Bacterial Etiology of Urinary Tract Infection in Delta Medical College and Hospital

Abstract

The urinary tract is one of the most common sites of bacterial infection, particularly in females; 20-30% of women have recurrent urinary tract infection (UTI) at some time in their life. UTIs in men are less common and primarily occur after 50 years of age. Although the majority of infections are acute and short-lived, they contribute to a significant amount of morbidity in the population. Severe infections result in a loss of renal function and serious long term sequelae. In females, a distinction is made between cystitis, urethritis and vaginitis. The purpose of this study was to look into the prevalence of bacteriuria in Delta Medical College. Specimens of urine were examined from 57 inpatients and out-patients and 53 students and staff. Conventional bacteriological techniques were used. Positive cultures were obtained from 21 samples from inpatients and out-patients and positive cultures were also obtained from 13 samples from students and staff. 22 samples from total positive samples showed symptomatic bacteriuria. The results were as follows: Escherichia coli, 25 cases; 73.53%: Klebsiella species, 9 cases; 26.47%. The most common organism was Escherichia coli, followed by Klebsiella species. Antibiogram of these isolates were also done to identify which antibiotic is effective against theses isolates. In this study I have used six types of antibiotics e.g. Amoxycillin, Gentamicin, Ciprofloxacin, Cephradin, Cotrimoxazole and Nalidixic acid. It was showed that Amoxycillin was most effective against Escherichia coli and Klebsiella species and Ciprofloxacin was most effective against Klebsiella species. Clinical studies were made to correlate the bacteriuria and symptoms. Bacteriological examination should be repeated to confirm the diagnosis if patient does not need immediate treatment. Escherichia coli were the commonest organism. This study was helpful for Clinicians and Microbiologists as a base line study of the bacterial etiology of urinary tract infections in Bangladesh.

Introduction

Urinary tract infection is a bacterial infection that affects any part of the urinary tract. It is a condition where one or more parts of the urinary system (The kidney, ureter, bladder and urethra) become infected [1]. UTIs are the most common of all bacterial infections and can occur at any time in the life of an individual. Almost 95% of cases of UTIs are caused by bacteria that typically multiply at the opening of the urethra and travel up to the bladder. Much less often, bacteria spread to the kidney from the blood stream. The main causal agent is Escherichia coli. Although urine contains a variety of fluids, salts and waste product [2]. It does not usually have bacteria in it. When bacteria get into the bladder or kidney and multiply in the urine, they may cause a UTI. The male and female urinary tracts are relatively same except for the length of the urethra. The most common type of UTI is acute cystitis often referred to as a bladder infection. An infection of the upper urinary tract or kidney is known as pyelonephritis and is potentially more serious. Patients should be hospitalized. Although they cause discomfort, urinary tract infections can usually be easily treated with a short course of antibiotics with no significant difference between the classes of antibiotics commonly used.

Methodology

Working place

All the experiments of this project work were carried out in the laboratories of the department of Microbiology, Delta medical college and Hospital.

Area of collection

All the samples were collected from the respective patients of Delta Medical Hospital. Samples were also collected from students and staff from delta Medical College.

Collection of mid-stream (MSU)

The patients were given a sterile, dry wide mouthed, leak –proof container and explained the importance of collecting a specimen with as little contamination as possible. Patients were voluntary agreed to give their sample. Female patients were instructed to clean the area around the urethral opening with clean water, dried the area and collected the urine with the labia held apart. About 20 ml of urine was collected in each container. During collection of samples, Hand gloves, mask, were worn and other precautionary practices were taken for personal safety. Sterile test tubes; sterile, dry, wide mouthed leak proof container, test tube rack, cotton were used to collect the samples aseptically. After collection, the tubes were properly capped, labeled with the date, the name, the identification number and the time of the collection. Then the container with specimen was transported to the Microbiological lab as soon as possible.

