PLASMID PROFILE AND CURING, BIOFILM DETECTION AMONG ESBL PRODUCING ISOLATES OF ACINETOBACTER SPECIES

Objective: The aim of the present study is to evaluate the plasmid profile and curing in extended spectrum beta-lactamases (ESBL) producers and know the association of biofilm formation among ESBLs.Methods: Standard disk diffusion method (Kirby Bauer's method) was performed to know the sensitivity and resistance patterns for commonly used antibiotics and clinical laboratory standard institute (CLSI) recommended confirmatory phenotypic method was used to confirming ESBL producers. 56 ESBL producers were subjected to plasmid profiling and curing. The tube (qualitative) and microtitre (quantitative) methods were performed to detect the biofilm formation.Results: In all ESBL producers, a single plasmid was found with approximately 21, 226 bp. After plasmid curing, the resistance patterns against the third generation cephalosporin group of antibiotics were transformed into susceptible for all bacterial isolates. In tube method, 11 (19.64%) isolates were strong adherent, 08 (14.28%) were either weakly adherent or non-biofilm producers. In the microtitre plate method, 09 (16.07%) were considered as positive, and 47 (83.92%) were considered as negative for biofilm formation.Conclusion: We found a single resistant plasmid in all isolates and after curing of plasmids, the transformation of resistance to susceptibility against third generation drugs were observed. So, we concluded that ESBL mediated antimicrobial resistance mechanisms in Acinetobacter spp. is maybe plasmid-mediated in the tested isolates. However, there was no association found between biofilm producers and ESBLs, this baseline information will be helpful to study more in this area.Keywords: Acinetobacter spp, Biofilm detection, ESBL, Plasmid profile and curin

Prevalence of various β-lactamase (ESBL, AmpC and MBL) producing multidrug resistant clinical isolates of Acinetobacter spp. in a tertiary care hospital.

Background: Extensive use of β- lactam group of antibiotics to treat the gram negative bacterial infections the antibiotic resistance are increasing and Acinetobacter species are budding as multidrug resistant (MDR) gram negative bacteria showing various β-lactamases mediated resistance. Materials and Methods: The presence of β-lactamases in the multi drug resistant clinical isolates of Acinetobacter species were screened by β-lactamase assay method. The presence of ESBL was detected by phenotypic confirmatory test recommended by Clinical and Laboratory Standards Institute (CLSI), AmpC was detected by CX/BA inhibitor method and carbapenem (IMP or MER)/EDTA combined disc method and Modified Hodge tests were performed to detect MBL mediated resistance mechanism. Results: We found high level of beta lactamase mediated resistance mechanism as part of multidrug resistance. Among 180 MDR isolates, 147 isolates were shown positive for beta lactamase assay method and these 147 isolates were further subjected to confirm presence of beta lactamase such as AmpC 105 (71.42%) followed by ESBL 84 (57.14%), MBL 43 (29.25%). Co-existence of ESBL and MBL also identified in 13 (8.84%) isolates. Conclusion: β-lactamase mediated resistance mechanisms are accounting very high in the multidrug resistant isolates of Acinetobacter species. Therefore early detection is necessary to screen and confirm beta lactamase mediated resistant strains to avoid treatment failure and prevent the spread of MDR

Association of ABO blood groups with Chikungunya virus

Chikungunya virus (CHIKV) an emerging arboviral infection of public health concern belongs to the genus Alphavirus, family Togaviridae. Blood group antigens are generally known to act as receptors for various etiological agents. The studies defining the relationship between blood groups and CHIKV is limited and hence it is necessary to study these parameters in detail. In the present study 1500 subjects were enrolled and demographic data (Age, Gender, Blood group, CHIKV infection status, and CHIKV infection confirmation mode) was collected from them. The risk of acquiring CHIKV disease and its association with factors such as blood group, age and gender was analyzed statistically. The data of this study showed a possible association between blood group, age and gender of the study population with CHIKV infection. It is observed that CHIKV infections were higher in individuals with Rh positive blood group when compared to their Rh negative counterparts.CHIKV infections were found to be higher in Rh positive individuals of AB and A blood groups than that of Rh negative counterparts. Results also indicated that infections were higher in adults belonging to the age group > 30 years and also higher in males as compared to females enrolled in this study. These data present further evidence for the association of the blood groups, age and gender to susceptibility to CHIKV infection. Further studies are needed to confirm these findings. This is the second study showing the possible association of blood groups with chikungunya

OCCURRENCE OF VARIOUS BETA-LACTAMASE PRODUCING GRAM NEGATIVE BACILLI IN THE HOSPITAL EFFLUENT.

