RAJIVGANDHIUniversitYOFHEALTH SCIENCES
KARNATAKA,BANGALORE
ANNEXURE-II SYNOPSISFORREGISTRATIONOF SUBJECTS FOR DISSERTATION
1. / NAME OF THE CANDIDATE AND ADDRESS [IN BLOCK LETTERS] / DR.VIJAYA.SPOST GRADUATE STUDENT
DEPT OF MICROBIOLOGY
NAVODAYAMEDICALCOLLEGE
RAICHUR.
2 / NAME OF THE INSTITUTION / NAVODAYAMEDICALCOLLEGE,
RAICHUR-584103
3 / COURSE OF STUDY & SUBJECT / MD IN MICROBIOLOGY
4 / DATE OF ADMISSION TO THE COURSE / 24TH MAY 2010
5 / TITLE OF THE TOPIC / OCCURRENCE AND DETECTION OF AMP C BETA LACTAMASES AMONG GRAM NEGATIVE CLINICAL ISOLATES AT NAVODAYA MEDICAL COLLEGE HOSPITAL AND RESEARCH CENTRE, RAICHUR.
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/ BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY:
Amp C Beta Lactamases are Group I cephalosporinases according to Bush- Jacoby Medeiros system and belong to class C β lactamases in Ambler system1.They confer resistance to a wide variety of β-lactam antibiotics including alpha methoxy β-lactams [cephamycins] such as cefoxitin and cefotetan, narrow and broad spectrum cephalosporins, aztreonam and are poorly inhibited by β lactamase inhibitors such as clavulanic acid 2 .
They are usually chromosomally encoded Amp C enzymes in several bacterial species belonging to the family Enterobacteriaceae, including Enterobactercloacae, Enterobacteraerogenes, Citrobacter freundii, Morganella morganii, Serratia marcescens and Escherichiacoli, which are frequently encountered as nosocomial pathogens. Since the first report of transferable plasmid mediated class C β lactamases, such as MIR-1 in the late 1980s, their increasing presence worldwide is of great concern3.
Plasmid mediated AmpC β lactamases differ from chromosomal AmpCs in being uninducible and are typically associated with broad multidrug resistance. Many clinical laboratories are not fully aware of the importance of Plasmid mediated AmpC β lactamases. This lack of understanding is responsible for a continuing failure to prevent the rapid world wide dissemination of pathogens possessing these β lactamases. Prevalence of this resistance mechanism appears to be increasing and has been responsible for nosocomial outbreaks of multidrug resistant Gram negative pathogens that require expensive control efforts4.
Detecting Amp C isolates is clinically important, not only because of their broader cephalosporin resistance, but also because carbapenem resistance can arise in such strains by further mutations, resulting in reduced porin expression5.
Current detection methods for organisms producing plasmid mediated Amp C β lactamases are technically demanding for clinical laboratories to perform on a routine basis. As a result, organisms producing these types of β lactamases often go undetected and therefore have been responsible for several nosocomial outbreaks. The detection of organisms producing these β lactamases is thus important for enhanced infection control and to ensure effective therapeutic options6.
The present study has been undertaken to determine the occurrence of Amp C enzyme producing Gram negative clinical isolates at NavodayaMedicalCollegeHospital and Research Centre, Raichur
6.2 REVIEW OF LITERATURE:
- Philip E Coudron, Ellen S Moland and Kenneth S Thomson (2000) tested a total of 1,286 consecutive, non-repeat isolates of Escherichiacoli, Klebsiellapneumoniae and Proteus mirabilisspecies and found that 45(3.5%) yielded a cefoxitin zone diameter less than 18mm (screen positive) and 16(1.2%) demonstrated Amp C bands by isoelectric focusing. Based on the species, of 683 E.coli, 371 K.pneumoniae and 232 P.mirabilis isolates tested, 13(1.9%), 28(7.6%) and 4(1.7%) respectively demonstrated decreased zone diameters and 11(1.6%), 4(1.1%) and 1(0.4%) respectively, demonstrated Amp C bands. Out of the 45 cefoxitin resistant isolates, the three dimensional extract test accurately identified all 16 Amp C producers and 28 of 29(97%) isolates as non-Amp C producers. Most isolates (86%) in the latter group were K.pneumoniae isolates. These data confirms that E.coli, K.pneumoniae and P.mirabilisharbor plasmid mediated Amp C enzymes7.