Standard procedure of mid-stream urine collection

Washing hands, before urine collection, cleaning the area around penis or vagina. A man should retract the fore skin, if present and cleaning the head of his penis thoroughly with medicated swab. A woman should spread open the fold of the skin around vagina with one hand, then use her other hand to clean the area around her vagina and urethra thoroughly with medicated swab. She should wipe the area from front to back to avoid spreading bacteria to the vagina that is normally found around the anus. After the urine has passed for several seconds, placing the collection container in the stream and collecting about 20 ml of this mid-stream urine without stopping the flow. Should not be touched the rim of the container to the genital area and should not have toilet paper, hair, feces or menstrual blood in the urine sample. If urine is not collected at home and cannot get it to the lab within an hour, the sample should be refrigerated at 4 degree celsius and stored up to 18 hours [3].

The Equipment which were used for culture and sensitivity test in Microbiology laboratory are as follows

Incubator: Bacteria are grown in media usually at 37 degree celcius. So, incubator is used in microbiology laboratory to grow bacteria in media. Incubator is an electrical instrument in which temperature is maintained.

Autoclave: Autoclave is a sterilization instrument by maintaining temperature and pressure. With the help of this instrument the media, petridish, specimen collection bottles, swab stick can be sterilized at 121 degree celsius and 15 pound pressure, keeping for 15 minutes.

Hot air oven: With the help of this instrument conical flask, measuring cylinder, jar, petridish, test tubes are sterilizedat 180 degree celsius for one hour. Temperature can be raised up 24 degree celsius with the aid of Hot air oven.

Sterile petridish: Sterile petridish is used for culture and sensitivity test.

Sterile conical flask: Sterile, dry conical flask is used for taking the powder of media and distilled water to make a media by heating this sterile conical flask.

Sterile measuring cylinder: Sterile, dry measuring cylinder is used for measuring distilled water.

Tripod stand: The sterile, dry conical flask, containing the media is heated with the flame of the spirit lamp by standing on the tripod stand.

Spirit lamp: The conical flask, containing the media is heated with the flame of the spirit lamp. Bacteriological loop is also sterilized with the flame of the spirit lamp.

Bacteriological loop: It is a platinum ring of aluminum rod. With the aid of this loop bacteria are inoculated in media.

Electric balance: It is used to measure the powder of the media.

Reagents were used in this study

Mac-Conkey agra: Base for the isolation and differentiation of Enteric organisms.

Directions: 50.0 gm of the powder was taken in sterile conical flask. 1 L of distilled water was measured by measuring cylinder and poured into the powder. It was mixed thoroughly. Then it was heated with frequent agitation and boiled for one minute to completely dissolve the powder. Auto clave was done at 121 degree celsius for 15 minutes. Overheating was avoided.

Approximate formula per liter:

Pancreatic digest of gelatin: 17g

Paptones: 3g

Lactose: 10g

Bile salt: 1.5 g

Sodium chloride: 5g

Agar: 13.5g

Neutral red: 0.03g

Crystal violet: 0.001g

Mueller Hinton agar: Base for antimicrobial Disk diffusion susceptibility testing:

Direction: 38 g of the powder was taken in sterile conical flask. 1L of Distilled water was measured by measuring cylinder and poured in to the powder. It was mixed thoroughly. Then it was heated with frequent agitation and boiled for one minute to completely dissolve the powder. Autoclave was done at 121 degree celsius for 15 minutes. Overheating was avoided.

Approximate formula per liter:

Beef extract powder: 2.0g

Acid digest of casein: 17.5g

Starch: 1.5g

Agar: 17.0g

Nutrient Agar: Base for the cultivation a wide variety of microorganism.

Direction: 28 g of the powder was taken in sterile conical flask. 1 L of Distilled water was measured by measuring cylinder and poured in to the powder. It was mixed thoroughly. Then it was heated with frequent agitation and boiled for one minute to completely dissolve the powder. Autoclave was done at 121 degree celsius for 15 minutes. Overheating was avoided.

