead to serious complications, hence the origin and the course of cystic artery is important. Objective: To evaluate the variations in origin of cystic artery, relation with the Calot’s triangle, common hepatic duct and cystic duct. Method: It is cross-sectional study was conducted over a period of 2 years on 40 Indian formalin fixed cadavers from Departments of Anatomy. Result: Out of 40 cadavers, cystic artery (CA) was found to be originating from the right hepatic artery in 28 cases (70%), from the left hepatic artery in 2 cases (5%), from gastro duodenal artery in 4 cases (10%), from proper hepatic artery in 3 cases (7.5%), from Superior mesenteric artery in 2 cases (5%), 1 case from right gastric artery (2.5%). It was found that in all cadavers cystic artery and cystic lymph node was found as a content of Calot’s triangle. In 54 cases (90%) cystic arteries were posterior to common hepatic duct and in (10%) it was anterior to the common hepatic duct. In two cases (3.33%), cystic arteries were found anterior to the cystic duct. Superficial and deep branch of the cystic artery was found in one case. Conclusion: Anatomical knowledge of cystic artery is necessary to prevent any iatrogenic complications during surgery. EnglishCystic artery, Calot’s triangle, Hepatic ductINTRODUCTION The chief source of blood supply to the gallbladder and the cystic duct is the cystic artery. Cystic artery commonly arises from the right hepatic artery in the angle between the common hepatic duct and cystic duct. Cystic artery may arise from left hepatic artery, the proper hepatic artery, the common hepatic artery, the gastro duodenal artery, the superior pancreaticoduodenal artery and the superior mesenteric artery.1 The cystic artery after taking origin it passes through the Calot’s triangle in 75–80% cases. Calot’s triangle is a triangular space, bounded by the cystic duct, the common hepatic duct and inferior surface of the liver. The most important content of the triangle is the cystic artery and cystic lymph node.2 Knowledge of normal anatomy and its variations in the cystic artery and relation with common hepatic duct and cystic duct is important during excision of the gallbladder.3 When cystic artery is outside the Calot’s triangle and it crosses anterior to common hepatic duct it may create complications during surgery. Awareness of anatomical variations of the hepatobiliary arterial system is essential to avoid complication within that surgical field. Blood vessel damages during laparoscopic cholecystectomy, including cystic artery hemorrhage or bile leakage results in open surgery in up to 1.9% of cases, 4 and causing mortality 0.02%. 5 Anatomical variations of the cystic artery and cystic duct are common and these variations have always attracted both the anatomists and surgeons. METHOD This is cross sectional study carried out on 40 embalmed cadavers of Indian origin over a period of 2 years in the department of anatomy. The study is approved by ethical committee. By using dissecting instruments (scissors, forceps, scalpel, etc) the abdomen was opened and then the fat in the liver, gall bladder and duodenum cleared .We defined the boundaries of Calot’s triangle then traced the origin and course of cystic artery and variations were noted. Statistical analysis done and percentage calculated. In this study we noted the following parameters, 1 Source of origin of the cystic artery. 2 Relations of the cystic artery to the Calot’s triangle (inside/outside). 3 Relations of the cystic artery to the cystic duct and common hepatic duct noted. (Anterior/ Posterior/ Not related). 4 Content of Calot’s triangle. RESULT A total of forty cadavers were observed to study anatomical variations. The Calot‘s triangle was bounded by the Common hepatic duct medially, cystic duct laterally and liver superiorly. 1. Variation in the origin of the cystic artery: Out of 40 cadavers, cystic artery (CA) was originating from the right hepatic artery in 28 cases (70%), from the left hepatic artery in 2 cases (5%), from gastro duodenal artery in 4 cases (10%), from proper hepatic artery in 3 cases (7.5%) , from Superior mesenteric artery in 2 cases (5%), 1 case from right gastric artery (2.5%). 