MOLECULAR CHARACTERIZATION OF PATHOGENIC ORGANISMS ISOLATED FROM URINARY TRACT INFECTIOUS

PROTOCOL FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

4TH T BLOCK, JAYANAGAR

BANGALORE, KARNATAKA

BY

MANSOOR P.P

M. PHARM PART-I

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,

BHARATHICOLLEGE OF PHARMACY,

BHARATHI NAGAR.

UNDER THE GUIDANCE OF

Dr. K. N. SHANTI, Ph.D.

PROFFESSOR,

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,

BHARATHICOLLEGE OF PHARMACY,

BHARATHI NAGARA,

KARNATAKA-571422.

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the Candidate and Address (In Block Letters) / MANSOOR P.P
S/O USMAN KOYA P.P
SAFIYA MANZIL
PARAVANNA
MALAPPURAM
KERALA-676 502
2. / Name of the Institution / BHARATHICOLLEGE OF PHARMACY,
BHARATHI NAGARA,
MANDYA- 571422.
3. / Course of Study and Subject / MASTER OF PHARMACY IN PHARMACEUTICAL BIOTECHNOLOGY.
4. / Date of Admission of Course / 30-JUNE-2008
5. / Title of Topic / MOLECULAR CHARACTERIZATION OF PATHOGENIC ORGANISMS ISOLATED FROM URINARY TRACT INFECTIOUS
6. / Brief Resume of the Intended Work
6.1 Need for the study
6.2 Review of the literature
6.3 Objectives of the study / ENCLOSURE-I
7 /

Materials and Methods

7.1 Source of data

7.2 Method of collection of data

7.3 Does study require any investigations or interventions to conduct on patients or other human or animal? If so, please describe briefly.

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

/ ENCLOSURE-II
8 / List of References / ENCLOSURE-III

ENCLOSURE-I

6. BRIEF RESUME OF THE INTENDED WORK:

6.1 NEED FOR STUDY:

Urinary tract infection (UTI) is one ofthe most common infectiousdiseases diagnosed in patients. It is estimated that 3–5million office visits occur yearly owing to lower UTIin theUnited States6. The urinary system consists of the kidneys, ureters, bladder, and urethra. The urinary tract is normally sterile and does not have bacteria present. If bacteria enter the urinary tract, they multiply and cause a urinary tract infection. Escherichia coli is the most common bacteria causing urinary tract infections5. Most pathogens responsible for UTI are enterobacteriaceae with a high predominance of Escherichia coli. This is especially true of spontaneous UTI in females (cystitis and pyelonephritis). Other strains are less common, including Proteus mirabilis and Staphylococcus saprophyticus8. The urinary tract Humans is a common site of Pseudomonas aeruginosa infection, which frequently follows catheterization, cytoscopy and operations on the bladder.

Enterobacteriace does not express a detectable phenotype when recovered from the urinary tract or after in vitro subculture14. Infections caused by antibiotic-resistant Enterococci are increasing and are becoming difficult to manage due to the diminishing number of available antibiotics to treat them4. The microorganisms are well-diversified character, to identifying the microorganisms by phenotypical is not much effective. Molecular characterization will help to analyses the diversified character and help full for selecting the antibiotics to cure the diseases. This research helps to analyses the pathogenicity and antimicrobial property by genetic level.

6.2REVIEW OF LITERATURE:

1. Ambulatory Care Visits to Physician Offices, Analyzed in many cases, bacteria first travel to the urethra. When bacteria multiply, an infection can occur. An infection limited to the urethra is called urethritis. If bacteria move to the bladder and multiply, a bladder infection, called cystitis, results. If the infection is not treated promptly, bacteria may then travel further up the ureters to multiply and infect the kidneys. A kidney infection is called pyelonephritis. Microorganisms called Chlamydia and Mycoplasma may also cause UTIs in both men and women, but these infections tend to remain limited to the urethra and reproductive system. Unlike E. coli, Chlamydia and Mycoplasma may be sexually transmitted, and infections require treatment of both partners.2

2. Acar, and Goldstein, Reported approximately70.0% of the E. coli resistant to quinolones were collectedfrom community-acquired UTIs. It could reflect the overuse ofthe quinolones for treatment of community-acquired UTI. Someauthors have advocated that quinolone resistance is higher indeveloping countries than in developed nations because of theuse of less active quinolones, such as nalidixic acid, and/orthe use of low dosages of more potent compounds such as ciprofloxacinresulting in selection of mutant isolates.1

