RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

Annexure II

Proforma for registration of subject for dissertation

1. / Name of the candidate / Dr. Amit .R. Ugargol
2. / Name of the institution / St. JohnsMedicalCollege, Bangalore
3. / Course of study and subject / Post graduation in Microbiology MD (3yrs)
4. / Date of admission to the course / 18th March 2009

5. TITLE OF THE TOPIC:

Characterisation, Antibiotic resistance pattern and detection of Metalloβ-lactamases and Amp C in Pseudomonas aeruginosa in a tertiary care hospital.

6. BRIEF RESUME OF THE STUDY

6.1 NEED FOR STUDY:

Pseudomonas aeruginosa is a common nosocomial pathogen.It is a major cause of multidrug resistance infections in hospitalized patients1.It shows a high propensity for development of resistance against antibiotics2. Antibiotic resistance and production of virulence factors increases the morbidity and mortality associated with Pseudomonal infections.This leads to rising costs of care resulting from prolonged hospital stay and the need for more expensive drugs.It therefore becomes imperative to study the clinical correlation between virulence factors and antibiotic resistance pattern of P aeruginosa.

This study will enable detection of different virulence markers in various clinical isolates.

Correlation of virulence markers with the antibiotic resistance pattern will enable us with better understanding of pathogenecity and epidemiology of Psedomonas aeruginosa.

6.2 REVIEW OF LITERATURE :

An Overview of Pseudomonas aeruginosa:

Pseudomonas aeruginosa is an opportunistic bacteria which is ubiquitous in nature and moist environmental hospital sites (sinks,toilets,mechanical ventilators and dialysis equipment)2,3. It is a common nosocomial pathogen and a major cause of drug resistant infection in immunosuppressed and hospitalized patients.Pseudomonas aeruginosa exhibits resistance to a variety of antimicrobials including beta lactams.Metallo-β-lactamase(MBL) producing P aeruginosa is an emerging threat and a cause of concern in treating hospitalized patients2.

Virulence and pathogenecity:

P aeruginosa produce an array of virulence factors which play an important role in the pathogenecity3.Cell associated factors such as alginate,facilitate adherence and forms a mucoid exopolysaccharide capsule, protecting them from host phagocytic activity as well as antibiotics.

Along with alginate there are other extracellular factors produced by P aruginosa,such as Exotoxin A (inhibits protein synthesis in host), Pyocyanin (produce toxic oxygen metabolites causing tissue damage), elastases (disrupt elastin which is a component of blood vessels and lung parenchyma),Phospholipase C (breaks down lipids and lecithin

leading to tissue damage)3.

Culture:

P aeruginosa is an obligate aerobe that grows readily on many types of culture media,

sometimes producing fruity odour. Some strains hemolyze blood. P aeruginosa forms

smooth round colonies with a fluorescent greenish color. It often produces the nonfluorescent bluish pigment pyocyanin, which diffuses into the agar1,3.

In patients with cystic fibrosis cultures often yield mucoid colonies which is due to overproduction of alginate which is a exopolysaccharide.In such patients alginate appears to provide the matrix for the organisms to live in a biofilm.

Drug resistance:

P aeruginosais a leading cause of nosocomial infections ranking second among the gram negative pathogens.Resistance to antimicrobials is an increasing clinical problem and is a recognized public health threat2. It has two main mechanisms of resistance,intrinsic and acquired3,4.

Intrinsic resistance is mainly by the action of efflux pumps present in the cell wall which pumpout the antibiotics from the cytoplasm.Acquired resistance is due to the presence of β-lactamases namely Metallo beta lactamase (MBL, Type B Ambler classfn) and AmpC (Ambler Type C).MBL use metal ion Zn to catalyze hydrolysis of βlactam.According to an Indian study P aeruiginosa was isolated from respiratory tract(41.8%),urinary tract(25.5%),wound(20%),blood(12.7%), 80% of these isolates produced MBL5.

