Download rajiv gandhi university of health sciences karnataka, bangalore

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.
7
MATERIALS AND METHODS
7.1
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.
7.2 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
+
- Non Citrobacter species
Citrobacter species
Arginine dihydrolase
-
+
Citrobacter species
Non Citrobacter species
H2S (BY TSI METHOD)
-
+
Other Citrobacter species
Indole
+
C.koseri
C.amalonaticus
malonate
+
C.koseri
Citrobacter freundii complex
C.sedlakii C.freundii C.werkmanii C.youngae C.braakii
C.freundii complex
C.sedlakii C.freundii C.werkmanii C.youngae C.braakii
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
Ampicillin
Amikacin
Cefazolin
Cefaperazone
Cefotaxime
Ceftazidime
Cefuroxime
Ciprofloxacin
Cotrimoxazole
Gentamicin
Imipenem / Meropenem
Netilmicin
Piperacillin
Piperacillin / Tazobactam
DISC STRENGTH (mcg)
10 ( Himedia )
30 ( Himedia )
30 ( Himedia )
75 ( Himedia )
30 ( Himedia )
30 ( Himedia )
30 ( Himedia )
5 ( Himedia )
1.25 / 23.75 (Himedia)
10 ( Himedia )
10 ( Oxoid )
30 ( Himedia )
100 ( Himedia )
100/10 (Himedia )
5.Extended Spectrum β lactamases :ESBL enables bacteria to become resistant to newer cephalosporins. Betalactamase 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 : o 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.
o 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
8
LIST OF REFERENCES:1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. B Thapa , P Adhikari , K Mahat , MR Chhetri and LN Joshi ; Multidrugresistant nosocomial citrobacter in a hospital kathmandu : Nepal Med coll J
2009;11(3):195-199.
7. 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.
8. 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.
9. 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.
10.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.
11.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.
9
Signature of candidate
:
10
Remarks of guide
:
11
Name and designation of :
12
11.1 Guide
:
11.2 Signature
:
11.3 Co-guide
(if any)
:
11.4 Signature
:
11.5 Head of the
Department
:
11.6 Signature
:
This study can be done in the Department
Dr. Savitha Nagaraj M.D (microbiologist)
St.John’s Medical College
Bangalore.
NIL
Dr. Muralidharan S. , M.D(microbiologist)
St.John’s Medical College
Bangalore.
12.1 Remarks of the Chairman & Principal :
12.2 Signature
: