RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES …
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
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|1 |Name of the candidate and address |Harish .H |
| | |M.Sc MLT ,1st year St.John’s Medical college ,Bangalore|
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|2 |Name of the Institution |St.John’s Medical College, Bangalore |
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|3 |Course of study and subject |M.Sc MLT(Microbiology) |
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|4 |Date of Admission to course |05-09-2011 |
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|5 |Title of the topic |
| |Title: Biochemical identification of Citrobacter species &their antibiotic susceptibility pattern from clinical samples. |
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|6 |BRIEF RESUME OF THE INTENDED WORK : |
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| |6.1 NEED FOR STUDY |
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| |Citrobacter species are known to cause urinary tract infections and wound infections and are implicated as an occasional cause of |
| |gastroenteritis particularly in infants and young children.1 |
| |Citrobacter species need to be identified to species level as they could be confused with other genera belonging to the Enterobacteriaceae |
| |family2. |
| |Speciation is important to know the prevalence of various species from clinical samples. |
| |Antibiotic susceptibility pattern of these organisms is also important for treatment and epidemiological purpose. |
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| |6.2 REVIEW OF LITERATURE |
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| |Members of genus Citrobacter are named for their ability to use citrate as their sole carbon source. Of the approximate dozen species |
| |Citrobacter freundii, Citrobacter koseri & Citrobacter amalonaticus are linked to human disease3. |
| |In human beings Citrobacter species cause significant morbidity and mortality and cause a variety of infectious process ranging from urinary |
| |tract and wound infections to more invasive diseases including septicemia and neonatal meningitis4. |
| |Members of genus Citrobacter are often found in feces of humans and may be isolated from variety of clinical specimens5 . Citrobacter species|
| |are implicated as an occasional cause of gastroenteritis particularly in infants and young children1. |
| |Among approximate 11 different species under this genus, Citrobacter freundii and Citrobacter koseri are the major species implicated in |
| |infections. They do not often give rise to serious infections except Citrobacter koseri , which has been responsible for several outbreaks of|
| |neonatal meningitis 5 ,6. |
| |Citrobacter koseri is usually resistant to Ampicillin and Ticarcillin and Citrobacter freundii is usually resistant to Ampicillin and first |
| |generation cephalosporins1. |
| |In addition like members of other genera, isolates of Citrobacter may be resistant to many classes of antibiotics as a result of plasmid |
| |encoded resistant genes 1, 7 . |
| |Citrobacter species are known to harbour Extended Spectrum β lactamase |
| |(ESBL), and AmpC Genes on both chromosome and plasmid 6,8 . |
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| |Studies done on urine isolates of Citrobacter showed 30% ESBL production among them by modified double disc method. Another study in North |
| |India demonstrated 62% ESBL production among the isolates. 4,8.. |
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| |6.3 OBJECTIVES OF THE STUDY |
| |Speciation of Citrobacter isolated from clinical samples. |
| |To study the antibiotic susceptibility pattern by Kirby – Bauer Disc diffusion Method as per CLSI Guidelines 20119. |
| |To determine the presence of Extended Spectrum β lactamase (ESBL), in these isolates. |
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| |MATERIALS AND METHODS |
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| |Clinical samples sent to the microbiology laboratory of St.John’s Medical |
| |College and Hospital growing Citrobacter will be included . |
| |Total 20 isolates of Citrobacter will be included in the study. |
| |These isolates will be sourced from all clinical samples like urine, pus, |
| |blood, sputum, stool and sterile fluids. |
|7 | |
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| |7.2 Inclusion Criteria :- All isolates suspected to be Citrobacter will be included . |
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| |7.3 Exclusion Criteria :- All isolates presumptively identified as Citrobacter species and do not conform to Citrobacter speciation and |
| |identification ,will not be included in further characterization. |
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| |7.4 Method |
| |The isolates that are biochemically identified as Citrobacter species will be further subjected to other biochemical tests for speciation and|
| |confirmation. Initial identification will be done by Mannitol, Motility (MM), Triple sugar iron (TSI), Indole (P), Citrate (C) and Urease |
| |(U). |
| |These isolates will be stored in Nutrient agar deeps at 40 C till further processing. |
| |Antibiotic susceptibility will be done for all by Kirby – Bauer Disc Diffusion Method, as per CLSI Guidelines 20119. |
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| |IDENTIFICATION:- |
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| |PRELIMINARY IDENTIFICATION BY FOLLOWING TESTS 4 |
