Determination Of Antibiotic Susceptibility Biology Essay

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The study comprised one thousand one hundred and fifty three female subjects visiting Jinnah Hospital Karachi, FON General Hospital Karachi, APWA Hospital and Maternity Home Karachi, Al-Khidmat Welfare Hospital Karachi and females residing in different localities of Karachi. The subjects who had history of renal stones or having history of use of antibiotics, hospitalization or catheterization during previous one month were not included in the study. The subjects were categorized into two groups on the basis of symptoms suggesting UTI noted at the time of completion of subject information questionnaire like urgency, frequency, dysuria, nocturia, flank pain and a foul odor of urine etc.

Group A: included subjects having at least one of the symptoms suggesting UTI.

Group B: included subjects having no symptom suggesting UTI.

SUBJECT INFORMATION QUESTIONNAIRE

At the time of collection of urine specimens, all the subjects were interviewed and a questionnaire (Appendix I) was completed which consisted of medicodemographic details such as name, age, physiological age group, marital status, pregnancy, diabetic status, blood pressure and symptoms of UTI etc.

CATEGORIZATION OF SUBJECTS WITH RESPECT TO MEDICODEMOGRAPHIC CHARACTERISTICS

Following medicodemographic characteristics were taken into consideration for categorization of all subjects to group A and B.

Age:

The subjects were categorized into age groups of <15, 15-25, 25-35, 35-45, 45-55, 55-65 and >65 years.

Physiological age groups:

On the basis of menstruation, the subjects were classified into following three groups:

Premenarcheal: Subjects before the onset of first menstruation (menarche) were included in this group.

Reproductive: Subjects from postmenarche till the onset of menopause were included in this group.

Postmenopausal: The subjects after the onset of menopause were included in this group.

Marital status:

The subjects were classified into three groups: single, married, widow/divorced.

Pregnancy:

For the categorization on the basis of pregnancy, only married females were included and grouped as pregnant and non-pregnant.

Diabetes:

All the subjects were grouped into diabetic (patients having history of diabetes) and non-diabetic.

Blood pressure:

On the basis of history of blood pressure, the subjects were classified into three groups; hypotensive subjects, hypertensive subjects and subjects with normal blood pressure.

COLLECTION OF SPECIMENS

Before collection of the urine specimen, the subjects were instructed to clean the genital area carefully with non-antiseptic soap and water. Then, freshly voided midstream urine specimen was collected in a sterile wide mouthed container.

SPECIMEN TRANSPORTATION

Urine specimens were immediately transported to the Department of Microbiology, University of Karachi, Karachi to be processed within one hour of collection. If delay was suspected, the preservative boric acid (0.1g/10 ml of urine) was added to prevent multiplication of bacteria (Cheesbrough, 2000).

ANALYSIS OF SPECIENS

All the urine specimens were subjected to urinalysis, culture for quantitation and qualitative assessment of bacteria.

URINALYSIS

Urine analysis included physical, chemical and microscopic examinations. The Combur 10 test urine strips (Boehringer-Knoll Limited) were used for urine analysis and results of albumin and glucose were considered for the study. If required, then results for albumin and glucose were checked by heat and acidification test and Benedict's qualitative and quantitative test.

Heat and acidification test:

After centrifugation of urine, 3 ml of supernatant was transferred to a 10 x 100 mm test tube. The upper part of urine column was boiled thoroughly by heating the side of the tube over flame while moving it back and forth in the flame to avoid bumping. If turbidity appeared it might be due to albumin, urates or phosphates. Whether the turbidity appeared or not, 2-3 drops of 50% (v/v) acetic acid was added and heated again over flame. The turbidity was noted visually and graded as '0' to '4+'. The results were interpreted as follows (Sonnenwirth and Jarett, 1980).

S. No.

Observations

Interpretation

1

If turbidity did not appear after first and second heating.

Albumin, urates or phosphates were absent.

2

If turbidity appeared after first heating.

Albumin, urates or phosphates may be present.

3

If turbidity appeared after first heating and disappeared after addition of 50% acetic acid and did not appear after second heating.

