RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

1. NAME OF CANDIDATEAnd Address (in block letters)

Dr. VISHWAJEET BARDOLOI

ZOO ROAD TINIALI,

PADMA PATH,

BY LANE – 8,

HOUSE NO.- 9,

GUWAHATI – 781024

ASSAM

2. NAME OF THE INSTITUTIONS.S. INSTITUTE OF MEDICAL SCIENCES

AND RESEARCH CENTRE

DAVANGERE-577005.

3. COURSE OF STUDY & SUBJECTPOST GRADUATE DEGREE,

M. D. MICROBIOLOGY

4. DATE OF ADMISSION TO COURSE 29th MAY 2012

5. TITLE OF TOPIC BIOFILM PRODUCING BACTERIAL ISOLATES FROM URINARY TRACT INFECTIONS AND URINARY CATHETER : A COMPARATIVE STUDY

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6. BRIEF RESUME OF THE INTENDED WORK:

6.1 NEED FOR THE STUDY :

Catheter associated urinary tract infections (CAUTI) account for up to 40% of all

nosocomial infections and 80% of all nosocomial Urinary Tract Infections (UTIs).1 Most

cases of CAUTI are associated with biofilm formation which is a representative type of

biofilm associated infection usually composed of multi drug resistant microorganisms. 2

Bacteria in biofilm are protected from antimicrobial chemotherapy as well as host

defence mechanisms, establishing chronic persistent infections, septicemia and death if

not treated. Biofilms are composed of clusters of diverse microorganisms and

extracellular matrix (primarily polysaccharide materials), formed on both the

extraluminal and intraluminal surfaces of urinary catheters.3

Acinetobacter, Pseudomonas, Klebsiella, Staphylococcus, Enterobacter and E. coli

are the most common causes of nosocomial infections, and the same may be the common

cause of colonization and biofilm production in indwelling urinary catheters.4

Very few studies are done regarding biofilm producing properties of isolates from

nosocomial urinary tract infection without indwelling catheter and community acquired

urinary tract infections.5

Scanning electron microscopy is the gold standard method of demonstration of

biofilms. Several phenotypic and genotypic methods have been used to demonstrate

biofilm forming properties of isolates from CAUTI and surface of catheters, as an

indirect evidence of presence of biofilms. The Phenotypic methods namely, tissue culture

plate method, Congo red agar method, tube methods are simple, rapid and fairly sensitive

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for detection of biofilm forming properties.6,7

Molecular methods consisting of sonication and PCR, though more sensitive and

specific are limited by the high cost.

Although, several studies on biofilm producing properties of different bacterial

isolates from catheter associated urinary tract infection are conducted, limited data is

available on concurrent isolation of such bacteria from internal and/or external surface of

indwelling urinary catheter with biofilm. Comparative studies on biofilm producing

properties of isolates from CAUTI and community acquired urinary tract infections

without indwelling urinary catheter, are very few. No systematic study has been

conducted so far in and around Davangere regarding the role of bacteria with biofilm

producing properties in patients with indwelling urinary catheter and community

acquired urinary tract infections.

6.2. Review of Literature :

Microbial attachment to surfaces was first described by Zobell in 1943, but the word

“BIOFILM” made its first appearance in scientific literature in 1970s.8

Stickler DJ et al (1996), reported biofilm production and blockage of catheter leading

to cystitis and septicemia in patients with long term indwelling urinary catheter.9

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Christensen GD et al. (1985) have investigated visual assessment of bacterial

adherence to culture tubes, microtitire plates, mouse and rat animal models for biofilm

producing properties. They investigated sensitivity and specificity of different phenotypic

methods for testing the biofilm producing properties of bacterial isolates.10

Macleod SM and Stickler DJ (2007) have investigated bacterial flora of biofilms on

Foley catheter by conventional culture methods. Out of 106 Foley catheters with

biofilms, 76 were colonized by two or more species and single organism was found in

remaining 30 catheters.3

Frank DN et al. (2009) have identified different bacterial isolates from biofilms on

internal and external surface of Foley catheter tip by molecular phylogenetic analysis of

16S ribosomal RNA gene sequences by PCR. The predominant types of bacteria

identified were, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus

epidermidis, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecalis and

Propioniobacterium acnes.4

Murugan S et al. (2011) have reported biofilm producing properties of E. Coli isolates

from urinary tract infection from patients attending tertiary care hospitals at Palakkad