This study was designed to understand the prevalence of antibiotic resistant Gram negative bacilli producing various beta lactamases in hospital effluents. A total of 121 Gram negative bacilli were isolated and identified by standard biochemical tests from 31 hospital effluent samples. Antibiotic susceptibility profiling of isolated bacteria was assessed by Kirby Bauer disc method. Detection of various (ESBL, AmpC and MBL) producing isolates were further carried out by various methods. From the isolated bacteria, E.coli was predominant (37.19%) followed by Pseudomonas spp. (22.31%) Klebsiella spp. (19.83%) Non fermentative gram negative bacilli (NFGNB-10.74%), Enterobacter spp. (6.61%) and others (3.30 %). The present study suggests that although waste water treatment reduces the number of bacteria however, there is chance of antimicrobial resistant organisms in the hospital effluent. Hospitals should take sanitary measures to prevent the spread of multi drug resistant bacteria including beta-lactamases resistant strains transfer between hospital and the environment. The indiscriminate use of antibiotics in hospitals should be reduced

SEROPREVALENCE OF SCRUB TYPHUS AMONG FEBRILE PATIENTS – A PRELIMINARY STUDY

Aim: Scrub typhus is a vector borne zoonotic diseases re-emerging in India. But there are no reports from Andhra Pradesh till date due to low index of suspicion and lack of diagnostic facilities. This emphasizes the need for a study on this re-emerging Zoonosis. Methodology: A total of 280 sera samples with pyrexia of unknown origin were screened by using Weil-Felix test, rapid immunochromatographic method and ELISA. Result: Among the tested 280 febrile ill patients, 158 (56.42percent) was found to be positive by Weil-Felix test, 163 (58.21percent) was positive by ELISA and 160 (57.14percent) was positive by immunochromatographic test. Conclusion: By this study it can be inferred that awareness about vector borne zoonotic diseases is crucial. Clinical practice, epidemiological surveys and definite diagnostic tests have to be developed to study this neglected area, since this condition is very much prevalent in this part of the country. Keywords: Febrile illness; Spotted fever group; Typhus fever group; Scrub typhus; Weil-Felix test; Immunochromatographic test; ELISA

Multifaceted phytogenic silver nanoparticles by an insectivorous plant drosera spatulata Labill var. bakoensis and its potential therapeutic applications

The current investigation highlights the green synthesis of silver nanoparticles (AgNPs) by the insectivorous plant Drosera spatulata Labill var. bakoensis, which is the first of its kind. The biosynthesized nanoparticles revealed a UV visible surface plasmon resonance (SPR) band at 427 nm. The natural phytoconstituents which reduce the monovalent silver were identified by FTIR. The particle size of the Ds-AgNPs was detected by the Nanoparticle size analyzer confirms that the average size of nanoparticles was around 23 ± 2 nm. Ds-AgNPs exhibit high stability because of its high negative zeta potential (− 34.1 mV). AFM studies also revealed that the Ds-AgNPs were spherical in shape and average size ranges from 10 to 20 ± 5 nm. TEM analysis also revealed that the average size of Ds-AgNPs was also around 21 ± 4 nm and the shape is roughly spherical and well dispersed. The crystal nature of Ds-AgNPs was detected as a face-centered cube by the XRD analysis. Furthermore, studies on antibacterial and antifungal activities manifested outstanding antimicrobial activities of Ds-AgNPs compared with standard antibiotic Amoxyclav. In addition, demonstration of superior free radical scavenging efficacy coupled with potential in vitro cytotoxic significance on Human colon cancer cell lines (HT-29) suggests that the Ds-AgNPs attain excellent multifunctional therapeutic applications