6.3 OBJECTIVES OF THE STUDY:

1)To isolate and identify Pseudomonas aeruginosa from various clinical

Samples.

2) Detection of virulence factors Alginate, Phospholipase ‘C’ in these isolates.

3) To determine the antibiotic susceptibility pattern of these isolates.

4) To detect MBL and Amp C production in the isolates.

7. MATERIALS AND METHODS:

7.1 SOURCE OF DATA:

250 (5% precision and 95% confidence interval) isolates of P aeruginosa obtained from various clinical samples received at the diagnostic laboratory; department of microbiology, St. John’s Medical College Hospital, Bangalore will be studied. The period of study will be from September 2009-January2011.

Inclusion criteria:

1)Pseudomonas aeruginosa isolates from all in-patient clinical samples received in the microbiology laboratory.

Exclusion criteria:

1)Pseudomonas aeruginosa isolated from out -patient clinical samples.

2)Pseudomonas aeruginosa isolated from urine with a colony count of <105 cfu/ml.

3)Pseudomonas aeruginosa isolated from pus samples without pus cells in the Gram stained direct smear.

4)Pseudomonas aeruginosa isolated from a single blood culture.

7.2 METHODS OF COLLECTION AND DATA:

Collection and processing of sampleswill be done according to the recommended procedures 6,7,8 .

Isolation and identification of Pseudomonas aeruginosa6:

Following tests will be used

1)Grams stain.

2)Detection of motility.

3) Colony morphology

4)Oxidase test(filter paper method).

5)Growth at 420C.

6)Acetamide test.

7)Sugar utilisation tests.

8)Nitrate reduction test.

9)Arginine hydrolysis.

10)Lysine and ornithine decarboxylation.

11)Oxidative-fermentative test (Hugh and Leifson).

Drug susceptibility tests:

Susceptibility to the following antibiotics will be studied using Kirby Bauer

method. Zone sizes will be measured and interpreted according to CLSI guidelines

1)Piperacillin 100µg.

2)Piperacillin-tazobactam 100/10µg.

3)Cefoperazone 75µg.

4)Ceftazidime 30µg.

5)Meropenem 10µg.

6)Gentamicin 10µg.

7)Amikacin 30µg.

8)Netilmicin 30µg.

9)Ciprofloxacin 5µg.

Determination of Minimum Inhibitory Concentration (MIC):

MIC is the lowest concentration of the antibiotic completely inhibiting the growth of the organisms. Minimum inhibitory concentration for Meropenem will be determined by Micro broth dilution and Agar Dilution Method.

Test to detect MBL production:

Imipenem(IMP)-EDTA Combined disk test9: Test organisms are inoculated on to plates with Mueller Hinton agar as recommended by the CLSI.Two 10µg imipenem disks(Becton Dickinson) are placed on the plates and appropriate amounts of 10µl of EDTA solution is added to one of them to obtain desired concentrations(750µg).The inhibition zones of imipenem and imipenem EDTA disks are compared after 16-18 hrs of incubation in air at 250C.If the increase in inhibition zone with the imipenem and EDTA disk is ≥7mm than imipenem disk alone,test was considered is MBL positive9. Sensitivity and specificity being 81% and 98% respectively9.

Test to detect Amp C Production:

Disk antagonism test (DAT)10: The disk antagonism test is used to detect inducibility of β-lactamase.Disks of inducing agent cefoxitin and test are used to detect the inducibility of β-lactamase.

Disks of inducing agent cefoxitin and cephalosporins(Cefoperazone,Ceftazidime) are placed on the surface of the test bacterial lawn on MHA of the suspected inducible AmpC β-lactamase producers separated by 15mm.The plates are examined after over night incubation at 37°C.If blunting of the cephalosporin disks adjacent to the cefoxtin disks occurred,the organisms are considered to produce inducible AmpCβ-lactamase.

Tests to detect virulence factors in clinical isolates of P aeruginosa:

1)Phospholipase-C:The method of Haberman and Hardt is employed for

estimation of phospholipase C activity determination11.