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| |M M T P C U M M T P C U |
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| |+ + +/+ + + +/+ + + +/+ - + +/- |
| |H2S |
| |Presumptive Citrobacter freundii Complex Presumptive Citrobacter Species |
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| |Ornithine decarboxylase |
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| |+ Citrobacter species - Non Citrobacter species |
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| |Arginine dihydrolase |
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| |+ - |
| |Citrobacter species Non Citrobacter species |
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| |H2S (BY TSI METHOD) |
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| |- + |
| |Other Citrobacter species Citrobacter freundii complex |
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| |Indole C.sedlakii C.freundii C.werkmanii C.youngae C.braakii |
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| |+ - |
| |C.koseri C.freundii complex |
| |C.amalonaticus |
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| |malonate C.sedlakii C.freundii C.werkmanii C.youngae C.braakii |
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| |+ - |
| |C.koseri C.amalonaticus |
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| |ADDITIONAL BIOCHEMICAL TESTS : - |
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| |1% Sucrose Fermentation. |
| |1% Dulcitol Fermentation. |
| |1% Melibiose Fermentation. |
| |1% Salicin Fermentation. |
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| |Methods1. |
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| |1.ARGININE DEHYDROLASE : - |
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| |Many species of bacteria possess enzymes capable of decarboxylating specific amino acid in the test medium. |
| |The decarboxylase enzyme removes a molecule of CO2 from amino acid to form alkaline reacting amines. |
| |The decarboxylase activity of Enterobacteriaceae is most commonly measured in clinical microbiology laboratory with a Moller decarboxylase |
| |broth. |
| |The end point of reaction is production of alkaline pH shift in medium and development of blue purple colour after incubation with test |
| |organism. |
| |Pyridoxal phosphate is included in test medium and acts as coenzyme to further enhance of decarboxylase activity. |
| |Appropriate controls will be used |
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| |2.ORNITHINE DECARBOXYLASE : - |
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| |A tube of decarboxylase base added with ornithine are inoculated heavily and overlaid with sterile mineral oil. |
| |On incubation at 350 c both tubes will turn from pale gray to yellow as glucose is fermented. |
| |Following this the Ornithine in the test tube will be decarboxylated resulting in Ornithine broth turning a violet colour. |
| |Although most positive tests may be detected within 6 – 8 hours. The test is read after 24 hours of incubation. |
| |Appropriate controls will be used. |
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| |3.Sugar Fermentation :- |
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| |The carbohydrate to be tested is first sterilized and added aseptically to basal medium to final concentration of 0.5 - 1%. |
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| |The formula of typical basal medium contains |
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| |Trypticase ( BBL) - 10gm |
| |Sodium chloride - 05gm |
| |Phenol red - 0.018gm |
| |Distilled water - 1 liter. |
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| |4.ANTIBIOTIC SUSCEPTIBILITY TEST 9: - |
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| |Antibiotic susceptibility will be done according to central laboratory standard institute (CLSI) by Kirby – Bauer Disc diffusion Method. |
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| |ANTIBIOTIC |
| |DISC STRENGTH (mcg) |
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| |Ampicillin |
| |10 ( Himedia ) |
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| |Amikacin |
| |30 ( Himedia ) |
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| |Cefazolin |
| |30 ( Himedia ) |
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| |Cefaperazone |
| |75 ( Himedia ) |
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| |Cefotaxime |
| |30 ( Himedia ) |
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| |Ceftazidime |
| |30 ( Himedia ) |
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| |Cefuroxime |
| |30 ( Himedia ) |
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| |Ciprofloxacin |
| |5 ( Himedia ) |
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| |Cotrimoxazole |
| |1.25 / 23.75 (Himedia) |
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| |Gentamicin |
| |10 ( Himedia ) |
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| |Imipenem / Meropenem |
| |10 ( Oxoid ) |
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| |Netilmicin |
| |30 ( Himedia ) |
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| |Piperacillin |
| |100 ( Himedia ) |
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| |Piperacillin / Tazobactam |
| |100/10 (Himedia ) |
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| |5.Extended Spectrum β lactamases :- |
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| |ESBL enables bacteria to become resistant to newer cephalosporins. Beta- lactamase mediated resistance may be overcome by combining |
| |beta-lactam antibiotics with beta- lactamase inhibitors which bind irreversibly to the beta lactamases and render them inactive thus sparing |