Original Inventions based on Chemical scaffolds and electro-physical activity-derived biosimilars interacting with specialties in biology yielding platforms for analysis in virology and antiviral compounds

Background: Original inventions in developing countries, in terms of number of patents filed, granted and that are taken to useful applications as well as in terms of publications of high impact remain relatively lower [1,2] compared to that of developed nations. The reasons could be attributed to lack of importance given to basic research in funding, the number of institutes involved, limited technical support or expertise available etc [1]. Though such initiatives may take a long time to yield fruits, one of the parallel steps we considered worth was to take the original inventions from Japan, born out of basic research in one field, taken through an application-oriented inter disciplinary interactive research in healthcare, thereby paving way for novel solutions. Thus was conceived, the Inventions- Inter-Disciplinary Interactions and Solutions (IIDIAS), an academic session as a part of the one-day International stem cell meet organized every year in the month of October by Nichi-In Centre for Regenerative Medicine (NCRM), an academic Institute based in Chennai, India. In the IIDIAS session, based on original invention(s) presented as a prelude in brief, original interdisciplinary interactive research work based on the original invention by NCRM and/or its collaborators are presented by the faculty of the relevant institute. That will be followed by an interactive session in which the potential solutions based on the above accomplishments would be discussed. The IIDIAS session 2014 was based on the following two inventions: A bio-film-based biosimilar invented by an electro-physicist A unique polymer invented by a chemical engineer Inventions and Interdisciplinary Interactions: Invention –I: The bio-film-based biosimilar invented by an electro-physicist: An electro physicist with the Kyoto University, Japan, Dr Nobuyuki Yamaji observed during his experiments that plants and mammalian tissues secrete a layer of fluid after getting hit by lightning or electric current [3]. The significance of the secretion was unknown to him. Later when he met his friend, Dr. Sunao Kubota, who was a physician and professor of General Surgery in St. Marianna University, he shared his work and Dr. Kubota began analyzing the nature of the secretions. Dr. Kubota hypothesized that the secretion's role may be to provide a temporary barrier to the entry of pathogens through the area of skin dehiscence caused by the electrical damage, until healing process helps to cover the dehiscence. Then for the next decade Yamaji and Kubota worked to isolate the ingredients of the secretion and they together developed a citric acid-based biosimilar capable of killing a wide range of pathogens which are known to cause common as well as severe infections including nosocomial infections such as MRSA, VRE etc [4] without having to use alcohol-based disinfectants. This biosimilar named as Clinister, had the ability to kill even H5N1 influenza virus and is used in Japan as a spray disinfectant in public places [3]. Inter-Disciplinary-Interaction based on the Invention-I: Evaluation of citric acid-based granules in controlling Chickungunya Virus‏: Clinister has not only been proven as a disinfectant but is also a powerful anti-bacterial and anti-fungal agent that strongly restricts the recurrence of the microbes for a long term. In the present study, Clinister was evaluated for its virostatic efficacy against Chikungunya virus (CHIKV) in vitro for the first time. It showed expressive effect on virus grown in BHK cell cultures by inhibiting the development of virus induced cytopathic effect at an effective concentration of 1.5mg/ml in comparison to 70% ethanol, a common disinfectant. The effects on the virus were further checked by Polymerase Chain Reaction (PCR) at different regular post infective intervals from 0 to 72 hrs. The observations suggested that, other than the anti-microbial activity, Clinister also have specific effect on viruses that may cater the need to prevent the spreading of contagious viruses during epidemics which may occur in the future. However it is essential to