2)Alginate production:Tube Method: 2-3 colonies are inoculated with 5ml of BHI broth in glass tubes.Cultures are incubated at 370C for 18-20 hrs and the cultures are aspirated.Tubes are stained with saffranine.The presence of visible stained film on the wall of the tube is considered to be positive for slime production.If the wall of the glass tube remain unstained,the strain is considered as a non slime producer12.

Results:

The results will be analyzed using descriptive statistics like mean,median and proportion.After cross tabulation association between variables will be assessed using Chi square test.

7.3 Does the study requires any investigation or intervention to be conducted on patients or other humans or animals. If so please describe briefly.

No

8. REFERENCES;

  1. Fauci,Braunwald,Kasper,Hauser,Longo,Jameson,Loscalzo.HARRISON’S PRINCIPLES of Internal Medicine.17ed.USA:R.R.Donnelley and Sons;1958.Vol(1).
  2. A.C. Gales,N. Jones,J. Turnidge,R. Rennie and R.Ramphal.Charecterization of P aeruginosa isolates,Occurrence rates,Antimicrobial susceptibility patterns and Molecular typing in the Global SENTRY Antimicrobial Surveillance Program.Clin Infect Dis 2001;32(Suppl2):146-55.
  3. Jawetz,Melnick,Adelberg’s,Geo.F.Brooks,Karan.C.Caroll,Janet.S.Buteletal.Medical Microbiology.24th ed.USA :The McGraw-Hill Companies;1954.
  4. Patrick R Murray ,Ken Rosenthal,George S Kobayashi,Michael A Pfaller.Medical Microbiology.6th ed.USA:Mosby,Inc;1998.
  5. M Shanthi,Uma Sekar.Multi-drug resistant P aeruginosa and Acinetobacter baumannii infections among hospitalized patients:Risk factors and out comes.J Asso Physicians 2009;57:
  6. Elmer W Koneman, Stephen D Allen, William M Jenda et. al. Color Atlas and Textbook of Diagnostic Microbiology, 6th edition 2006. Publishers J.B. Lippincot Company.
  7. Baron E J., Peterson L R and Finegold S M. Bailey and Scott’s Diagnostic Microbiology. 9th edition 1994, Mosby Publications.
  8. Collee JG and Marr W. Mackie and Mc Cartney’s Practical Medical Microbiology; 14th, edition 1996.Chapter 5. Specimen collection, culture containers and media.

9 B Behra,P Mathur,A Das,A Kapil,V Sharma.An evaluation of four different

phenotypic techniques for detection of metallo-β-lactamase producing

Paeruginosa.Indian J Med Microbiol 2008;26(3):233-37.

10 Suranjana Arora,Manjusri Bal.AnpC β-lactamase producing bacterial isolates from

Kolkata hospital.Indian J Med Res 2005 Sep;122:224-233.

11 Rahul Mittal, Rakesh K. Khandwaha, Varsha Gupta*, P.K. Mittal & Kusum Harjai.

Phenotypic characters of urinary isolates of Pseudomonas aeruginosa& their

association with mouse renal colonization.Indian J Med Res 2006 Jan;123:67-72.

12 S Vishnu Prasad,Mamta Ballal,P.G.Shivananda.Slime production a virulence marker

In P aeruginosa strains isolated from clinical and environmental specimens.A

Comparative study of two methods.Indian J Path Microbiol 2009 Apr:

52(2):191-193.

9. Signature of the candidate:

10. Remarks of the guide:

11. Name and designation of:

(In block letters)

11.1 Guide : DR. SRIKANTH, M.D.

PROFESSOR,

DEPT. OF MICROBIOLOGY,

ST. JOHNSMEDICALCOLLEGE,

BANGALORE- 560034

11.2 Signature :

11.3 Co guide (if any) :

11.4 Signature :

11.5 Head of the Department : DR. MURALIDHARAN, M.D.

11.6 Signature:

12.1 Remarks of the Chairman and Principal:

12.2 Signature:

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