RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE- 41 PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION.
1. NAME OF THE CANDIDATE ASHNA JOHN
2. ADDRESS M.Sc. MLT (MICROBIOLOGY) ST. JOHN’S MEDICAL COLLEGE AND HOSPITAL, BANGALORE-34
3. COURSE OF STUDY M.Sc. MLT
4. SUBJECT MICROBIOLOGY
5. DATE OF ADMISSION 05-09-2011
6. TITLE IDENTIFICATION AND CHARACTERIZATION OF TRIBE PROTEEAE FROM URINE AND PUS SAMPLES. 7.1 NEED FOR THE STUDY
Tribe Proteeae which includes Proteus, Providencia and Morganella are widely distributed in nature. Tribe Proteeae are associated with nosocomial infection, however they are not always properly identified and speciated. Tribe Proteeae is the commonest cause of urinary tract infection, especially Proteus mirabilis. Providencia species also cause nosocomial infection in wounds and burns. Morganella has been implicated as the cause of diarrhea and wound infection. All of these organisms are ubiquitous in the environment. Individual case reports and nosocomial outbreak reports indicate that they are capable of causing major infectious diseases. However there is a need to study atypical Proteus and newer genera (e.g. Morganella and its species) as well as Proteus penneri. Study of these isolates along with gram positive infections also needs to be done. This study helps in proper identification and antibiotic sensitivity, so that appropriate measures are instituted. One of the technical problems in the study of Proteus is its swarming nature and hence a comparison of Mac Conkey’s agar, Cystine Lactose Electrolyte Deficient agar (CLED), use of chemicals and solvents will be exploited.
7.2 REVIEW OF LITERATURE
The genus Proteus along with the two other genera (Morganella and Providencia) belongs to the tribe Proteeae.1 All members of tribe Proteeae with few exceptions are motile, gram negative, non-capsulated, pleomorphic and are assigned to the Enterobacteriaceae family mainly on the basis of shared biochemical characteristics.2The formation of the enzyme phenyl alanine deaminase converts phenyl alanine to phenyl pyruvic acid (PPA reaction) is the characteristic feature of the tribe Proteeae by which they are differentiated from other members of the family Enterobacteriaceae.1 The genus Proteus currently consists of species Proteus mirabilis, Proteus penneri, Proteus vulgaris, Proteus myxofaciens and Proteus hauseri.3 Proteus species are found in multiple environmental habitats, including long term care facilities and in hospitals as well as from patients with underlying diseases or compromised immune systems.4 Proteus mirabilis is the second most common cause of urinary tract infection and is also an important cause of nosocomial infections.5 Proteus mirabilis causes serious kidney infections which can involve invasion of host urothelial cells.6 Proteus vulgaris and Proteus penneri are usually associated with hospital acquired infection. They are isolated from patients with chronic debilitating diseases and from those who are immune compromised.1 Proteus species shows a characteristic swarming motility, which is observed on non-inhibitory agar medium as a wave- like movement across the entire surface of the agar medium.7 The genus Morganella currently consists of one species, Morganella morganii with two subspecies morganii and sibonii.3 Morganella morganii is commonly found in human and animal feces. It is most often found as an opportunistic pathogen in patients who are hospitalized, particularly those on prolonged antibiotic therapy. Morganella morganii causes urinary tract infections which are often associated with alkaline urine. The bacteria have also been occasionally reported to cause sepsis, pneumonia, wound infection and pericarditis.1 Morganella species do not show true swarming on solid nutrient media.8 The genus Providencia consists of five species Providencia alcalifaciens, Providencia heimbachae, Providencia rettgeri, Providencia rustigianii, and Providencia stuartii.9 Providencia stuartii and Providencia rettgeri are commonly found in patients with long term indwelling urinary catheters. Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii also may cause invasive diarrhea.1 Strains of Providencia species do not swarm on solid nutrient media.3 The choice of a specific antimicrobial agent depends on antibiotic susceptibility pattern of isolated strains. Proteus mirabilis is susceptible to nearly all antimicrobials except tetracycline. Proteus mirabilis is sensitive to ampicillin, broad- spectrum penicillins such as ticarcillin, piperacillin, first-, second-, and third generation cephalosporins, imipenem and aztreonam; Proteus vulgaris and Proteus penneri are sensitive to trimethoprim and sulfamethoxazole, quinolone, imipenem and fourth generation cephalosporins. Morganella morganii strains are usually susceptible to cefepime, imipenem, piperacillin and aminoglycosides and resistant to ceftazidime and other third generation cephalosporins. Providencia species is resistant to tetracyclines, older penicillins, Cephalosporins, flouroquinolones and amino glycosides. It is susceptible to late generation cephalosporins, aztronam and carbapenems.1 7.3 OBJECTIVES
1. Identification and characterization of tribe Proteeae from clinical samples (mainly urine and pus).
2. Determination and antibiotic susceptibility of the above isolates by disc diffusion method (Kirby Bauer method).
3. Standardization of different methods of inhibition of swarming.
8.0 MATERIALS AND METHODS
8.1 SOURCE OF DATA
Study design
Prospective cross sectional study.