study the specific targets of Clinister in viruses at a molecular level, and the same is under progress. Invention – II : A unique polymer invented by a chemical engineer A chemical engineer Prof. Yuichi Mori jointly with polymer scientist Dr Hiroshi Yoshioka developed a thermoreversible polymer (TGP) hydrogel composed of thermoresponsive polymer block [poly (Nisopropylacrylamide- co-n-butyl methacrylate) (poly NIPAAm-co-BMA)] and the hydrophilic polymer block [polyethylene glycol (PEG)] (commercial name :Mebiol gel) [5]. This Mebiol gel provides a suitable in vitro environment enabling culture expansion of cells in the lab without the use of biological components such as amniotic membrane or feeder layers. TGP has been earlier employed for the three-dimensional culture of many cell types like corneal limbal stem cells [6], chondrocytes [7], embryonic stem cells [8], hepatocytes [9], induced pluripotent stem (iPS) cells [8] and bone marrow mononuclear cells [10]. TGP has been used for transportation of corneal endothelial precursor cells over long distances without cool preservation [11], for micro-encapsulation of islet cells [12] and as a wound dressing [13]. TGP has been intralesionally applied along with stem cells in an animal model of spinal cord in injury [10]. TGP is a unique polymer capable of maintaining stem cells in an undifferentiated manner for a longer period of time [14]. It does not affect the gene expression profile [15] and the karyotype of the cells is maintained even after long term culture [16]. The safety and efficacy of TGP as a valuable scaffold material has been established in the various in vitro and translational studies [5]. Inter-Disciplinary-Interaction based on the Invention-II: Mebiol Gel as a 3D scaffold for hepatocytes and viral replication Mebiol Gel was used as a 3D scaffold for growing hepatocyte cell line. Mebiol Gel based hepatocytes had better differentiation and the cells were susceptible to hepatitis C virus replication. The Mebiol Gel based 3D culture system can be used as a better cell culture system for viral studies. Potential Solutions: The works presented in the IIDIAS session have portrayed that original invention in any field when subjected to multi-disciplinary interaction after carefully evaluating the potentials can lead to novel solutions. Though the experts in various disciplines in the reported work have got to interact by chance or due to geographical proximity to each other, when a networking platform to throw such ideas to a forum with mutually rewarding opportunities given for discussion and planning of interactive researches is made possible, novel solutions may not be impossible, if the target platform is well understood within the limitations of each stake holder. Creation of a niche or a suitable environment for such multi disciplinary interaction may prevail and work to its best in a physical state of interaction but advances in information technology has broken the need for such physical interaction among peers to bring out novel solutions. Interactions across specialties in the IIDIAS session help in identifying roles for the invention in other fields which may not be directly related to healthcare. The probable solutions and advantages by combining the electro-physical activity-derived biosimilar and the chemically synthesized polymer described in the study include: A novel platform for in vitro culture of HCV having been described, similar platforms for not so easily cultivable viruses can also be developed. The 3D viral culture technology can be exploited to study the replication process and other intricacies of the virus in in vitro culture. When in vitro expansion of viruses in an appropriate manner becomes feasible, anti-viral agents, either virostatic or virucidal, herbal or synthetic biosimilars can be analysed to come out with effective anti viral agents. A biosimilar-based compound having been proven against Chickungunya virus, the same could be tried against life threatening viruses in appropriate and safe environments. Clinister being a food additive-based material, is considered to be safe to human beings [4] and hence its inclusion as an additive to cell culture medium could be studied further in order to check whether the use of common antibiotics and anti-fungals which