Albumin and urates were absent, phosph-ates were present.

4

If turbidity appeared after first heating and disappeared after addition of 50% acetic acid and reappeared after second heating.

Albumin and phosph-ates were present.

5

If turbidity appeared after first heating and decreased after addition of 50% acetic acid and persisted after second heating.

Albumin and phosph-ates were present.

6

If turbidity appeared after first heating, persisted after addition of 50% acetic acid and disappeared after second heating.

Urates were present.

7

If turbidity appeared after first heating and did not decrease or disappeared after addition of 50% acetic acid and persisted after second heating.

Albumin was present, urates or phosphates were absent.

8

If turbidity did not appear after first heating and appeared after second heating.

Albumin was present, urates or phosphates were absent.

Benedict's qualitative test:

Five ml Benedict's qualitative reagent was taken in a test tube. Eight drops (0.4ml) of urine were added in the tube and mixed. This tube was placed in boiling water bath for five minutes. After 5 minutes the tube was removed from water bath and change in colour was observed and graded from "0'' to "4+" as follows (Cheesbrough, 2000).

Appearance of reaction

Grades

Sugar concentration (%)

Blue, clear or cloudy

Nil

0

Green with no precipitate

Traces

<0.5

Green with precipitates

+

About 0.5

Brown and cloudy

++

About 1.0

Orange and cloudy

+++

About 1.5

Red and Cloudy

++++

 2.0

Benedicts quantitative test:

When the Benedict's qualitative test was positive, Benedict's quantitative test was performed.

Five ml Benedict's quantitative solution was taken in a 50 ml flask and approximately 1 gm sodium carbonate was added to it. Then, 10-20 ml distilled water was added. The flask was heated to boil and drop-wise urine was added with occasional shaking till the appearance of white precipitates. Volume of urine required for the appearance of white precipitates was noted and amount of glucose (%) was calculated by the following formula (Sonnenwirth and Jarett, 1980).

Urinary glucose (%) = 1/x

where, x = volume of urine required.

Microscopic examination:

Microscopic examination was performed for the detection of unorganized deposits (amorphous forms and crystals) and organized deposits (pus cells, erythrocytes, casts, epithelial cells, bacteria and others). The urine specimen was thoroughly mixed and 10 ml of urine was transferred to a conical centrifuged tube. It was centrifuged at 1500 rpm for 5 minutes. After centrifugation supernatant was discarded with a single smooth action and sediment was resuspended in remaining drop of supernatant with several firm finger strokes. A drop of sediment was transferred onto a microscopic glass slide and overlaid with coverslip. Several fields were examined immediately before evaporation for search of erythrocytes, pus cells, epithelial cells, cast, crystals etc. If pus cells, erythrocytes or casts were found, the count was performed in several fields. For pus cells and erythrocytes, the count was recorded as per high power field (HPF) and for casts as per low power field (LPF) (Sonnenwirth and Jarett, 1980).

CULTURE

Media:

MacConkey's agar (Oxoid) and Blood agar media were used for quantitation and primary isolation of microorganisms. Blood agar base (Oxoid) was used for the preparation of blood agar medium. All the plates were incubated at 35-37oC for 18-24 hours to check the sterility.

Quantitation for significant bacteriuria:

Quantitation was performed by standard calibrated loop method. A standardized loop delivering 0.002 ml of urine was used. It was dipped vertically into the urine. Then urine was spreaded onto the surface of a Blood agar medium for total count and MacConkey agar medium for count of Gram-negative bacteria. After incubation at 35-37oC for 18-24 hours, plates were examined and colonies were counted. The total number of bacteria per ml of urine was obtained by multiplying the number of colonies with 500 (volume factor) (Cheesbrough, 2000). Delivery of loop was periodically checked by pour plate method (Baron et al., 1994).