Approximate formula per liter:

Agar: 15.0 g

Veg. peptone: 5.0 g

Sodium chloride: 5.0 g

Veg. Extract: 1.5 g

Yeast Extract: 1.5 g

Triple Sugar iron

Triple Sugar Iron is only a screening device. The important components of this medium are ferrous sulfate and the three sugars glucose, lactose and sucrose. The glucose is present in one –tenth the concentration of the other two sugars. The medium in the tube has a solid, poorly oxygenated area on the bottom, called the butt and an angled, well –oxygenated area on top, called the slant. The organism was inoculated into the butt and across the surface of the slant.

Examination of urine

First day: Physical, chemical and microscopic examinations were carried out in doing this study.

Physical examination includes: Color, appearance, specific gravity

Chemical examination includes: Reaction, the presence of proteins, the presence of reducing substance, the presence of ketone body, bilirubin, bile salts, and blood.

Microscopic examination of urine: In Microbiology laboratory microscopic examination is done to find out presence of organized and unorganized deposits, epithelial cells, pus cells, red blood cells, pus cells and epithelial cells. In severe condition calcium oxalate, crystals, triple phosphate crystals are also examined by microscopic examination. But in my study I did microscopic examination to find out pus cells, red blood cells and epithelial cells. Urine was centrifuged at 3000 rotation per minute for five minutes in centrifuged machine. Slide was cleared with cotton and then numbered according to the identification number of the patient. Then one drop of urine was poured on to the slide from the test tube and covered this drop by cover slip. Then slide was examined under 40x objective of Microscope.

Inoculation and incubation

For urine sample: First of all equipment was autoclaved which were needed in doing this study the previous day. Then a bacteriological loop was sterilized, which held about 0.001ml of urine. According to conventional method streak plate technique was used to inoculate the samples on Mac-Conkey media and Blood agar for isolation of microorganisms and all the plates were incubated at 37oC for 24 hours.

Counting and recording: Plates containing less than 104 colonies were counted as not significant bacteriuria and also pus cells were not significantly present. Plates containing more than 105 colonies were counted as significant bacteriuria with significantly presence of pus cells.The counts were recorded as colony forming unit (cfu)/per g. The number of bacteria per ml of original culture was calculated by multiplying the number of colonies counted by the dilution factors divided by the volume of sample used for spreading. That is, Number of cells per ml = number of colonies x dilution factor/volume of sample used =cfu/g.

Isolation of microorganism

Following microbiological media were used for isolation of different pathogenic bacteria from the samples:

MacConkey agar (MAC): MacConkey agar was used for the isolation of gram-negative bacteria and used for differentiate between lactose fermented & non-lactose fermenter.

Nutrient agar (NA): contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.

Motility test: Motility test was used for differentiate between motile and non-motile bacteria.

Storage of the isolated microorganisms

After inoculation, the isolated microorganisms were refrigerated at 4 degree celsius for further testing.

Identification of microorganisms

Identification of bacteria: Both primary and confirmative identification of bacteria was performed.

Primary identification of bacteria: Primary identification of bacteria was performed based on the selectivity of media, change of media after growth, morphological characteristics of the colonies and microscopic characteristics.

Colony morphology: Morphological characteristics including size, shape, surface, texture, edge, elevation, color, opacity etc from different types of colonies in different culture media were observed and recorded.

Confirmative identification of bacteria: Confirmative identification was accomplished based on biochemical identification.

Several biochemical tests were performed according to the manual of methods for general Bacteriology by American Society of Microbiology (ASM, 1981) to identify the bacteria. The following biochemical tests were included.

Triple sugar iron (TSI) test: This media was used for initial identification of gram-negative bacilli. Three primary characteristics of a bacterium detected by this media ability to produce gas.

Eosine-methylene blue (EMB) test: this media was used for E.coli bacteria by green metalic sheen.

Antibiotic susceptibility test

Antibiotic susceptibility test of the identified organisms was determined by Kirby- Bauer method using Mueller-Hinton agar medium (Barry and Thornberry, 1985). Antibiotic potency of antibiotic discs used was given.

Potency of antibiotic discs used (Oxoid, UK) (Table 1).