(Kerala) by Tube method and Congo Red Agar method. By tube method, 84.37% of

E.coli (81/96) isolates were found to have biofilm producing properties. Congo Red Agar

method did not correlate well with tube method in demonstrating similar property.2

However, Adilson Oliveria and Maria de Lourdes RS Cunha have reported that the

Congo red agar method is highly specific (100%) and sufficiently sensitive (89%)

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compared to PCR based on the detection of icaA, icaD or icaC genes.11

Comparative study of biofilm producing properties of isolates from CAUTI and UTI

without indwelling catheter by Abdallah et al (2011) at Ain Shams University hospital at

Cairo, Egypt reported that 43.3% of isolates from CAUTI were having biofilm producing

properties compared to 30% from urinary tract infection without indwelling catheter.

However results were not statistically significant.5

Soto et al. (2007) have reported that 74% of the strains causing relapses in community

acquired urinary tract infections in women were having biofilm producing properties.

They also stated that biofilm production may be the key determinant for persistence of

uropathogenic E. Coli (UPEC) in vaginal reservoir or bladder epithelial cells or both.12

Murugan S et al (2011) have concluded that biofilms are highly resistant to

antimicrobial treatment. Antimicrobial resistance of biofilm producing bacteria include

mechanisms like trapping of antibiotics in the exopolysaccharide matrix causing a

diffusion barrier by restricting the rate of molecule transfer to the interior of the biofilm,

escaping of bacteria within biofilms from host defence mechanisms, and alteration of

metabolism and decrease in bacterial growth rate due to Quorum sensing and genotypic

adaptations.2

Macleod SM and Stickler DJ (2007) have used Scanning electron microscope to

evaluate multiple sections of the catheter length and demonstrated extensive encrustation

around eye holes and throughout the lumen of urinary catheters. They also demonstrated

that Proteus mirabilis out numbers the other organisms causing CAUTI with biofilm.3

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Trautner BW et al (2004) have reviewed strategies for prevention of CAUTI with

biofilm. Closed drainage and catheter removal are effective strategies and they have

enumerated possibly effective and ineffective strategeties for prevention of CAUTI with

biofilm.13

Trautner BW et al have reviewed novel approaches for preventing biofilm formation

on indwelling urinary catheter, viz, Disruption of quorum sensing, Iron-scavenging

catheters and bacterial interference.13

6.3. Objectives of study:

1) Isolation, identification and characterization of biofilm producing properties of

bacterial isolates from catheter associated urinary tract infection (CAUTI),

community acquired urinary tract infections and external and internal surface of

distal 2cm catheter tip near the eye hole of the catheter.

2) Comparison of biofilm producing properties of bacterial isolates from catheter

associated urinary tract infection (CAUTI), indwelling urinary catheter and

community acquired urinary tract infections regarding biofilm forming properties.

7.MATERIALS AND METHODS:

7.1 Source of data:

A prospective observational study consisting of 100 patients admitted in different

Intensive Care Units (ICUs) with catheter associated urinary tract infection (CAUTI) and

100 patients with community acquired urinary tract infections (without a indwelling

urinary catheter) confirmed by semi quantitative urine culture at S.S. Institute of Medical

Sciences and Research Centre will be conducted.

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7.2 Method of collection of data (including sampling procedure, if any)

Urine specimen from catheter and specimens from external and internal surface of the

cut catheter tip (2 cm) will be collected simultaneously.

SPECIMEN COLLECTION (Urine):

All non-duplicated urine specimens from catheter associated urinary tract infection

(CAUTI) will be collected as per standard laboratory procedures.14

CASE DEFINITION:

Catheter associated urinary tract infection (CAUTI) is defined as an infection in a

patient with urinary catheter meeting the National Health care Safety Network (NHSN)

definition of a urinary tract infection.15

SPECIMEN COLLECTION (From catheter tip):

Catheter will be disconnected and removed with aseptic precautions. Distal 2

centimeters, near the eye of Foley catheter will be cut with a sterile scalpel. External

surface of catheter will be scraped and the material is inoculated into 10 ml of Brain

Heart Infusion broth (Hi Media, Mumbai. India).16

Catheter will be cut longitudinally with a surgical blade with aseptic precautions.

