RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES ,BANGALORE-41

KARNATAKA

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1.NAME OF THE CANDIDATE
AND ADDRESS / ANCY M .P (SR.ANN PAUL)
POST GRADUATE (M.Sc.MLT)
DEPARTMENT OF MICROBIOLOGY
ST.JOHNSMEDICALCOLLEGE
HOSPITAL,BANGALORE-34
2. NAME OF THE INSTITUTION / ST.JOHNSMEDICALCOLLEGE AND
HOSPITAL,BANGALORE-34
3. COURSE OF STUDY AND
SUBJECT / 1ST YEAR M.Sc.MLT.(MICROBIOLOGY)
4. DATE OF ADMISSION TO
COURSE / 1ST SEPTEMBER 2010
5. TITLE / DETECTION OF METALLO BETA
LACTAMASE IN MULTIDRUG
RESISTANT PSEUDOMONAS SPECIES.

6. BRIEF RESUME OF THE INTENDED WORK

6.1. NEED FOR STUDY

  • Pseudomonas aeruginosa is one of the most common pathogens causing nosocomial infection. Acquired drug resistance is frequent in nosocomial isolates of non fermenting Gram negative bacilli and often involves more than one antimicrobial class.
  • Carbapenems have been the one of the successful antibiotic used in the treatment of infection caused by beta lactam resistant Gram negative bacilli1.
  • However, of late Carbapenems resistance has been observed frequently in non fermenting bacilli like pseudomonas aeruginosa. Resistance to Carbapenems is due to decreased outer membrane permeability, increased efflux system, alteration of penicillin binding proteins and Carbapenems hydrolyzing enzyme-Carbapenems. Carbapenemase are class B metallo beta lactamase2.
  • The emergence of these metallo beta lactamase in Gram negative bacilli is becoming a therapeutic challenge. Therefore rapid detection of metallo beta lactamases production is necessary to modify therapy.
  • Plasmid mediated metallo beta lactamase genes spread rapidly to other species of gram negative bacilli3. The occurrence of an metallo beta lactamase positive isolate in hospital environment posses not only a therapeutic problem but also a serious concern for infection control management.
  • In order to select the correct antibiotic for treatment and to prevent dissemination of such infection it is needed to study the antimicrobial resistant pattern.
  • Several phenotypic techniques have been studied, all taking advantage of the enzyme’s zinc dependence by using chelating agents, such as EDTA1.
  • Therefore we plan to undertake this study using Imipenem EDTA combined disc test. This will be helpful to determining the usefulness of the test for the detection of metallo beta lactamase production in Imipenem resistant clinical isolates of pseudomonas species.

6.2. REVIEW OF LITERATURE

Pseudomonas is a large and complex genus of Gram negative bacteria of importance, as it include species with both clinical and environmental implication. The genus pseudomonas first proposed by Migula in 1894. Pseudomonas species are aerobic, non spore forming Gram negative rods which are straight or slightly curved and 0.5 to 1.0 by 1.5 to 5.0 micrometer in size. They are usually motile with one or several polar flagella. They possess a strictly aerobic respiratory metabolism with oxygen as the terminal electron acceptor; in some cases nitrate can be used as an alternative electron acceptor that allows anaerobic growth4.

Many species produce water soluble pigments which diffuse through the culture medium. Majority of them are saprophytic being found in soil , water, sewage or wherever decomposing organic matter is found. The genus Pseudomonas belongs to the family Pseudomonadaceae which contain over 200 species. Human disease has been caused by pseudomonas aeruginosa, pseudomonas maltophila, Pseudomonas mallei, Pseudomonas pseudomallei, Pseudomonas cepacia, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas multivorans and Pseudomonas putida. The most important among these is Pseudomonas aeruginosa. Recently Pseudomonas mallei, Pseudomonas pseudomallei and Pseudomonas cepacia have been assigned a new genes Burkholderia which also belongs to family Pseudomonadaceae5.Pseuomonas aeruginosa is the most important agent causing nosocomial infections5 and a classic opportunistic pathogen with innate resistance to many antibiotic and disinfectants6.Pseudomonas aeruginosa exhibit intrinsic resistance to various antimicrobial agents including beta lactam antibiotics7.

Bacterial resistance has greatly hampered effective treatment of patients in clinical setting. The ability to produce beta lactamase enzyme is the major cause of resistance of bacteria to beta lactam antibiotics and has been the subject of extensive microbiological, biochemical and genetic investigation8.