2. Number of cystic artery and its branches: The single cystic artery was found in 39 cases. In one case, a branch of cystic artery (superficial and deep branch) was found. In one case, we observed double origin of cystic artery, one from right hepatic artery and other from proper hepatic artery. 3. Relation of the cystic artery to the Calot’s triangle: Cystic artery was found to be inside the Calot’s triangle in 38 cases (95%) while in 2 cases (5%) it was outside the Calot’s triangle (Fig. 2). 4. Relation of the cystic artery to the common hepatic duct: (Table: 1) 5. Relation of the cystic artery to the cystic duct: Out of 40 cadavers, cystic artery was found to be passing anterior to the cystic duct in 2 cases (5%) and in remaining cases it was not related to the cystic duct. 6. Content of calots triangle: In each case, we had noted the cystic lymph node as a content of calots triangle along with cystic artery. DISCUSSION The performance of a safe cholecystectomy depends on thorough knowledge about the normal anatomy and anatomical variations that may contribute to the occurrence of major postoperative complications. The famous triangle originally described by Calot’s in 1891.6 It contain the branch of right hepatic artery , the cystic artery, the cystic lymph node, connective tissue and lymphatic. During cholecystectomy this triangle is dissected to identify the cystic artery and cystic duct before ligation and division. The explanation for the variations in the cystic artery is found in the developmental pattern of the biliary system. Embryologically, the simple branching pattern of the gastro duodenal and hepatobiliary vasculature is profoundly altered by the growth of the liver and pancreas and by the assumption of a curved form in the stomach and duodenum. These factors operate to complicate the branching of the coeliac axis and proximal segment of the superior mesenteric artery. Considering that the liver is derived from a portion of the primitive duct supplied primordially by the coeliac and mesenteric arteries, it may receive rami from both of these sources. The same is true from the gallbladder. The liver and gallbladder develop from a foregut endodermal hepatic diverticulum, which usually carries a rich supply of vessels from the abdominal aorta and its initial branches. Most of the vessels picked up from the abdominal aorta during development degenerate leaving in place the mature vascular system. Because the pattern of degeneration is highly variable, the origin and branching pattern of the vessels to these organs also vary considerably.7 Considering the complexity of this developmental scheme it is easy to understand the large degree of arterial variations within this vascular system.8 Knowledge of the different anatomical variations of the arterial supply of the gallbladder, liver and stomach is of great importance in hepatobiliary and gastric surgical procedures.9 In the present study cystic artery was found to be originating from the right hepatic artery in 28 cases (70%), from the left hepatic artery 2 cases (5%), proper hepatic artery 3 cases (7.5%) and gastro duodenal artery in 4 case (10%) and superior mesenteric artery 2 cases (5%), right gastric artery in one case (2.5%). Findings of the present study were compared with values given by other co-worker, (Table: 2) In our study we found that single cystic artery in 39 cases and double cystic artery in one case. Similarly, double cystic artery was found by Balija13 in 21.1% cases and by Suzuki14 in 11.1% cases. Bincy M. George et al15 reported a case of double cystic artery and both arteries arose from the hepatic artery proper. In our study in one cadaver we noted that the superficial and deep branches of cystic artery in Calot’s triangle, and similar findings by Khalilur Rahman.16 Saidi et al17 in 102 Nairobian liver dissections, found double cystic artery in 8 cases (7.8%) and Futara et al.18 reported a frequency of 10 % in Ethiopians. Loukas et al19 described double cystic arteries arising from both the right hepatic artery and the posterior superior pancreaticoduodenal artery coexisting with an accessory left hepatic artery arising from a left gastric artery. But in our study we found out double origin of cystic artery one from right hepatic artery and other from proper hepatic artery. Comparison of presence