| |the beta-lactam antibiotic9,10,11. |
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| |ESBL (Extended Spectrum β lactamase) DETECTION : - |
| |ESBL can be detected by combined disc test. |
| |Isolates showing resistance to third generation cephalosporins by disc diffusion. |
| |will be subjected to the combined disc test. |
| |A difference of 5 mm between the zone of the cephalosporins with and without clavulinic acid is taken as positive for ESBL.(Himedia discs). |
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| |Ceftazidime - 30mcg |
| |Ceftazidime + Clavulinic acid |
| |Cefotaxime - 30mcg |
| |Cefotaxime + Clavulinic acid |
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| |7.5 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please |
| |described briefly. |
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| |No, only samples received in the laboratory will be studied. |
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| |7.6 Has ethical clearance been obtained from your in case of 7.5 |
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| |Not applicable |
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| |LIST OF REFERENCES:- |
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| |Washington c.winn, Stephan. D, Allen, William. M.J, Elmer W, Koneman, Gray w.procop , Paul c.schreekenberger , Gill L.woods : |
| |Enterobacteriaceae: Konemans color atlas of diagnostic microbiology. 6th Edn, Lippin cotts Williams and wilkins. p211 – 302. |
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| |P B Crichton. Enterobacteriaceae, Escherichia, Klebsiella, Protus and other genera. In: collee JG, Fraser AG, marmion BP, Simmons A, Editor. |
| |Mackie and McCartney – Practical Medical Microbiology. 14th ed. India. Churchill Livingstone 2006:361-84. |
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|8 |Michael S.donnenberg . Enterobacteriaceae ; Principle and practice of infectious diseases, eds Gerald L.Mandel ,john E , Bennette , Raphael |
| |dolin , 6th edn, vol.2 , Elsevier Churchill living stone , Pennsylvania p2567 – 2586. |
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| |Michael Janda J,Sharon L. Abbott,Wendy.Cheung K.W, and Deborah F.Hanson: Biochemical Identification of Citrobacter in Clinical Laboratory. |
| |J.Clin. Microbiol. Aug 1994. p 1850-1854. |
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| |Patrick.R.Murray, Barry Holmes and Hazel M.Aucken ; Citrobacter Enterobacter, Klebsiella, Plesiomonas , Serratia and other members of |
| |Enterobacteriaceae ; Eds, S.Peter borriello , Patrick.R.Murray and Guido Funke , 10th Edn, vol 2 , Hodder Arnold p1474 – 1498. |
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| |B Thapa , P Adhikari , K Mahat , MR Chhetri and LN Joshi ; Multidrug-resistant nosocomial citrobacter in a hospital kathmandu : Nepal Med |
| |coll J 2009;11(3):195-199. |
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| |George samonis, DAH HIS HO, Grace F. Gooch , Kenneth V.I. Rolston, and Gerald P. Bodey : In vitro susceptibility of citrobacter species to |
| |various antimicrobial agents; Antimicrob. Agents chemother. May-1987, vol31:5.p.829-830. |
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| |Meher rizvi, Nazish Fathima, Indu sukla, Abida Malik; Epidemology of Extended Spectrum β lactamases in serratia and citrobacter species in |
| |North India. Indian journal of pathology and microbiology – 53(1), jan- mar 2010. |
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| |Clinical and Laboratory Standards Institute .Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. |
| |M100-S20 Vol.30. No 1.Approaved Standard, Wayne, Pennsylvnia, 2011. |
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| |Shobha k.L, Gowrish Rao.S, Sugandhi Rao, Sreeja C.K: Prevalence of extendend spectrum Beta-Lactamases in Urinary isolates of Escherichia |
| |coli, Klebsiella and Citrobacter species and their antimicrobial susceptibility pattern in Tertiary care hospital. Indian Journal of the |
| |Practicing Doctor 2007; 3:1-2. |
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| |Srujan Mohanty, Ritu Singhal, Seema Sood,Benu Dhawan, Bimal K.Das & Arti Kapil; Comparitive in vitro activity of beta-lactum/beta-lactamase |
| |inhibitor combinations against Gram negative bacteria; Indian J Med Res 122, Nov 2005, p 425-428. |
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| |Signature of candidate : |
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| |Remarks of guide : This study can be done in the Department |
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| |Name and designation of : |
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| |11.1 Guide : Dr. Savitha Nagaraj M.D (microbiologist) |
| |St.John’s Medical College |
| |Bangalore. |
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| |11.2 Signature : |
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| |11.3 Co-guide : NIL |
| |(if any) |
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| |11.4 Signature : |
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| |11.5 Head of the : Dr. Muralidharan S. , M.D(microbiologist) |
| |Department St.John’s Medical College |
| |Bangalore. |
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| |11.6 Signature : |
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| |12.1 Remarks of the Chairman & Principal : |
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| |12.2 Signature : |
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