- Vikas Manchanda and Narendra P.Singh in their study( 2003) showed that among the 135 clinical isolates of Gram negative bacilli, 20.7% harboured Amp C enzymes using a modified three dimensional test. Maximal incidence was found among Acinetobacter spp.(42.8%) followed by Klebsiella pneumoniae isolates(33.3%).No Amp C harbouring isolates revealed decreased susceptibility to cefoxitin8.
- Shahid.M et al (2004) have compared conventional three dimensionalextract test with other modified procedures. All the methods gave positive results, but the best resultswere given by the spot inoculation method. It is simple, cost-effective,16 isolates may be tested on a single culture plate. Thus it is a less labour intensive and more economic technique than other reported phenotypic methods9.
- S Singhal et al(2005) screened a total of 272 isolates for Amp C β lactamases by modified double disk approximation method(MDDM) and further confirmed by three dimensional extract method and Amp C disc test. A total of 61(23%) showed resistance to cefoxitin. The occurrence of Amp C β lactamases was found to be 8%(22) of the total isolates and the two detection methods for Amp C β lactamase showed concordant results. The confirmation for Amp C β lactamase can be carried out routinely in tertiary care hospitals by Amp C disc test, as it is a simple and rapid procedure10.
- Philip.E.Coudron (2005) evaluated non-beta lactam inhibitor based methods for detecting plasmid mediated Amp C β lactamases in Klebsiella spp. Escherichia coli and Proteus mirabilis. Using CLSI methodology and disks containing cefotetan alone and in combination with 400μg of boronic acid, 9 of 10 positive control strains and 54 of 55 Amp C. PCR positive clinical isolates were detected11.
- Suranjana Arora and Manjusri Bal(2005) finding showed 6.7% Amp C β lactamase and 1.4% inducible Amp C β lactamase producing clinical isolates from Kolkata12.
- Parul Sinha et al (2008) subjected a total of 250 E.coli isolates to Amp C disc test to detect Amp C β lactamases producing strains and found that the prevalence of Amp C β lactamase producers were 24%(60/250)13.
1. To study the antibiotic susceptibility patterns of Gram negative clinical isolates.
2. To screen the AmpC beta lactamase producing Gram negative isolates.
3. To detect the AmpC β lactamase producing isolates by phenotypic confirmatory test.
4. To evaluate other simple methods to detect AmpC β lactamases and compare with
the phenotypic confirmatory method.
MATERIALS AND METHODS :
7.1 SOURCE OF DATA:
It is a prospective study of about 200 consecutive, non-repeat Gram negative isolates from different clinical samples (Urine, pus, sputum and other samples) in the Dept. of Microbiology, NavodayaMedicalCollegeHospital and Research Centre, Raichur from Dec 2010 toNov 2011.
7.2 METHODS OF COLLECTION OF DATA:
- Various clinical samples like urine, pus, sputum, blood etc. arecollected from patients
- The samples will be processed and all the Gram negative bacteria isolated and identified by Standard laboratory procedures14.
- Antibiotic susceptibility tests will be done by Modified Kirby bauer disk diffusion method according to CLSI guidelines15.
- Screening the Gram negative isolates for AmpC β lactamases by Modified disc Approximation method using cefoxitin(30μg), ceftazidime(30μg) and cefotaxime(30μg) discs. The isolates showing reduced susceptibility to ceftazidime/cefotaximeand cefoxitin or showing blunting of ceftazidime or cefotaxime zone of inhibition adjacent to cefoxitin (Inducible Amp C) will be considered as “Screen positive”10.