Qualitative assessment

Different types of isolated colonies were picked and streaked on fresh media plates. All plates were incubated at 35-37oC for 18-24 hours. After incubation, the isolated colonies were transferred to nutrient agar slants or blood agar slants to get pure cultures for storage in refrigerator at 4oC. All the pure cultures were subjected to characterization by using different tests conforming to required standard diagnostic criteria (Baron et al. 1994; Holt et al., 1994; Cheesbrough, 2000). The criteria included study of morphological, cultural, biochemical and physiological characteristics.

Statistical analysis

Chi-square (2) test (Essex-Sorlie, 1995) was applied for the comparison of predominant organism isolated from symptomatic and asymptomatic UTI with respect to different medicodemographic characteristics.

DETERMINATION OF ANTIBIOTIC SUSCEPTIBILITY

Antibiotic susceptibility testing was performed using disc diffusion method as described by National Committee for Clinical Laboratory Standards (Presently called as Clinical Laboratory Standards Institute) (Cheesbrough, 2000).

Media:

Mueller Hinton Agar (MHA) (Merck) was employed for determination of antimicrobial susceptibility test and Mueller Hinton Broth (MHB) (Merck) was used for preparation of inoculum.

Preparation of plates for antimicrobial susceptibility

MHA was poured into sterile petri plates to get a depth of 4 - 6 mm. These plates were incubated at 35-37 oC for 18-24 hours to check the sterility of plates. The plates were stored at 4 oC and were used within two weeks. Prior to use, plates were dried in an incubator to facilitate the removal of excess surface water.

Antibiotic discs

Different antibiotic discs i.e. streptomycin, gentamicin, kanamycin, neomycin, tetracycline, ofloxacin, ciprofloxacin, amoxicillin-clavulanate, norfloxacin, rifampin, nalidixic acid, doxycyclin, clindamycin, sulfisoxazole, nitrofurantoin and trimethoprim-sulfamethoxazole were used for antibiotic susceptibility test (Appendix II).

Preparation of turbidity standard

McFarland Nephlometer standard tube number 0.5 was used to standardized the turbidity of test inoculum. Since inoculum was prepared in MHB, 1% (v/v) sulphuric acid in MHB and 1.175% (w/v) aqueous solution of barium chloride (BaCl2.2H2O) were prepared in order to estimate bacterial cell density (Sonnenwirth and Jerett, 1980; Baron et al., 1994). For the preparation of turbidity standard 0.05 ml 1.175% barium chloride solution was added to 9.95 ml 1% sulphuric acid solution slowly and with constant agitation. The tube was sealed and stored in dark at room temperature (Baron et al., 1994).

Inoculum

A loopfull from pure growth of organisms was transferred to 5 ml of MHB. The broth was incubated at 35-37oC for 18-24 hours. After incubation, the turbidity of the culture was compared with 0.5 McFarland Nephlometer Standard to get approximate cell density 150 x 106 CFU/ml. The standardized inoculum suspension was inoculated within 15-20 minutes.

Inoculation of medium

A sterile cotton swab was immersed into the standardized inoculum suspension. Excess broth was drained by pressing and rotating the swab against the inside of the suspension tube. Then, it was streaked evenly in three directions on the surface of agar plates. A final circular motion was made around the agar rim with the cotton swab. These plates were allowed to dry for 3-5 minutes.

Disc placement

Antibiotic susceptibility discs were placed on the surface of inoculated MHA plates by using a sterile forcep. After placement the discs were pressed gently to the agar surface.

Incubation

The inoculated plates were incubated at 35-37oC for 18-24 hours.

Interpretation

Interpretative criteria for each antibiotic tested were those recommended by the NCCLS (Cheesbrough, 2000). Inhibition zone diameters were measured with a ruler under reflected light. The susceptibility or resistance was interpreted on the basis of criteria mentioned in Appendix II.

SCREENING OF ANTIMICROBIAL ACTIVITIES OF AQUEOUS INFUSIONS AND DECOCTIONS OF EMBLICA OFFICINALIS AND CORIANDRUM SATIVUM

Screening of antimicrobial activities of aqueous infusion and decoctions of dried fruits of E. officinalis and seeds of C. sativum was performed by well diffusion method.