Samples for study & duration
A total of 50 consecutive isolates of tribe Proteeae from urine and pus specimens received in the department of microbiology. St. John’s medical college hospital, Bangalore will be studied during the period Jan. 2012- Dec. 2012. The strains will be sub cultured on Mac Conkey’s agar to obtain significant count.
8.2 INCLUSION CRITERIA All isolates from clinical samples (mainly pus and urine) presumptively identified as Tribe Proteeae will be included.
8.3 EXCLUSION CRITERIA Old cultures. 8.4 METHOD OF ANALYSIS
Collection of samples will be done according to the recommended procedure10, and it will be processed in laboratory The various tests for the identification of tribe Proteeae are given below; 1. Gram stain
2. Growth in
Sheep blood agar
Mac Conkey’s agar
CLED agar
3. Preliminary test
Oxidase test
Motility test by hanging drop
MMTPCU
Phenyl pyruvic acid (PPA)
LIA
4. Extended biochemical tests
Mannose
Adonisol
Maltose
Trehalose
Xylose
Inositol
Lysine
Arginine
Ornithine 5. Antibiotic susceptibility tests
Susceptibility to the following antibiotics will be studied using Modified Kirby Bauer method. ATCC Escherichia coli 25922 is used as control. Zone sizes will be measured and interpreted according to CLSI guidelines. The antibiotics used will be
Ampicillin - 10 µg
Cefazolin - 30 µg
Gentamicin - 10 µg
Amikacin - 30 µg
Piperacillin-tazobactam - 100/10 µg
Cefuroxime - 30 µg
Cefoxitin - 30 µg
Cefotaxime - 30 µg
Ciprofloxacin - 05 µg
Levofloxacin - 05 µg
Imipenem - 10 µg
Meropenem - 10 µg
Piperacillin - 100 µg
Ceftazidime - 30 µg
Chloramphenicol - 30 µg
Tetracycline - 30 µg
Norfloxacin - 10 µg 6. Minimum Inhibitory Concentration (MIC) by agar dilution-using levofloxacin and ciprofloxacin
7. Inhibition of swarming
Increasing the concentration of the agar.
CLED.
By using alcohol.
STATISTICAL ANALYSIS
The continuous quantitative variable will be compared by using the chi-square
test. The sensitivity to the antibiotics will be compared in percentage
9.0 Does the study require any investigation or interventions to be conducted on patients or other human beings or animals if so describe briefly.
No.
10.0 Has ethical clearance been obtained from your institution in case of 9.0
No. 11.0 REFERENCES
1. Subhash Chandra Parija. Coliforms. In: Textbook of Microbiology. Elsevier. 2009; 330- 334.
2. Jim Manos and Robert Belas. The genera Proteus, Providencia and Morganella In; Prokaryotes. 2006; 6thedn: 245-269.
3. J. Michael Miller, Francis W. Brenner et.al. Classification identification and clinical significance of Proteus, Providencia and Morganella. J. Clin Microbiol reviews. 2009; 13: 534-546.
4. Yah.S.C, Eghafona N.O.et.al. Bioline. African J. Microbiol. 2007; 6: 1757- 1762.
5. Laure Pagani, Robert Migliavacca et.al. Emerging Extended- Spectrum β- lactamases in Proteus mirabilis. J. Clin Microbiol. 2002; 40: 1549- 1552.
6. C. Allison, N. Colemanto et al. Ability of Proteus mirabilis to invade human urothelical cells is coupled to mortality and swarming differentiation. Abstract ASM publication. 1992; 6: 11, 4740-4746.
7. R.M. Mordi and M.I. Momoh. Incidence of proteus species in wound infection and their sensitive pattern in the university of Benin Teaching Hospital. African J. Biotechnology. 2009; 8: 725-730.
8. Bernard W. Senior. Proteus, Morganella and Providencia. In: Topley and Wilson’s Microbiology and microbial infection Bacteriology vol 2. S. Peter Borriello, Patrick R. Murray and Guido Funke. Hodder Arnoid. 2005;10thedn: 1435- 1452.
9. Washington C Winn, Stephen D Allen, William M Janda, Elmer W Koneman, Gray Prowp, Paul C Scherckenberg and Gail L Woods. The Enterobacteriaceae. In: Konemans Colour atlas and Text book of Diagnostic microbiology. Lippincott Williams and Wilkins 2006; 6thedn: 267-269
10. J. Gerald Colle, Andrew G. Fraser, Barrie P. Marmian, Antony Simmons. Laboratory strategy in the diagnosis of infective syndromes. In: Mackey and McCartney Practical Medical Microbiology. 1996;14thedn: 68-90. SIGNATURE OF CANDIDATE
NAME AND DESIGNATION OF THE DR. SRINIVASA. H GUIDE PROFESSOR DEPARTMENT OF MICROBIOLOGY ST. JOHN’S MEDICAL COLLEGE BANGALORE – 34
REMARKS OF THE GUIDE
SIGNATURE OF THE GUIDE
HEAD OF THE DEPARTMENT DR. S.MURALIDHARAN HEAD OF THE DEPARTMENT DEPT: OF MICROBIOLOGY ST. JOHN’S MEDICAL COLLEGE BANGALORE – 560034
REMARKS OF THE H.O.D
SIGNATURE OF THE H.O.D