of cystic artery within or outside the Calot’s triangle by other authors; (Table: 3) Variation in relation of cystic artery anterior or posterior to common hepatic duct (Table: 4) In our study we found that anterior relation of cystic artery to cystic duct in 2 cases (5 %), Flinsky10 found anterior relation to cystic duct in 2.94% and by Desler et al8 1%,Gawali et al12 (10%). In every cases there is a lymph node termed as Calot’s node was found in the Calot’s triangle. Lymph node was also found by Sujuki14. Hemorrhage and bile leakage are the most common causes for conversion of laparoscopic to open surgery and usually occur due to variation of structures of the hepatobiliary triangle.21 CONCLUSION The success of laparoscopic, open cholecystectomy and other procedures are depending upon the anatomical knowledge of cystic artery and billiary duct system and it is necessary to prevent any iatrogenic complications during surgery Englishhttp://ijcrr.com/abstract.php?article_id=220http://ijcrr.com/article_html.php?did=2201. Strandring S, (Ed). Gray’sAnatomy. The anatomical basis of clinical practice. 40th edition. Elsevier Chuchill Livingstone. Edinburg, 2008: 1218-1219. 2. Chen TH, Shyu JF, Chen CH, Ma KH, Wu CW, Lui WY, et al. Variations of the cystic artery in Chinese adults. Surgical Laparoscopy, Endoscopy and Percutaneous Techniques. 2000; 10(3):154-157. 3. Nagral S. Anatomy relevant to cholecystectomy. J Min Access Surg 2005; 1(2):53-8. 4. Thompson JE Jr, Bock R, Lowe DK, Moody WE 3rd. Vena cava injuries during laparoscopic cholecystectomy. Surg Laparosc Endosc 1996;6: 221-223 5. Deziel DJ, Millikan KW, Economou SG, Doolas A, Ko ST, Airan MC. Complications of laparoscopic cholecystectomy: a national survey of 4,292 hospitals and an analysis of 77,604 cases. Am J Surg 1993; 165: 9-14. 6. Haubrich, W S 2002, Calot of the triangle of Calot, Gastroenterology, 2000; 123(5): 144. 7. Hiatt JR, Gabbay J, Busuttil RW. Surgical anatomy of the hepatic arteries in 1000 cases. Ann Surg 1994; 220:50-2 8. Daseler EH et al. The cystic artery and constituents of the hepatic pedicle. A study of 580 specimens. Surgery, gynecology and obstetrics, 1947; 85:47–63. 9. Flint ER. Abnormalities of the right hepatic, cystic and gastroduodenal arteries and of the bile ducts. Br J Surg. 1923; 10: 509–519. 10. Flisinski P, Szpinda M, Flisinski M. The cystic artery in human foetuses. Folia morphologica 2004;63(1):47-50. 11. Michels NA. Variational anatomy of the hepatic, cystic, and retroduodenal arteries. Am. Med. Assoc 1953b; 66: 20-34. 12. Gawali Rohan A., A study on variations of origin and course of cystic artery and its relation to the calot’s triangle Int J Med Phar Sci | Vol 5; Issue 3; October 2014 13. Balija, M, Huis, M, Nikolic, V, and Stulhofer, M, Laparoscopic visualization of the cystic artery anatomy, World J Surg, 1999; 23(7): 703-07 14. Suzuki.M, Akaishi.S,. Rikiyama.T, Naitoh.T, Rahman.M, Matsuno. S. Laparoscopic cholecystectomy, Calot’s triangle, and variations in cystic arterial supply. Journal Surgical Endoscopy. NewYork. Springer. 2000 February; 14(2): 141-44. 15. Bincy M. George, Somayaji SM. Multiple variations of the subhepatic hepatobiliary variations. International Journal of Anatomical Variations 2010; 3: 39- 40. 16. Khalilur Rahman MD1, Selina Anwar Presence of Cystic Artery in the Calot’s Triangle and Its Relation with Common Hepatic Duct – a Postmortem Study, Bangladesh J. Anat. 2012; 10(2) : 50-56 17. Saidi H, Karanja TM, Ogengo JA. Variant anatomy of the cystic artery in adult Kenyans. Clin Anat 2007;20:943-5. 18. Futara G, Ali A, Kinfu Y. Variations of the hepatic and cystic arteries among Ethiopians. Ethiop Med J 2001;39: 133– 142. 19. Loukas M, Fergurson A, Louis RG Jr, Colborn GL. Multiple variations of the hepatobiliary vasculature including double cystic arteries, accessory left hepatic artery and hepatosplenic trunk: a case report. Surg Radiol Anat 2006;28:525-8. 20. De Silva M, Fernando D. Anatomy of the Calot’s triangle and its relevance to laparoscopic cholecystectomy. Ceylon medical journal, 2001; 46(1):33–4. 21. Kano N, Yamakawa T, Ishikawa Y, Sakai S, Honda H, Kasugai H, et al. Laparoscopic cholecystectomy: a report of 409 consecutive cases and its future outlook. Surgery Today. 1994; 24(5):399-402.