- The “Screen positive” isolates will be detected for AmpC β lactamase by Modified three dimensional enzyme extract test [Phenotypic confirmatory test]. The crudeenzyme extracts will be prepared by freeze-thawing centrifuged cell pellets of broth cultures. It will then be inoculated into wells on a Mueller-Hinton agar(MHA)plate lawn cultured with E.coli ATCC 25922 and a cefoxitin disc will be placed onit. Linear slit is made from the well directing towards the disc, 3mm away from it. The plates will be read after overnight incubation at 37ºC. Any enhanced growth of the test strain showing disruption of the cefoxitin inhibition zone at the end-point of the slit, will be considered as positive for AmpC β lactamases5.
- AmpC disc test will be done to detect AmpC β lactamases. A lawn culture of E.coliATCC 25922 will be prepared on MHA plate. Sterile discs (6mm) is moistened with sterile saline and inoculated with several colonies of the test organisms. This disc will be placed beside a cefoxitin disc on the inoculated plate.The plates will be incubated overnight at 37ºC, aerobically. A positive test will be indicated as flattening or indentation of cefoxitin inhibition zone near the test disc13.
- Phenyl boronic acid (PBA) disc enhancement method will be done and compared with three dimensional enzyme extract test as a gold standard. Here two cefoxitin discs(30μg) is placed on a MHA plate lawn cultured with a 0.5McFarlandturbidity adjusted suspension of the test strain. To one of the discs 400μg of PBA is added.After overnight incubation in ambient air at 37ºC, the zones of inhibition is measured. Enhancement of 5mm of zone around a cefoxitin disc with PBA, in comparison with a disc with cefoxitin alone is taken as a positive reaction for AmpC production5.
7.3 Does the study require any investigations to be conducted on patients or
Animals? Specify.
The study involves collection of clinical samples from patients. It does not require any
other invasive procedures. No animal experiments required for the study.
7.4 Has the ethical committee clearance been obtained from your institution ?
Yes.
LIST OF REFERENCES :
- Rice LB, Bonomo RA.Mechanisms of Resistance to Antibacterial Agents,Chapter 71.In:Murray PR , Baron EJ, Jorgensen JH, Landry ML, Pfaller MA,editors. Manual of Clinical Microbiology, 9th Edition;volume 1. ASM Press;Washington,D.C, U.S.A : 2007.p.1128-9.
- Subha A, Renuka V, Ananthan S. AmpC β lactamase producing multidrugresistant strains of Klebsiella spp.and Escherichia coli isolated from children under five in Chennai. Indian J Med Res 2003 Jan;117:13-18.
- Yagi T, Wachino J, Kurokawa H, Suzuki S, Yamane K, Doi Y et al.Practical Methods using Boronic acid compounds foridentification of class C β lactamase producing Klebsiella pneumoniae and Escherichiacoli.J Clin Microbiol 2005 June;43(6):2551-2558.
- Ratna AK, Menon I, Kapur I, Kulkarni R.Occurrence and detection of AmpC β lactamases at a referral Hospital in Karnataka.Indian J Med Res2003 July;118: 29-32.
- Vandana KE, Honnavar P.AmpC Beta Lactamases among ESBL producing Escherichia coli and Klebsiella pneumoniae. If you don’t Look, you won’tFind.Journal of Clinicaland Diagnostic Research. 2009 Aug;(3):1653-1656.
- Black JA, Thomson KS, Pitout JDD.Use of β Lactamase Inhibitors in Disk tests to detect Plasmid Mediated AmpC β Lactamases. J Clin Microbiol 2004 May;42(5): 2203-2206.
- Coudron PE, Moland ES,Thomson KS.Occurrence and Detection of AmpC Beta Lactamases among Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolates at a Veterans Medical Center. J Clin Microbiol 2000 May;38(5):1791-1796.