Material from luminal surface of the catheter is scraped with scrapers or with metal brush

and specimen will be inoculated into 10 ml of Brain Heart Infusion broth. (Hi Media,

Mumbai. India). Catheter tip is put into 10 ml of Brain Heart Infusion broth aseptically

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and vortexed for 1 minute. All three specimens will be incubated for 3 hours and plated

onto Mac Conkey’s agar and 5% sheep blood agar.

COMMUNITY ACQUIRED URINARY TRACT INFECTION:

5 to 10 ml of early morning midstream urine specimen will be collected with aseptic

precautions as per standard laboratory procedures.14 Isolates from one hundred

community acquired urinary tract infections with significant bacteriuria will be included

in the present study.

Bacterial isolates from urine from patients with indwelling catheter, 2cm of cut

catheter tip and midstream urine specimen from community acquired urinary tract

infection will be isolated and indentified by standard laboratory procedures.14

Antibiotic susceptibility testing will be done by Kirby-Bauer’s disc diffusion method

as per CLSI guidelines.17

QUALITATIVE DETERMINATION OF BIOFILM PRODUCTING PROPERTY:

Bacterial isolates from CAUTI and isolates from external and internal surface of

catheter will be subjected to two Qualitative phenotypic methods for biofilm producing

property.

1. TUBE METHOD 7 :

The tube method consists of inoculating 10ml of Brain Heart Infusion broth with

3 to 4 colonies of bacterial isolates from blood agar plate and incubating the broth

culture tube overnight (18 hours) at 37˚C. The culture tubes are then emptied of

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their contents, washed with deionised water several times and stained with

safranin 0.1%. Slime production is judged to have occurred if a visible film lines

the walls of the tube and the isolate will be interpreted as biofilm producer. Ring

formation at the liquid-air interface is not considered indicative of slime

production.

2. CONGO RED AGAR METHOD (CRA) 6 :

Biofilm forming colony morphology will be detected for organisms on Congo

Red Agar plates.

Bacteria will be cultured in 10ml Brain Heart Infusion broth at 35˚C for 24 hours

without shaking, and will be then plated onto CRA plates. Incubation will be carried out

at 35˚C for 24 hours and an additional 24 hours at room temperature before recording the

colony morphology. Crusty black colonies with dry filamentous appearance will be

recorded as biofilm producers, smooth pink colonies as non producers and intermediate

colony morphology (pink with dark centers resembling bull’s eyes) as potential biofilm

producers.

CONTROLS FOR BIOFILM FORMING PROPERTY:

Biofilm producing reference strains of Acinetobacter baumannii (ATCC 19606) and

Pseudomonas aeruginosa (ATCC 27853) and non-biofilm forming reference strains of

Staphylococcus aureus (ATCC 25923) E. coli (ATCC 25922) will be used.

Inclusion Criteria :

1. Patients with nosocomial catheter associated urinary tract infection (CAUTI)

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admitted in different ICUs of SSIMS &RC will be included in the present study.

2. Patients submitting both Urine sample and catheter tip collected concurrently will

be included in the present study.

3. Patients with community acquired urinary tract infections without indwelling

urinary catheter will be included in the study.

Exclusion Criteria :

1) Patients with only urine specimen sent from catheter associated urinary tract

infection (CAUTI) will not be included in the present study.

2) Patients with only catheter tip sent for diagnosis of catheter associated urinary

tract infection (CAUTI) will not be included in the present study.

3) Isolates of CAUTI from community acquired urinary tract infection.

4) Isolates from nosocomial CAUTI in patients from other areas of the hospital other

than the ICUs.

Statistical analysis:

Data will be shown in terms of numbers and percentages, and will be analyzed by

Chi-square test/ Fisher exact test.

7.3. Does the study require any investigations or interventions to be conducted on

patients or other humans or animals? If so describe briefly?

Yes, the study requires experiments to be carried out on human beings. Informed

written consent will be taken and urine specimen is collected from the patients with

indwelling catheter. Concurrently, the catheter is also disconnected and the distal 2

10

cm of catheter is collected.

7.4. Has ethical clearance been obtained from your institution in case of 7.3?

Yes, it has been obtained on 02-11-2012

LIST OF REFERENCES:

1) Hartstein AI, Garber SB, Ward TT, Jones SR, Morthland VH.

Nosocomial urinary tract infection: a prospective evaluation of 108

catheterized patients. Infect Control. 1981;2:380-86.

2) Murugan S, Uma Devi P, Neetu John P. Antimicrobial susceptibility pattern of

biofilm producing Escherichia coli of urinary tract infections. Curr Res Bacteriol.

