RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES …



RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

ANNEXURE –II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

| | | |

|1 |Name of the candidate and address |Harish .H |

| | |M.Sc MLT ,1st year St.John’s Medical college ,Bangalore|

| | | |

|2 |Name of the Institution |St.John’s Medical College, Bangalore |

| | | |

|3 |Course of study and subject |M.Sc MLT(Microbiology) |

| | | |

|4 |Date of Admission to course |05-09-2011 |

| | |

|5 |Title of the topic |

| |Title: Biochemical identification of Citrobacter species &their antibiotic susceptibility pattern from clinical samples. |

| | |

|6 |BRIEF RESUME OF THE INTENDED WORK : |

| | |

| |6.1 NEED FOR STUDY |

| | |

| |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. |

| | |

| | |

| |6.2 REVIEW OF LITERATURE |

| | |

| |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 . |

| | |

| | |

| | |

| | |

| |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.. |

| | |

| |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. |

| | |

| |MATERIALS AND METHODS |

| | |

| |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 | |

| | |

| |7.2 Inclusion Criteria :- All isolates suspected to be Citrobacter will be included . |

| | |

| |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. |

| | |

| |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. |

| | |

| | |

| |IDENTIFICATION:- |

| | |

| |PRELIMINARY IDENTIFICATION BY FOLLOWING TESTS 4 |

| | |

| |M M T P C U M M T P C U |

| | |

| |+ + +/+ + + +/+ + + +/+ - + +/- |

| |H2S |

| |Presumptive Citrobacter freundii Complex Presumptive Citrobacter Species |

| | |

| | |

| |Ornithine decarboxylase |

| | |

| |+ Citrobacter species - Non Citrobacter species |

| | |

| |Arginine dihydrolase |

| | |

| |+ - |

| |Citrobacter species Non Citrobacter species |

| | |

| | |

| |H2S (BY TSI METHOD) |

| | |

| |- + |

| |Other Citrobacter species Citrobacter freundii complex |

| | |

| | |

| |Indole C.sedlakii C.freundii C.werkmanii C.youngae C.braakii |

| | |

| |+ - |

| |C.koseri C.freundii complex |

| |C.amalonaticus |

| | |

| |malonate C.sedlakii C.freundii C.werkmanii C.youngae C.braakii |

| | |

| |+ - |

| |C.koseri C.amalonaticus |

| | |

| | |

| | |

| |ADDITIONAL BIOCHEMICAL TESTS : - |

| | |

| |1% Sucrose Fermentation. |

| |1% Dulcitol Fermentation. |

| |1% Melibiose Fermentation. |

| |1% Salicin Fermentation. |

| | |

| |Methods1. |

| | |

| |1.ARGININE DEHYDROLASE : - |

| | |

| |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 |

| | |

| |2.ORNITHINE DECARBOXYLASE : - |

| | |

| |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. |

| | |

| | |

| | |

| | |

| | |

| |3.Sugar Fermentation :- |

| | |

| |The carbohydrate to be tested is first sterilized and added aseptically to basal medium to final concentration of 0.5 - 1%. |

| | |

| |The formula of typical basal medium contains |

| | |

| |Trypticase ( BBL) - 10gm |

| |Sodium chloride - 05gm |

| |Phenol red - 0.018gm |

| |Distilled water - 1 liter. |

| | |

| |4.ANTIBIOTIC SUSCEPTIBILITY TEST 9: - |

| | |

| |Antibiotic susceptibility will be done according to central laboratory standard institute (CLSI) by Kirby – Bauer Disc diffusion Method. |

| | |

| |ANTIBIOTIC |

| |DISC STRENGTH (mcg) |

| | |

| |Ampicillin |

| |10 ( Himedia ) |

| | |

| |Amikacin |

| |30 ( Himedia ) |

| | |

| |Cefazolin |

| |30 ( Himedia ) |

| | |

| |Cefaperazone |

| |75 ( Himedia ) |

| | |

| |Cefotaxime |

| |30 ( Himedia ) |

| | |

| |Ceftazidime |

| |30 ( Himedia ) |

| | |

| |Cefuroxime |

| |30 ( Himedia ) |

| | |

| |Ciprofloxacin |

| |5 ( Himedia ) |

| | |

| |Cotrimoxazole |

| |1.25 / 23.75 (Himedia) |

| | |

| |Gentamicin |

| |10 ( Himedia ) |

| | |

| |Imipenem / Meropenem |

| |10 ( Oxoid ) |

| | |

| |Netilmicin |

| |30 ( Himedia ) |

| | |

| |Piperacillin |

| |100 ( Himedia ) |

| | |

| |Piperacillin / Tazobactam |

| |100/10 (Himedia ) |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| |5.Extended Spectrum β lactamases :- |

| | |

| |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. |

| | |

| |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). |

| | |

| |Ceftazidime - 30mcg |

| |Ceftazidime + Clavulinic acid |

| |Cefotaxime - 30mcg |

| |Cefotaxime + Clavulinic acid |

| | |

| |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. |

| | |

| |No, only samples received in the laboratory will be studied. |

| | |

| |7.6 Has ethical clearance been obtained from your in case of 7.5 |

| | |

| |Not applicable |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| |LIST OF REFERENCES:- |

| | |

| |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. |

| | |

| |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. |

| | |

|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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| |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. |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| |Signature of candidate : |

| | |

| | |

| |Remarks of guide : This study can be done in the Department |

| | |

| | |

| |Name and designation of : |

| | |

| |11.1 Guide : Dr. Savitha Nagaraj M.D (microbiologist) |

| |St.John’s Medical College |

| |Bangalore. |

| | |

| |11.2 Signature : |

| | |

| | |

| |11.3 Co-guide : NIL |

| |(if any) |

| | |

| |11.4 Signature : |

| | |

| | |

| |11.5 Head of the : Dr. Muralidharan S. , M.D(microbiologist) |

| |Department St.John’s Medical College |

| |Bangalore. |

| | |

| |11.6 Signature : |

| | |

| | |

| |12.1 Remarks of the Chairman & Principal : |

| | |

| | |

| | |

| |12.2 Signature : |

| | |

| | |

| | |

|9 | |

| | |

| | |

| | |

|10 | |

| | |

| | |

|11 | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

|12 | |

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download