3. Sharon, et al., Found when bacteria get into the bladder or kidney and multiply in the urine, they cause a UTI. The most common type of UTI is a bladder infection which is also often called cystitis. Another kind of UTI is a kidney infection, known as pyelonephritis, and is much more serious. Although they cause discomfort, urinary tract infections can usually be quickly and easily treated with a short course of antibiotics.(Urinary Tract Infections". Retrieved on 2007-08-25). D-mannose would be present in sufficient concentration in the urine, it could interfere with the adherence of the bacterium E. coli to the epithelial cells lining the urinary tract (similar to the mechanism underlying the effect of cranberry juice.12

4. Bonacorsi, et al., Reported molecular approach indicates that uncircumcised male infants are at higher risk for infection with highly virulent uropathogenic E. coli strains than are females. Preputial colonization may have a key role in the selection of such strains.3

5. Linda LH, and Freston JW, Found diagnosis of urinary tract pathogens is based on the quantization of bacteria in the urine. The culture is taken from midstream, clean catch urine specimen. As rule yielding >105 CFU/ml is considered as positive (significant level of organisms).7

6. Gales et al, Worked on the potency and spectrum of various antimicrobial agents tested against 434 bacterial isolates causing urinary tract infection (UTI) in hospitalized patients in Latin America were evaluated. The genotypes of the extended-spectrum β-lactamase-producing and selected multi-resistant Isolates were also evaluated by molecular typing techniques. Escherlchla coil (60.4%) was the most common etiological agent causing UTI, followed by Klebsiella spp.(11.2%) and Pseudomonas aeruginosa (8.3%). In contrast, Enterococcus spp. isolates caused only 2.3% of UTIs. Fewer than 50% of E. coli isolates were susceptible to broad-spectrum penicillins. The resistance rates to ciprofloxacin and the new quinolones were also high among these isolates. The molecular characterization of ciprofloxacin-resistant E. coli showed that most of them have a double mutation in the gyrA gene associated with a single mutation in the parC gene. The Klebsiella pneumoniae isolates studied demonstrated high resistance rates to β-lactam drugs, including broad-spectrum cephalosporins. The carbapenems were the compounds with the highest susceptibility rate among these isolates (100.0% susceptible) followed by cefepime (91.7% susceptible). Meropenem, imipenem and cefepime were also the most active drugs against Enterobacter spp. Among P. aeruglnosa isolates, meropenem (MICA, 2 mg/L) was the most active compound, followed by imipenem (MICA, 4 mg/L), cefepime (MICA, 8 mg/L) and ceftazidime (MICA, 16 mg/L). The results presented in this report confirm that bacterial resistance continues to be a great problem in Latin American medical institutions.11

7. Norrby et al., Studied the comparative effectiveness of norfloxacin and co-trimoxazole in urinary tract infections. They observed norfloxacin extremely effective against Gram-negative urinary tract isolates, but Gram positive urinary tract isolates were more susceptible to co-trimoxazole than to norfloxacin.10

8. Hoda Helmi et al.,The molecular basis of carriage of resistance genes to high levelaminoglycosides and vancomycin and the genetic relatedness between the strains were also studied. A total of 120 isolates of antibiotic resistant Enterococci were investigated. Minimum inhibitory concentrations(MIC) to high-level aminoglycoside and glycopeptide antibiotics weredetermined by agar dilution andE-test methods respectively. Genesencodingaminoglycoside modifying enzymes (AMEs) and vancomycinresistance determinants were detected by polymerase chain reaction (PCR).4

6.3 OBJECTIVES:

  • To isolate and identification of pathogenic organism from urine samples
  • Estimation of antimicrobial activity
  • Molecular characterization of isolated organism

ENCLOSURE-II

7. MATERIALS AND METHODS:

  1. To isolate and identification of pathogenic organism from urine samples
  2. Collection of urine samples from hospital
  3. Isolate organism by agar plating method
  4. Identification of organism Bergyal’s manual
  5. Estimation of antimicrobial properties by difco agar method

2. Estimation of antimicrobial activity

3. Molecular characterization of isolated organism

  • Isolation of DNA
  • Restriction digestion of isolated DNA
  • Amplify the DNA by random primers
  • Analysis the result by using statica software

7.1 SOURCE OF DATA:

1. Bharathi college of pharmacy library,Bharathi Nagara.

2. E-Library from BharathiCollege of pharmacy.

3. IISCLibrary, Bangalore

7.2 METHOD OF COLLECTION OF DATA:

The preliminary data required for the experimental study were obtained from

1. Internet

2. Scientific Abstracts

3. Scientific Journals

4. Relevent Books

7.3 Does the study require any investigations or interventions to conduct on patients or other human or animal? If so, please describe briefly.