2011;4(2):73-80.

3) Macleod SM, Stickler DJ. Species interactions in mixed-community crystalline

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biofilms on urinary catheters. J Med Microbiol. 2007;56:1549-57.

4) Frank DN, Wilson SS, St Amand AL, Pace NR. Culture Independent

Microbiological Analysis of Foley Urinary Catheter biofilms. PLoS One; 4(11).

[Internet]. 2009 [cited 2012 Aug 12]; [about 5 p].

Available from :

www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone000781

5) Abdallah NMA, Elsayed SB, Mostafa MMY, El-gohary GM. Biofilm forming

bacteria isolates from urinary tract infection, relation to catheterization and

susceptibility to antibiotics. Int J Biotechnol Mol Biol Res. 2011;2(10):172-78.

6) Aricola CR, Compoccia D, Baldassarri L, Donati ME, Pirini V, Gamberini S

et al. Detection of biofilm formation in Staphylococcus epidermidis from implant

infections. Comparison of a PCR method that recognizes the presence of ica

genes with two classic phenotypic methods. J Biomed Mater Res.

2005;76A(2):425-30.

7) Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime

producing strains of Staphylococcus epidermidis to smooth surfaces. Infect

Immun. 1982;37:318-26.

8) Zobell CE, Allen EC. The Significance of Marine Bacteria in the Fouling of

Submerged Surfaces. J Bacteriol. 1935;29(3):239-51.

9) Stickler DJ. Bacterial biofilms and the encrustation of urethral catheters.

Biofouling. 1996;94:293-305.

10) Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton

DM, Beachey EH. Adherence of coagulase-negative staphylococci to plastic

tissue culture plates: a quantitative model for the adherence of staphylococci to

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medical devices. J Clin Microbiol. 1985;22(6):996-1006.

11) Oliveria A, Cunha Maria de Lourdes RS. Comparison of methods for the

detection of biofilm production in coagulase-negative staphylococci. Oliveria and

Cunha BMC Research Notes [Internet] 2010;3:260.doi:10.1186/1756-0500-3-

260. Available from:

http://www.biomedcentral.com/1756-0500/3/260

12) Soto SM, Smithson A, Martinez JA, Horcajada JP, Mensa J, Vila J. Biofilm

formation in uropathogenic E. coli strain: relationship with prostatitis,

urovirulence factors and and antimicrobial resistance. J Urol. 2007;177(1):365-

68.

13)Trautner BW, Darouiche RO. Role of biofilm in catheter-associated urinary

tract infection. Am J Infect Control 2004;32(3):177-83.

14) Collee TG, Diguid JP, Fraser AG, editors. Mackie and Mc Cartney practical

Medical Microbiology. 14th ed. Edinburg: Churchill Livingstone; 2006.

15) Horan TC, Andrus M, Dudeck MA, CDC/NHSN surveillance definition of

health care-associated infection and criteria for specific types of infections. Am J

Infect Control. 2008;37:309-32.

16) Revdiwala S, Rajdev BM, Mulla S. Characterization of bacterial etiologic

agents of biofilm formation in medical devices in critical care setup. Crit Care

Res Pract. [Internet].2012 [cited 2012 Aug 12];[about 5 p].

Available from :

www.hindawi.com/journals/ccrp/2012/945805/

17) Clinical and Laboratory Standards Institute (CLSI); Performance Standards

for Antimicrobial Susceptibility Testing; Twenty first Informational Supplement.

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M100-S21;31(1). USA Pensylvania:CLSI;2011.

9. SIGNATURE OF CANDIDATE

10. REMARKS OF 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. KG BASAVARAJAPPA M.D.

PROFESSOR & HEAD, DEPARTMENT OF MICROBIOLOGY, S.S. INSTITUTE OF MEDICAL SCIENCES AND RESEACH CENTRE, DAVANGERE – 577005.

Dr. YOGEESHA BABU KVM.D.

PROFESSOR,DEPARTMENT OF MICROBIOLOGY, S.S. INSTITUTE OF MEDICAL SCIENCES AND RESEACH CENTRE,DAVANGERE – 577005.

Dr. KG BASAVARAJAPPA M.D.

PROFESSOR & HEAD, DEPARTMENT OF MICROBIOLOGY, S.S. INSTITUTE OF MEDICAL SCIENCES AND RESEACH CENTRE,DAVANGERE – 577005.

12.12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL

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12.2 Signature.

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