- Manchanda V, Singh NP.Occurrence and detection of AmpC β Lactamases among Gram negative clinical isolates using a Modified three dimensional test at Guru Tegh Bahadur Hospital,Delhi,India.J Antimicrob Chemother 2003; 51: 415-418.
- Shahid M, Malik A, Agrawal M , Singhal S. Phenotypic detection of extended spectrum and AmpC β Lactamases by a new spot-inoculation method and modified three dimensional extract test : Comparison with the conventional three dimensional extract test. J Antimicrob Chemother 2004; 54:684-687.
- Singhal S, Mathur T, Khan S, Upadhyay DJ, Chugh S, Gaind R, Rattan A. Evaluation of methods for AmpC β Lactamase in Gram Negative clinical isolates from tertiary care Hospitals . Indian J.Med Microbiol 2005; 23(2) :120-124.
- Coudron PE. Inhibitor Based methods for detection of plasmid mediated AmpC
Microbiol 2005 Aug ; 42(8): 4163-4167. 12.Arora S, Bal M. AmpC β-Lactamase producing bacterial isolates from Kolkata
Hospital. Indian J Med Res 2005 September;122:224-233.
13.Sinha P, Sharma R, Rishi S, Sood S, Pathak D. Prevalence of extended spectrum beta
lactamase and AmpC beta lactamase producers among Escherichia coli isolates in a tertiary care hospital in Jaipur. Indian J of Pathol Microbiol 2008;51(3): 367-369.
14.Collee JG, Miles RS, Watt B. Tests for the Identification of Bacteria. Chapter7. In:
Collee JG, Fraser AG, Marmion BP, Simmons A,editors. Mackie and McCartney
Practical Medical Microbiology.14th Edition. Elsevier;India:2006. p.131-149.
15.Clinical and Laboratory Standards Institute, Performance Standards for antimicrobial
susceptibility testing ;17th informational supplement. M100-S17(2007) ,M2-A9,Vol 27
No.1: p 32-38 (ISBN-I-56238-625-5), CLSI, 940 West ValleyRoad,Suite
1400,Wayne, Pennsylvania 19087-1898 USA,2007.
SIGNATURE OF THE CANDIDATE:
REMARKS OF THE GUIDE:
RECOMMENDED FOR REGISTRATION
NAME AND DESIGNATION OF:
11.1 GUIDE DR. ACHUT RAO
PROFESSOR AND HEAD,
DEPT. OF MICROBIOLOGY,
NAVODAYAMEDICAL COLLEGE,
RAICHUR.
11.2 SIGNATURE:
11.3 CO-GUIDE:(IF ANY)
11.4 SIGNATURE:
11.5 HEAD OF THE DEPARTMENT:
DR.ACHUT RAO
PROFESSOR AND HEAD,
DEPT. OF MICROBIOLOGY,
NAVODAYA MEDICAL COLLEGE,
RAICHUR.
11.6 SIGNATURE:
12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL
RECOMMENDED FOR REGISTRATION
12.2 SIGNATURE OF THE PRINCIPAL
References:
1. Park.k, park’s textbook of preventive and social medicine, 20th edition m:s,Banarsidas Bhanot ,publishers 2009,page 447-455.
2k. ishore j,j kishore’s national health programs of India 8th edition century publication 2009 page90.
3. Reproductive and child health programme
plan for maternal health and emoe.pdf accessed on 2.08.2010.
4. Stephen s.lLime,Lalit dandona ,Joseph a.Hosington,Spencerl.James,Morgaret c.Hogan:emmanuela Gakidou, India’s janani suraksha yojana conditional cash transfer programme to increase birth in health facilities and impact evaluation, lancet 2010:375:2009-23.
5.
