A cross-sectional study was performed on the population of patients with ocular infection referred ophthalmology clinic of Kashan in 2017. The sample consisted of 180 patients with eye infections who were examined at this clinic during July 2017 to December 2017. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments. The protocol of this study was approved by Research Ethics Committee, KAUMS (IR.KAUMS.REC.1396.31), Iran and written informed consent was obtained from all patients. Subjects were selected by purposive sampling. The inclusion criteria were informed consent and diagnosis of conjunctivitis and the exclusion criterion was the withdrawal of consent.
After examination by an ophthalmologist and confirmation of conjunctivitis diagnosis, patients were briefly interviewed and then demographic information and conjunctiva samples were collected. For isolation and differential recognition of bacterial causes of conjunctivitis, the collected swab samples were placed in thioglycollate medium and transferred to the microbiology laboratory of Kashan University of Medical Sciences, where inoculation was performed on blood agar, chocolate agar, and MacConkey agar. The prepared plates were incubated at 37°C for 24-48 hours with visual mentoring of colonization. Anaerobic culture was also performed on the same mediums in the candle jars. For differential recognition, all bacterial isolates were evaluated in terms of appearance, the morphology of colonies, pigment production, gram staining, and biochemical characteristics. To identify different bacteria, tests such as optochin, coagulase, PYR, oxidase and culture media such as mannitol salt agar, DNAase, chocolate agar were used.
The susceptibility of the isolated bacteria to common antibiotics was determined by the Kirby-Bauer test. Bacterial colonies were dissolved in sterile physiological saline and turbidity was adjusted to 0.5 to 1 with the help of McFarland tubes. Then a sterile swab was used to inoculate this suspension to a plate with Mueller Hinton medium and the discs were inserted. After 24 hours of incubation at 37°C, the diameter of the inhibitory area around the discs was measured [15]. The antibiogram was developed for oxacillin, rifampin, ampicillin, amoxicillin, cefepime, ceftazidime, piperacillin ciprofloxacin, chloramphenicol, doxycycline, vancomycin, amoxicillin/ clavulanate, amikacin, tetracycline, cotrimoxazole, erythromycin and gentamicin as instructed in CLSI (2016). After 24 hours of incubation, the inhibitory area was compared with the standard chart [16].
The methicillin resistance (mecA) in staphylococci isolated from the patients was identified using the PCR technique. Specific primers with the following sequence were used to investigate the presence of mecA gene. The thermal cycle used in the thermocycler device first made an initial denaturation for 5 minutes at 95°C, then 30 cycles at 94°C for 15 seconds, 61°C for 15 seconds and 72°C for 30 seconds. Then final Extention was performed at 72°C for 5 minutes. The volume of the PCR reaction mixture was 25µl for each reaction, including PCR buffer (2.5µl), MgCL2 (0.8 mM), dNTPs (0.16 mM), Primers (16 pmol of each), Taq polymerase (1u/µl) and 2 microliters of purified bacterial DNA. Following the protocols of ethics and confidentiality, all participants were informed about the fundamentals and objectives of the study, confidentiality of their information, anonymity of the questionnaire, and their right to refuse to participate or withdraw from the study. After the data collection phase, data were tabulated in terms of bacteria type and demographic variables and then further analyzed using the chi-square test, Fisher’s exact tests, and one-way ANOVA at the P-value=0.05 significance level.