PREPARATION OF AQUEOUS INFUSIONS

Aqueous infusions of E. officinalis and C. sativum were prepared by steeping 20g in 100 ml sterile distilled water in separate sterile flasks. The flasks were kept for two days with occasional shaking. The contents of flasks were filtered.

PREPARATION OF AQUEOUS DECOCTIONS

Aqueous decoctions of E. officinalis and C. sativum were prepared by boiling 20g in 100 ml sterile distilled water for 15 minutes. The flasks were then plugged and removed from heat and allowed to cool. After cooling the contents of flasks were filtered.

Media

Mueller-Hinton agar (MHA) (Merck) was used as base medium for screening of antibacterial activity and Mueller-Hinton broth (MHB) (Merck) was used for preparation of inoculum.

Well diffusion technique

Screening of antibacterial activity was performed by well diffusion technique. The MHA plates were inoculated with 0.1 ml of the standardized inoculum (150 x 106 CFU/ml) of each test organism. The inoculum was spread evenly over the surface of medium with loop or sterile glass spreader. A standard cork borer of 8 mm diameter was used to cut uniform wells on the surface of the MHA and 100 µl of each infusion and decoction of E. officinalis and C. sativum was introduced in the well (Kivanc & Kunduhoglu, 1997).

Incubation

The inoculated plates were incubated at 35-37 oC for 18-24 hours and inhibition zone diameter was measured to the nearest millimeter (mm).

Statistical analysis

Mean inhibition zone diameter and standard deviations were calculated (Essex-Sorlie, 1995).

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC) AND MINIMUM BACTERICIDAL CONCENTRATION (MBC) OF INFUSION AND DECOCTION

When positive antimicrobial activity was noted in screening, then minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of aqueous infusion and decoction were determined by using tube dilution assay (Baron et al, 1994).

Dilution of standardized inoculum

The standardized inoculum (150 x 106 CFU/ml) was diluted 150 times to reduce the number of organisms to 1 x 106 CFU/ml. This was test organism suspension.

Positive growth control

One ml of test organism suspension (1 x 106 CFU/ml) was mixed with 1 ml MHB. This tube contained 5 x 105 CFU/ml.

Checking of bacterial cell density of positive growth control

To check bacterial cell density of positive growth control, 0.5 ml of test organism suspension from positive growth control tube was mixed with 0.5 ml MHB to get 25 x 104 CFU/ml. Then 0.001 ml from this tube was spread on MHA plate and incubated at 35-37oC for 18-24 hours. After incubation, colony count was performed. If number of colonies was 250 CFU, the bacterial cell density of positive growth control was considered equivalent to 5 x 105 CFU/ml.

Preparation of test for infusion/decoction

Aqueous infusion and decoction were considered as 100%. The different concentrations infusion and decoction were prepared as per following protocol.

Contents

Amount in ml

Aqueous infusion/decoction

1

0.8

0.5

0.25

0.1

0.05

0.025

Mueller Hinton Broth

-

0.2

0.5

0.75

0.9

0.95

0.975

Standardized inoculum

1

1

1

1

1

1

1

Total volume

2

2

2

2

2

2

2

Final concentration

50%

40%

25%

12.5%

5%

2.5%

1.25%

Negative growth control

Two sets of negative growth control were prepared. One set for infusion and another for decoction. After preparation of different concentrations of infusion or decoction 1 ml Mueller Hinton Broth was added instead of inoculum.

Incubation

All the tubes were incubated at 35-37oC for 18-24 hours.

DETERMINATION OF MIC

After incubation, the tubes were examined visually for turbidity. Different concentrations of infusion and decoction were matched with their respective negative growth controls. The lowest concentration which showed lack of turbidity visually was designated as MIC.

DETERMINATION OF MBC

Subcultures were performed on MHA medium by inoculating 0.01 ml of all those tubes which showed no turbidity on visual examination. After incubation at 35-37oC for 18-24 hours results were observed in terms of growth. The concentration of infusion and decoction which did not show the growth or showed colony count less than 0.1% of the original inoculum (5 x 105 CFU/ml) was designated as MBC (Baron et al., 1994).

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