Methodology

Data collection

Data was collected from hospitalized patients and non-hospitalized individuals in the community by questionnaires

 Study population

The study included two groups: group A and group B.

Group A: (hospitalized patients)

One hundred patients who were admitted in the following wards were screened from fecal samples for gastrointestinal carriage of VRE; medical intensive care units (ICUs), pediatrics ICU (surgical, neonatal, or general pediatrics), renal units, and hemato-oncology wards. Their ages range from 1 month up to 80 years. This study was carried out in 8 months during the period from July 2006 to February 2007.                          .

Group B (non-hospitalized individuals)

During the same 8 months, 100 healthy subjects with age ranging from 1 month up to 80 years were recruited from the community and asked to provide a stool specimen. Age, sex, and antibiotic use in the previous 2 years and other relevant data were recorded for all subjects by a questionnaire (see annex).

 Questionnaires

Two questionnaires were used to collect data from hospitalized and non- hospitalized interviews. The first questionnaire was used to evaluating behavior, attitudes and knowledge toward antibiotic usage, a questionnaire was administered to a total of 100 non-hospitalized individuals. The second questionnaire was introduced to 100 patients or their guardians to be filled. Data included, age, sex, medical history, hospital history (including transfers and length of stay), medication history, as well as questions focusing on the degree of illness. Another questionnaire was distributed to hospital physicians to assess to the extent of vancomycin use. 

 Specimen collection

Rectal swabs were taken from bed hospitalized patient and collected by culture swab. The specimens collected from non-hospitalized individuals were placed in wide-mouthed, water-tight, sterile plastic containers.

 Microbiological examination

 Enrichment

Stool specimens or rectal swabs from all subjects were enriched at 45 °C in buffered peptone water in an overnight culture.

 Culture

Bile esculin acid agar, Slantez and Bartley agar, MacConkey agar plates were inoculated with the enrichment broth (37 °C / 48 h). One suspect colony per sample was subcultured on Blood Agar and identified by Gram-staining, catalase-reaction, bacitracin resistance, growth at 45°C and growth in 6.5% sodium chloride broth. Any Gram-positive cocci, bile-esculin-positive, red colony on   Slantez and Bartley agar, was assumed to be enterococci .

 Identification of isolates

Identification of these isolates to species level was performed by API-20 Streptococcus system . For further identification, stock cultures were frozen at −70°C in phosphate-buffered saline with 40% glycerol .

 Antimicrobial susceptibly testing

 Vancomycin susceptibly testing

Resistance to vancomycin 5 μg, 10 μg and 30 μg for all enterococcal isolates was detected by the modified Kirby-Bauer method recommended by the WHO. An inoculum with a turbidity equivalent to that of a 0.5 McFarland standard and Mueller-Hinton agar was used. Plates were read after incubation at 37°C for 24 hour, and the zone of inhibition obtained was measured and compared to that of the manufacturer interpretation charts according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (now known as clinical and Laboratory Standard Institute (CLSI).

 Other antimicrobials susceptibility testing

For all isolates, susceptibility to antimicrobials listed above was performed by the disk diffusion technique.

Vancomycin MIC determination

MIC for Vancomycin was determined for all enterococcal isolates using the microdilution method on Mueller-Hinton broth (MHB) with serial twofold dilutions range between 256 and 0.125 µg/ml, and the results were interpreted according to the standards of the NCCLS.

A. Preparation of antibiotic stock solutions

A vial of Vancomycin hydrochloride 500 mg powder was diluted by 10 ml distilled water. For preparation of stock solutions, from the initial, 1.024 ml of diluted antibiotic was added to 8.976 of sterile distilled water. Suitable range of vancomycin concentrations for enterococci was chosen.

B. Preparation of Inoculum

Colonies were taken directly from the plate into MHB. The suspension should match density of 0.5 McFarland standard.

C. Preparation of the McFarland Standard

A 0.5 ml of 0.048 M BaCl₂ (1.17% w/v BaCl₂·2H₂O) added to 99.5 mL of 0.18 M H₂SO₄ (1% w/v) with constant stirring. The solution was distributed into screw-capped tubes of the same size and volume as those used to prepare the test inoculum. The tubes sealed tightly to prevent loss by evaporation. Stored protected from light at room temperature. The turbidity standard was vigorously agitated on a vortex mixer before use .

 

Preparation of antibiotic dilution range

Vancomycin stock solution was diluted as follows:

Table: Scheme for preparing dilutions of vancomycin used in broth dilution susceptibility test.

Antibiotic solution Step.       Conc.            Source

Volume    +     MHB vol.  =    final conc.

1	5120 µg/ml	stock	1 ml	9 ml	512 µg/ml	9
2 3 4	512 512 512	Step1 Step1 Step1	1 1 1	1 3 7	256 128 64	8 7 6
5 6 7	64 64 64	Step 4 Step 4 Step 4	1 1 1	1 3 7	32 16 8	5 4 3
8 9 10	8 8 8	Step 7 Step 7 Step 7	1 1 1	1 3 7	4 2 1	2 1 0
11 12 13	1 1 1	Step 10 Step 10 Step 10	1 1 1	1 3 7	0.5 0.25 0.125	-1 -2 -3

• Microtiter plates were labeled with the appropriate antibiotic dilutions.           50 μl of antibiotic dilution was added to two rows of wells.
• Fifty μl of test organism was dispensed into one row and 50 μl of control into the second row of wells.
• Inoculated and uninoculated wells of antibiotic-free broth was Included (the first controls for the adequacy of the broth to support the growth of the organism, the second is a check of sterility).

• Microplate was covered by parafilm and incubated at 35-37 ºC for 18- 20 hour in incubator.
• MIC was defined as the lowest concentration that exhibits no growth by visual reading, and the strains were considered susceptible for the vancomycin, if their MICs were below or equal to the critical concentration.

A rapid and inexpensive method for the detection of vancomycin resistance in enterococci by a colorimetric method using 2,3,5-triphenyltetrazolium chloride (TTC) as a redox indicator for antibiotic susceptibility testing of enterococci isolates.

By following the above procedures, 20 μl of (0.01%) 2,3,5-triphenyltetrazolium chloride was added to 30 μl of broth media containing test organism and 50 μl of antibiotic dilution were added to two rows of wells.  Microplate was covered by parafilm and incubated at 35-37 ºC for 18- 20 hour in incubator.

Reduction result in an easily identified color change occurring in cell densities meaningful for MIC testing. Color change occurs when surrounding medium is reduced as a result of bacterial depletion of dissolved oxygen and acid production.   

 Statistical analysis

Data generated from the study was tabulated as Microsoft Excel sheets and uploaded to Statistical Package for Social Sciences (SPSS version 12). Cross tabulation of variables were generated. Chi square was used to detect statistically significant correlation among variables Significance was defined as P ≤ 0.05.