may be detrimental to cell growth can be avoided yielding safer anti-bacterial and anti-viral agents for cell culture. Conclusion: Multidisciplinary interaction within and across various domains of science has several potentials in bringing out novel solutions and this has been proven by this study of interaction among physicists, chemists and biologists. The authors recommend that establishment of barrier-free interaction platforms within and among institutes and various specialties at varied stages of research activities as portrayed here like the IIDIAS session to bring about novel solutions such as the ones described in this article. References Sumathipala A, Siribaddana S, Patel V. Under-representation of developing countries in the research literature: ethical issues arising from a survey of five leading medical journals. BMC Med Ethics. 2004;5: E5. The World Bank (2008). Global Economic Prospects 2008: Technology Diffusion in the Developing World. Washington, DC: The World Bank. Retrieved February 18, 2008 from http://siteresources.worldbank.org/INTGEP2008/Resources/complete-report.pdf Abraham S. The "Electric Biology" Duo. Trade Secrets- Nature Biotechnology Blog 2011. Full text can be accessed at http://blogs.nature.com/tradesecrets/2011/12/06/the-electric-biology-duo. Kubota S, Matsuzawa K, Wada M, Yamaji N. Bactericidal Effect of a Disinfectant (Bio Io Nurse) Prepared by Adding Citric Acid and Low Concentration Alcohol to Strongly Acidic Electrolyzed Water. New Food Industry 2008; 50:9. Dedeepiya VD, William JB, Parthiban JKBC, Yoshioka H, Mori Y, Kuroda S, Iwasaki M, Preethy S, Abraham SJK. Scaffolds for Cell Transplantation in Neurology: The Suitability of a Thermoreversible Gelation Polymer—Our Perspectives. Journal of Spinal Surgery 2014;1(1). Sitalakshmi G, Sudha B, Madhavan HN, Vinay S, Krishnakumar S, Mori Y, Yoshioka H, Abraham S. Ex Vivo Cultivation of Corneal Limbal Epithelial Cells in a Thermoreversible Polymer (Mebiol Gel) and Their Transplantation in Rabbits: An Animal Model. Tissue Eng Part A. 2009;15(2):407-15. Yasuda A, Kojima K, Tinsley KW, Yoshioka H, Mori Y, Vacanti CA. In vitro culture of chondrocytes in a novel thermoreversible gelation polymer scaffold containing growth factors. Tissue Eng. 2006;12(5):1237-45. Kataoka K, Huh N. Application of a Thermo-Reversible Gelation Polymer, Mebiol Gel, for Stem Cell Culture and Regenerative Medicine. J Stem Cells Regen Med. 2010 ;6(1); 10-14. Parveen N, Khan AA, Baskar S, Habeeb MA, Babu R, Abraham S, Yoshioka H, Mori Y, Mohammed HC. Intraperitoneal Transplantation of Hepatocytes Embedded in Thermoreversible Gelation Polymer (Mebiol Gel) in Acute Liver Failure Rat Model. Hepatitis Monthly 2008; 8(4): 275-280. William BJ, Prabakaran R, Ayyappan S, Puskhinraj H, Rao D, Manjunath S, Thamaraikannan P, Dedeepiya VD, Kuroda S, Yoshioka H, Mori Y, Preethy, Abraham S. Functional Recovery of Spinal Cord Injury Following Application of Intralesional Bone Marrow Mononuclear Cells Embedded in Polymer Scaffold - Two Year Follow-up in a Canine. J Stem Cell Res Ther.2011; 1:110. Rao SK, Sudhakar J, Parikumar P, Natarajan S, Insaan A, Yoshioka H, Mori Y, Tsukahara S, Baskar S, Manjunath SR, Senthilkumar R, Thamaraikannan P, Srinivasan T, Preethy S, Abraham SJ. Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer. Indian J Ophthalmol. 2014;62(2):130-5. Shimizu S, Yamazaki M, Kubota S, Ozasa T, Moriya H, Kobayashi K, Mikami M, Mori Y, Yamaguchi S. In vitro studies on a new method for islet microencapsulation using a thermoreversible gelation polymer, N-isopropylacrylamide-based copolymer. Artif Organs. 1996;20(11):1232-7. Yoshioka H, Mori Y, Kubota S. Wound dressing of newly developed thermogelling thermoreversible hydrogel. Jpn J Artif Organs. 1998; 27: 503-506. Arumugam S, Manjunath S , Senthilkumar R, Srinivasan V, Rajendiran S, Yoshioka H, Mori Y, Abraham S. In vitro expansion and characterization of human chondrocytes using a novel Thermoreversible Gelation Polymer (TGP). Journal of orthopaedics. 2011; 8(3); 1-10. Hishikawa K, Miura S, Marumo T, Yoshioka H, Mori Y, Takato T, Fujita T. Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer. Biochem Biophys Res Commun. 2004;317(4):1103-7. Lei Y, Schaffer DV. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci U S A. 2013;110(52):E5039-48