-NO-

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

-NOT APPLICABLE-

ENCLOSURE-III

  1. LIST OF REFERENCES:

1)Acar J F. & Goldstein F W, Trends in bacterial resistance to fluoroquinolones. Clinical Infectious Diseases, 1997; 24: S67–73.

2)Ambulatory Care Visits to Physician Offices, Hospital Outpatient Departments, and Emergency Departments: United States. Vital and Health Statistics. 1999–2000; 13: 157.

3)Bonacorsi, Lefebvre S, Clermont O, HoudouinV, Bourrillon A, Loirat C, Aujard Y, Bingen E. Escherichia coli strains causing urinary tract infection in uncircumcised infants resemble urosepsis-like adult strains. The Journal of Urology, 2000; 173: 195-197.

4)Hoda Helmi, Laila AboulFadl, Samah Saad El-Dine and Inas El-Defrawy, Molecular Characterization of Antibiotic Resistant Enterococci. Research Journal of Medicine and Medical Sciences, 2008, 3(1): 67-75.

5)Jean M. Kallhoff, ARNP, Diseases and conditions urinary tract infection(UTI) Last updated, 2006

6)Johnson JR, Stamm WE,Urinary tract infections in women: Diagnosis and treatment Annals of Internal Medicine [ANN. INTERN. MED.]. 1989; 111(11): 906-917.

7)Linda LH, and Freston JW, Diseases: Manifestations and Pathophysiology. IN: Remington Pharmaceutica Sciences. 16th ed. Mack Publishing Company. Pennsylvania. 1980; 615

8)Meyrier A, Guibert.J: Diagnosis and drug treatment of acute pyelonephritis. Drugs1992, 44:356–367

9)Meyrier.A: Diagnosis and management of renal infections. Curr Opin Nephrol Hypertens 1996, 5:151–157.

10)Norrby and Urinary tract infection group, Co-coordinated multicenter study of Norfloxacin vs. Co-trimexazol treatment in symptomatic urinary tract infection. The J. of Infectious Diseases; 1987; 155 (2): 170-77.

11)Sader HS, Gales AC., Schomberg L, Jones R.N and SENTRY Participants Group. Comparative evaluation of frequencies and antimicrobial susceptibilities ofpathogens isolated from urinary tract infection (UTI) in North America (NA) and Latin America (LA): Results of the SENTRY Antimicrobial Surveillance Program, 1997.InProgram and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. American Society for Microbiology, Washington, DC. , 1998; 39: 44

12)Sharon N, Eshdat Y, Silverblatt FJ, Ofek, "Bacterial adherence to cell surface sugars". Ciba Foundation symposium,1981; 80: 119–41.

13)Toyota S, Fukushi Y, Katoh S, Orikasa S, Suzuki Y. "[Anti-bacterial defense mechanism of the urinary bladder. Role of mannose in urine]" (in Japanese). Nippon Hinyōkika Gakkai zasshi. The japanese journal of urology 1989; 80 (12): 1816–23.

14)Wullt B., Bergsten G, Connell H, Rollano, P. Gebretsadik N, Hull, R. And Svanborg C. P fimbriae enhance the early establishment of Escherichia coli in the human urinary tract. Mol. Microbiol. 2000. 38:456-464.

15)Eshdat Y, Silverblatt FJ, and Sharon N Dissociation and reassembly of Escherichia coli type 1 pili.J Bacteriol. 1981; 148(1)308-314

9.
10.

11.
12. /
Signature of the candidate:
(MANSOOR P.P)

Remarks of the guide: The research programme may helps to understand
the problems associated with urinary tract
infection and the remedy
Name And Designation of:
Dr. K.N. SHANTI, Ph.D.
11.1 Guide Professor,
Department of Pharmaceutical Biotechnology
BharathiCollege of Pharmacy,
Bharathinagara,
Karnataka-571422.
11.2 Signature
11.3 Co-Guide
Dr.GURUKAR MATHEW.S, Ph.D.
11.4 Signature
11.5 Head of the department Dr.GURUKAR MATHEW.S, Ph.D.
Professor,
Department of pharmaceutical Biotechnology,
Bharathi college of pharmacy,
Bharathinagara,
Karnataka-571422.
11.6 Signature
12.1 Remarks of the Chairman and Principal: Recommended for approval
12.2 Signature Dr. Tamizh Mani. T. Ph.D.
Principal,
BharathiCollege of pharmacy,
Bharathinagara,
Karnataka-571422.