Study site, period and socio-demographic data
The present study was a single institutional cross-sectional study carried out at Arsho Advanced Medical laboratory, Addis Ababa, Ethiopia from June 2015 to May 2016. Willingness to participate in the study, presumptive diagnosis of urinary tract infection and no history of antibacterial therapy within 2 weeks prior to their attendance were the inclusion criteria. The requisition form filled up by physicians was used as standard proforma to document socio-demographic information of study subjects. Age groups of patients were classified following WHO guideline .
Sample collection and inoculation of primary isolation culture media
Clean-catch midstream urine was collected from patients complaining of UTI; referred from different health institutions in the city with sterile wide-mouthed urine cup. Urine samples were inoculated onto Blood Agar base (Oxoid, Basingstoke, Hampaire, UK) to which 10% sheep blood is incorporated and Cysteine Lactose Electrolyte Deficient medium (Oxoid, Basingstoke, Hampaire, UK) using a calibrated loop with a capacity of 1 μl in safety cabinet. All inoculated plates were incubated at 37 °C for 18–24 h aerobically and the number of colonies was counted. Colony counts yielding bacterial growth of >105/ml of urine (≥100,000 colonies) were regarded as significant for bacteriuria. Urine samples yielded three and more bacterial species were not considered for further investigation. Pure isolates of bacterial pathogen were preliminary characterized by colony morphology, Gram-stain, and catalase test before inoculating them into AST-GN72 and AST-GP71 cards.
Inoculum size determination
Quality control bacteria and pure cultures of bacterial isolates were suspended in 3 ml of sterile saline (aqueous 0.45 to 0.50% NaCl, pH 4.5 to 7.0) in a 12 × 75 mm clear plastic (polystyrene) test tube to achieve a turbidity equivalent to that of a McFarland 0.50 standard (range, 0.50 to 0.63), as measured by the DensiChek (bioMe’rieux) turbidity meter. These suspensions were used for the inoculation of GN72 and GP71 identification cards while AST cards were inoculated after bacterial suspensions were further diluted following the instruction of the manufacture.
Identification and determination of antimicrobial susceptibility
Species identification and antimicrobial susceptibility testing of Gram-positive and Gram-negative bacteria were determined with the automated VITEK 2 compact system (bioMérieux, France) using AST, GN72 and GP71 cards. The VITEK 2 compact system (bioMe’rieux) is an integrated modular system that consists of a filling-sealer unit, a reader-incubator, a computer control module, a data terminal, and a multicopy printer. The system detects bacterial growth and metabolic changes in the microwells of thin plastic cards using a fluorescence-based technology.
AST-GN72 cards were used for the identification and susceptibility testing of none-spore-forming, fermenting, and non-fermenting Gram-negative bacilli while the AST-GP71 cards were used for the automated identification and susceptibility testing of non-spore-forming Gram-positive bacteria. The cards were automatically filled by a vacuum device and were automatically sealed and subjected to a kinetic fluorescence measurement in accordance with the manufacturer’s instructions. Brief, identification cards were inoculated with quality control bacteria and pure cultures of bacterial isolate suspensions using an integrated vacuum apparatus. A test tube containing the bacterial suspension was placed into a special rack (cassette) and the identification card was placed in the neighboring slot while inserting the transfer tube into the corresponding suspension tube. The filled cassette was inserted manually into the VITEK 2 compact reader-incubator module. After the vacuum was applied and air was re-introduced into the station, the bacterial suspension was forced through the transfer tube into micro-channels that fill all the test wells and inoculated cards were automatically sealed prior to loading into the carousel incubator. All card types were incubated automatically incubated 35.5 ± 1.0 °C. Each card was removed from the carousel incubator once every 15 min, transported to the optical system for reaction readings, and then returned to the incubator until the next read time. Data were collected at 15-min intervals during the entire incubation period and final identification results were obtained in approximately 18 h or less. All cards used were automatically discarded in a waste container.
AST-GN72 cards consists of 64 biochemical method and substrates for identification and a panel of 19 antibiotics for drug susceptibility testing. Antibiotics with their different concentration used for the determination of drug susceptibility profile of Gram-negative bacteria in this investigation were: ampicillin (4,8,32), amoxicilin/clavulanic acid (4/2,16/8,32/16), cefalotin (2,8,32), cefazolin (4, 16, 64), cefepime (2,8,16,32), cefoxitin (8,16,32), cefpodoxime (0.5, 1, 4), ceftazidime (1,2,8,32), ceftriaxone (1,2,8,32), cefuroxime (2,8,32), ciprofloxacin (0.5,2,4), gentamicin (4,16,32), levofloxacin (0.25,0.5,2,8), nitrofurantoin (16,32,64), piperacillin/tazobactam (2/4,8/4,24/4,32/4,32/8), tetracycline (2,4,8), tobramycin (8,16,64), trimethoprim/sulfamethoxazole (1/19,4/76,16/304).
Similarly, the AST-GP71 card consists of an array of biochemical tests for species characterization and antibiotics for drug susceptibility testing of Gram-positive bacteria. Antibiotics with their different concentration used for the determination of drug susceptibility pattern of Gram-positive bacteria in this investigation were:- cefoxitin screen (6), ciprofloxacin (1, 2, 4), clindamycin(0.5,1,2), daptomycin (0.5, 1, 2, 4, 16), erythromycin (0.25,0.5,2), gentamicin (8,16,64), inducible clindamycin resistance (CM 0.5, CM/E 0.25/0.5), levofloxacin (0.25,2,8), linezolid (0.5,2,8), minocycline (0.12,0.5,1), moxifloxacin (0.25,2,8), nitrofurantoin (16,32,64), oxacillin (0.5,1,2), quinupristin/dalfopristin (0.25,0.5,2), rifampicin (0.25,0.5,2), tetracycline (0.5,1,2), tigecycline (0.25,0.5,1), trimethoprim/sulfamethoxazole (2/38,8/152,16/304), and vancomycin (1,2,4,8,16).
For quality control of susceptibility tests E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and E. faecalis ATCC929212 strains were used.
All ethical considerations and obligations were duly addressed and the study was conducted after the approval of the Department Research and Ethical Review Committee (DRERC) of the Department of Medical Laboratory Sciences, College of Health Sciences, and Addis Ababa University. Informed written consent was obtained from participants before data collection. The respondent was given the right to refuse to take part in the study and to withdraw at any time during the study period. All the information obtained from the study subjects were coded to maintain confidentially. When the participants were found to be positive for bacterial pathogen, they were informed by the hospital clinician and received proper treatment. Assent form was completed and signed by family member and/or adult guardian for participants under the age of 16 years.