6. suraksha+yojana.co
7. Http;// India net /maternal mortality in India 1997-2003 doc
8. http;//com/jcm.org.in/article;asp?issn=0970-0218;year=2009;vol 34; issue=1,s
Page=15 page =18.avlast=laha
9. Erica Royston and sue Armstrong; preventing maternal deaths by world health organization 1989; 9-12 and 218.
8) Signature of the candidate :
9) Remarks of the guide :
10) Name and designation of the guide:
10.1) Signature of guide:
10.2) Head of department : DR. S. G. HIREMATH
Prof & Head of the dept
Dept of community medicine
Navodaya medical college ,Raichur.
10.3) Signature of Hod:
10.4) Remarks of Chairperson or Principal;
10.5) Signature of Principal;
questionnaires
1) Name : w/o
2) Age ;
3) caste/religion ; Hindu/Muslim/others 4) Type of family : joint/nuclear/extended family
5) Address ;
6) Women’s education ; illiterate/primary/secondary/higher secondary/graduate
7) Husband’s education ; illiterate/primary/secondary/higher secondary/graduate
8) Women’s occupation ; agriculture/coli/carpenter/house wife
9) Husband’s occupation ; agriculture/coli/driver/carpenter
10) Age of marriage:<18 years/>18 years
11) Age at first pregnancy:<18years/>18years
12) Period of registration of antenatal checkups:1st /2nd /3rd trimester
13) Place of registration of antenatal check ups govt/private hospital/no registration
14) Did the women attend her antenatal checkups :yes/no
15) Frequency of antenatal checkups :1/2/3/4 times
16) Did the women take it immunization :yes/no
17) Did women receive iron-folic acid tablets :yes/no
18) Place of delivery:govt/private hospital
19) Mode of delivery:normal/ceaserian
20) Person who conducted delivery :trained/untrained/relative) Person who conducted delivery : trained/untrained/relatives
Trained-doctor/anm/lhv/nurse/dia
21) Is postnatal care given :yes/no
22) Complication during antenatal,intranatal,postnatal period :present/absent
a)Complication during antenatal period ;edema of feet,/ blurring of vision
Giddiness/ spotting/white discharge/excessive weight
Gain/fatigue/jaundice
b) Complications during intranatal period;foul smelling leak/excessive bleeding/
fever/prolonged labour
c) Complication during postnatal period;bleeding/fever/burning micturation
painful swollen breast/excessive
bleeding
23) From where did you utilize the health facility for above complication: govt/private?
24) Essential obstetric care that is routine health checkups govt/private/no
25) Emergency obstetrics care that is ,during any complication govt/private
26) Are you aware of Janani suraksha yojana scheme :yes/no
27) Through whom did you come to know about janani suraksha yojana :
28) Did you utilize the service; yes/no
29) If no why :
30) Are you using any contraceptive methods: yes/no
If yes ,Which contraceptive they use
31) Contraceptive method used :ocp/iud/tubectomy/not utilized/others
32) If no ,reasons for not utilization
33) From which health facility you utilized the family planning method govt/private/not utilized
34) Reasons for non-utilization of family planning method :
31) R TI’S/STD’S symptoms among women :present/absent
Symptoms like vaginal discharge/lower abdomen pain/ genital ulcer/painful intercourse/burning micturation?
32) Where did you utilize the service for RTI’S/STD’S :govt/private/not utilized
9 / Signature of the candidate
10 / Remarks of the guide
11 / Name and designation of
(in block letters)
11.1 guide
11.2 Signature
11.3 Co- guide (if any)
11.4 Signature
11.5 Head of the department
11.6 Signature / DR. SOUBHAGYA LAKSHMI,
Professor OF BIOCHEMISTRY,
NAVODAYAMEDICALCOLLEGE,
rAICHUR – 584103.
dr.S.S. BANGAR M.D
PROFESSOR AND H.O.D.,
DEPARTMENTOF BIOCHEMISTRY,
NAVODAYAMEDICALCOLLEGE,
RAICHUR – 584103.
12 / 12.1 Remarks of the chairman and Principal
12.2 Signature
1