The introduction of Carbapenems into clinical practice represent a great advance for the treatment of beta lactam resistant bacteria. Due to their broad spectrum of activity and stability to hydrolysis by most beta lactamase, the Carbapenems have been the drug of choice for treatment of infections caused by penicillin or cephalosporin resistant Gram negative bacilli. However, this scenario is changing with the emergence of metallo beta lactamase producing strains, Pseudomonas8. Based on molecular studies, Carbapenems hydrolyzing enzymes are classified into four groups A,B,C&D. The metallo beta lactamase belong to group B and are enzymes requiring divalent cations as cofactors for enzyme activity, being inhibited by the action of a metal ion chelator9.

There are reports on metallo beta lactamase production in Pseudomonas aeruginosa from various countries like Brazil, Korea, Singapore, and France. Metallo beta lactamase was first reported as a zinc dependent enzyme in Bacillus cereus in mid 1960s. In 1991,Japan reported the first plasmid mediated metallo beta lactamase in Pseudomonas aeruginosa. There are frequent reports of metallo beta lactamase production in Pseudomonas aeruginosa from the Asian and the Pacific countries , namely Hong Kong, Taiwan and Japan9.

Various methods have been recommended for screening metallo beta lactamase . These include the modified Hodge test, double disc synergy test using Imipenem and EDTA discs or ceftazidime and EDTA discs , EDTA impregnated Imipenem or meropenem discs. For minimum inhibitory concentration detection of Imipenem , the E-test strip and micro dilution plate method is recommended2. In 2002 from India, Navaneeth et al. first reported metallo beta lactamase production by Pseudomonas aeruginosa to be 12%7 Since then , the incidence of metallo beta lactamase production by Pseudomonas aeruginosa has been reported to be 10 to 13% from various clinical specimens across the country10. A study conducted by Mary et al, reported 42% metallo beta lactamase production by Pseudomonas8. Another study conducted by Sarkar et al reported 54.54%metallo beta lactamase production by Pseudomonas11.Another study conducted on 2006 by Varaiya Ami et al reported 14.3% metallo beta lactamase production from Pseudomonas 12.

According to various studies1, 2, 7 ,9 ,13 production of metallo beta lactamase can be detected by chelating agent like EDTA by enhancement of inhibition zone around Imipenem impregnated with EDTA compared to those without EDTA.

6.3. OBJECTIVES OF THE STUDY

  1. To isolate and identify the Pseudomonas species from clinically significant samples.
  2. To determine the antibiotic sensitivity pattern by disc diffusion method .
  3. To detect the metallo beta lactamase in Pseudomonas showing multidrug resistance.

7. MATERIAL AND METHOD

A total of 100 consecutive Pseudomonas isolated from clinically significant samples from patients admitted to St.Johns Medical college and Hospital Bangalore showing multidrug resistance will be studied during the period of one year from January 2011-December 2011.

7.1. METHOD OF COLLECTION OF DATA

All clinical samples of patients received in diagnostic laboratory for aerobic culture and sensitivity in Microbiology department of St. Johns Medical college and Hospital will be processed according to standard recommended procedure.

7.2 INCLUSION CRITERIA

Isolates of Pseudomonas species showing resistant to any one of the following or all of the following drugs will be included in this study.

  • Aminoglycosides
  • Third generation Cephalosporins
  • Quinolones.

7.3 EXCLUSION CRITERIA

Isolates of pseudomonas species showing sensitive to all of the three groups of following drugs Aminoglycosides, Third generation Cephalosporins and Quinolones are excluded from this study.

7.4 METHODS

Processing of all samples except blood;

  1. Gram stained direct smear examination .
  2. Inoculation into blood agar and MacConkey’s agar
  3. Identification of species by following biochemical tests.
  4. Oxidase test
  5. Motility test
  6. Triple sugar iron agar test
  7. Indole test
  8. Citrate utilization test
  9. Urease test
  10. Catalase test
  11. Oxidation and fermentation test with glucose ,maltose, mannitol, xylose .
  12. Decarboxylation test(Arginine, Lysine)
  13. Nitrate reduction test
  14. Esculin hydrolysis

Processing of blood sample

5ml of blood is collected by venepuncture under aseptic conditions and transferred into blood culture bottle containing brain heart infusion broth (biphasic). Incubate 37 degree Celsius for 24 hour and check for the growth and then process.

