The study was conducted in both Rafedia and Al-Watany hospitals in addition to Patient’s Friends Society (Mostawsaf Al-Rahmeh) in Nablus,Palestine,during the COVID-19 pandemic.
The study was done on 100 purposive urine samples from UTI patients in Rafedia, Al-Watany hospitals, and the Patient’s Friends Society (Mostawsaf Al-Rahmeh) in Nablus, Palestine.
To achieve that ethically, approval was sought from Institutional Review Board (IRB), and Directorate of Health Education, all the research protocols were performed under supervision of the laboratory supervisors in the hospitals and our Department Microbiology Lab at An-Najah National University [no. 17B1].
The urine samples were collected in a special sample container with ice for sample preservation during transporting from the hospitals to the ANNU lab, with an average of 8–10 samples per day taking our and community safety into consideration .
Samples were inoculated immediately after arriving at the university lab on blood and MacConky agar plates after mixing them at room temperature (25), the plates were placed in the incubator for 24 hours at 35–37°C.
On the next day, the bacteria from blood or MacConky plates were distinguished into gram-positive (the once that grown just on the blood agar) and negative (the once that grown on both blood and MacConky plates), and cultured on Uri select media for identification, after one day in the incubator the grown bacteria have been identified according to its characteristics and color referring to instructions and pictures from the manufacturer of the media, the ones that were questionable and not clearly identified from gram-positive; a catalase and coagulase tests were conducted to correctly identify them. while the ones from gram-negative group; an Analytical Profile Index (API) method were used to identify them. Confused bacterial community have been confirmed under the microscope (e.g. one of the samples was confused wheather it was S.agalactiae or Candida albicans, but under the microscope a coccus bacteria in strips was visualized so it was S.agalactiae, another one was suspected to be E.coli or K.pneumoniae; under the microscope a motile bacteria was visualized so its E.coli).
Before performing the antibiotic sensitivity test, a subculture for the samples on nutrient agar has been done to get newly, fresh growing bacteria to perform Disk Diffusion Method (DDM).
A bacterial suspension from the bacteria on nutrient agar with absorbance between 0.08–0.12 was done, from this suspension, using aseptic technique; broth culture of a specific organism was collected with a sterile swab, the excess liquid was removed from the swab by gently pressing or rotating it against the walls of the tube.
A lawn culture was obtained by streaking the swab on Mueller-Hinton agar, and in order to have a uniform culture; the plate was streaked with the swab in one direction, then it was rotated 120° and was streaked again, was rotated another 120° and was streaked one last time.
After that, the antibiotics were placed on the plates, 5 antibiotics were used for gram-positive group (Ampicillin (AMP 10µg), Amikacin (AK 30µg), Imipenem (IPM 10µg), Gentamicin (CN 10µg), ciprofloxacin (CIP 5µg)) and another 5 for gram negative group (Imipenem (IPM 10µg), Gentamicin (CN 10µg), Amikacin(AK 30µg), Ceftazidime(CAZ 10µg), Ciprofloxacin(CIP 5µg)).
After one day the results were obtained in tables by measuring the diameter of the zone of inhibition around each disc of the antibiotics, and referring to disc diffusion supplemental tables, results were classified as resistance(R), intermediate(I), and susceptible(S) [24].
All the procedures were performed under aseptic conditions, and all COVID-19 safety rules were followed in all places, which involve masks, lab coats, and gloves.
The samples and plates were discarded according to the international safety role in autoclavable bags, see Fig. 1.