  • Gram stain from growth
  • Subculture into blood agar and MacConkey’s agar for isolation.
  • Identification of species by biochemical method.
  1. Antibiotic susceptibility test

This will be done by Kirby-bauer disc diffusion method for

  • Gentamycin (10mcg)
  • Amikacin (30mcg)
  • Cefotaxime (30mcg)
  • Cefoperazone (75mcg)
  • Ceftazidime (30mcg)
  • Ciprofloxacin (5mcg)
  • Imipenem (10mcg)

5. Detection of metallo beta lactamase by two screening method.

1.Minimum inhibitory concentration of Imipenem by Agar dilution method.

2.Imipenem –EDTA combined disc1.

7.5 Does the study require any investigation or interventions to be conducted on patients or other human beings or animals? If so, please describe briefly.

- No

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

Not applicable.

Statistical analysis

This is a descriptive study and result will be expressed in term of percentage.

8.REFERENCE:

  1. Behera P, Sharma V, Kapil A,et al . An evaluation of four different phenotypic techniques for detection of metallo beta lactamase producing Pseudomonas aeruginosa. Indian J Med Microbiol 2008;26(3): 233-237.
  2. Varaya Ami, Kulkarni Nikhil, kulkarni Manasi, Bhalekar Pallavi & Dogra Jyotsana . Incidence of metallo beta lactamase producing Pseudomonas aeruginosa in ICU patients. Indian J Med Res 2008;127:398-402.
  3. Irfan s, Zarfar A, Guhar D, Ahsan T, Hasan R. Metallo beta lactamase producing clinical isolates of Acinebacter species and Pseudomonas aeruginosa from intensive care unit patients of a tertiary care hospital.Indian J Med Microbiol 2008; 26(3): 243-245.
  4. Edith Blongel-Hill, Deborath A. Henry and David P.Speert. Pseudomonas.In: Patrick R.Murray,Ellen jo Baron,James H.Jorgensen,Marie Louise Landry, Michael A. Pfaller , editors. Manual of clinical Microbiology 9th ed.Vol .1;Washington,D C:1970;734-735.
  5. Baveja C P. Pseudomonas Stenotrophomonas,burkhoderia . In: Text book of Microbiology, Arya publication, 3rd ed. 2009;291-292.
  6. J.R.W.Govan. Psudomonas, Stenotrophomonas, Burkholderia.In:J.G.Collee , A. G. Fraser, B.P. Marmion, A. Simmons,editors. Practical medical microbiology. 14th ed.New Delhi: 2007; 413-414.
  7. Navaneeth B.V, Sridharan.D, Sahay D. &Belwadi M.R.S. A preliminary study on metallo beta lactamase producing Pseudomonas aeurogonosa in hospitalized patients. Indian J Med Res 2002;116:264-267.
  8. Jyudason V Mary, Kandathil A J &Balaji V. Comparison of two methods to detect cabapenemase &metallo beta lactamase production in clinical isolates. Indian J Med Res 2005;121:780-783.
  9. Hemalatha V, Sekar Uma &Kamat Vijayalakshmi. Detection of metallo beta lactamase producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2005;122:148-152.
  10. Taneja N, maharwal S, Sharma m.Imipenem resistance in nonfermenters causing nosocomial urinary tract infection. Indian J Med Sci 2003;57(7): 294-299.
  11. Sarkar Bharathi, Biswas Debasis, Prasad Ramjee, Sharma J P. A clinicomicrobiological study on the importance of Pseudomonas in nosocomially infected ICU patients, with special reference to metallo beta lactamase production .Indian J Pathol Microbiol 2006;49(1):44-48.
  12. Varaiya Ami, kulkarni manasi, Bhalekar Pallavi, Dogra Jyotsana. Incidence of metallo beta lactamase producing Pseudomonas aeruginosa in diabetes and cancer patients. Indian J Pathol Microbiol 2008;51(2): 200-202.
  13. Singh P Sakshi, Shariff Malini, Barua Tanushree, Thukral S S.Comparative evaluation of phenotypic test for identification of metallo beta lactamases producing clinical isolates of Pseudomonas aeruoginosa .Indian J Med Res 2009;129:713-715.

9. / Signature of the candidate:
10. / Remarks of the guide:
11. / Name and designation of
11.1 Guide : Dr.Muralidharan.S
Professor and head,
Dept.of Microbiology,
St.John’s Medical College Bangalore.
11.2 Signature :
11.3 Co- guide (If any) :
11.4 Signature :
11.5 Head of the Department : Dr.Muralidharan.S.
MD (Microbiologist)
St.John’s Medical college
Bangalore 11.6 Signature :
12. / 12.1 Remarks of chairman and principal:
12.2 Signature :

1

1