Bacterial etiology and antibiotic susceptibility of conjunctivitis patients isolates from central of Iran CURRENT STATUS: POSTED

Background: Conjunctivitis is a very common ocular disease, which can be caused by a wide variety of microorganisms. Identification of the bacterial causes of this infection in a specific geographical area can help physicians devise more efficient therapeutic strategies for this disease in the local population. This study bacterial etiology and antibiotic susceptibility of conjunctivitis patients isolates from central of Iran. Methods : This study was performed of 180 patients referred to department of ophthalmology in Kashan University with symptoms of conjunctivitis from July 2017 to December 2017. Samples were collected from the conjunctival sac and cultured in bacterial culture mediums. Antibiotic susceptibility of the bacteria isolated was done using the Kirby-Bauer method. Methicillin resistance in staphylococci isolated from the patients was identified using PCR technique. Results : In the present study, Pseudomonas aeruginosa was most resistant to ampicillin. In the case of Staphylococcus epidermidis and Staphylococcus aureus , the highest resistance was observed against erythromycin and the least resistance was against rifampicin and linezolid. Conclusion : In this study, Staphylococcus aureus and Staphylococcus epidermidis is the most common cause of conjunctivitis in all age groups, however, this condition decreases with age and is also influenced by other factors such as season and weather conditions. The results of this study can be helpful in planning more prudent treatment strategies for patients with conjunctivitis in Kashan.

Staphylococcus, Streptococcus pneumoniae, and Haemophilus influenzae [6]. Bacterial conjunctivitis epidemics often occur in winter and early spring [3]. If conjunctivitis is caused by viral or bacterial agents, the infection can become contagious. Accurate diagnosis of the type of infection and its etiologic factors and prescription of suitable antibiotics may shorten the duration of the disease as well as transmission time [6].
In a study of 92 conjunctivitis patients at Labbafinejad Hospital in Tehran, 58.5% of cultures returned positive, of which 57.1% were for aerobic and 7.4% for anaerobic organisms. Among aerobic organisms, Staphylococcus epidermidis was the most common (30%). This study recommended that given the multitude of bacterial causes of conjunctivitis, it is best to postpone the antibiotic treatment until after identifying the pathogenic microorganism [7]. In a study on the prevalence of microbial causes of keratoconjunctivitis in patients examined in Feyz hospital of Isfahan, out of 196 samples tested, 75 had bacterial causes with the most common pathogens being Staphylococcus aureus (28.1%), Staphylococcus coagulase negative (16.8%), Bacillus (6.6%), Pseudomonas aeruginosa (5.1%), Enterobacter (4.2%), Klebsiella (3.6%), and group D Streptococcus (3.6%). In this study, the highest antibiotic sensitivity was observed in Staphylococcus aureus and Pseudomonas under exposure to ciprofloxacin and tobramycin respectively [8].
In a study carried out by Tesfaye et al. (2013), where they examined the bacterial profile and antibiotic susceptibility pattern of external ocular infections, 148 of the cultured 198 samples returned positive, among which the dominant gram-positive and gram-negative organisms were Staphylococcus aureus and Pseudomonas aeruginosa respectively. This study reported that grampositive bacteria were mostly susceptible to ciprofloxacin and vancomycin and gram-negative bacteria were largely susceptible to amikacin and ciprofloxacin [9]. In a study by Stan [11]. A study conducted on 548 samples collected from conjunctivitis patients reported that 17% of cases had methicillin-resistant staphylococci [12]. In another study on 3640 patients with extraocular infection, 1088 of the cases (30%) had methicillin-resistant Staphylococcus aureus of hospital origin and 2552 (70%) of them had methicillin-resistant staphylococci originating from the general population [13]. Research has shown that the overuse of antibiotics has greatly increased the antibiotic resistance of eye infecting bacteria and is turning the issue into a serious challenge for the fight against bacterial infections [14]. This highlights the importance of performing regular antibiotic susceptibility tests to monitor the sensitivity of infectious bacteria to different antimicrobial agents. Considering the increased resistance of staphylococci to methicillin, which leads to increased use of vancomycin, and since there are already several reports of vancomycin resistance, it is necessary to identify methicillin-resistant strains of staphylococci to prevent the overuse of these antibiotics [15].
Given the multitude of bacterial causes of conjunctivitis and the cost of doing an independent culture test for every conjunctivitis patient, it is necessary to identify the most common causes of these infections in different populations to help physicians devise a general treatment plan for that population accordingly. The increasing antibiotic resistance of many bacterial strains only adds to the importance of monitoring the antibiotic susceptibility of the bacteria involved in conjunctival infections for prescribing effective antibiotic treatments without contributing to the antibiotic resistance problem. This study aimed to identify the bacterial causes of conjunctivitis and their antibiotic resistance patterns and particularly the methicillin-resistant strains of staphylococcal 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- The frequency distribution of bacterial infections in the sampled conjunctivitis patients in terms of their age and gender is presented in Tables 1 and 2, respectively. As can be seen, no significant relationship was found between gender and infectious agents. Also, no significant relationship was found between age groups and infectious agents.
The frequency distribution of bacterial infections in the patients in terms of observed symptoms is provided in Fig.1. According to the observations, no significant relationship was observed between symptoms and infectious agents.
The study also investigated the antibiotic resistance of organisms isolated from conjunctivitis patients and their antibiotic resistance patterns. The results of this investigation are presented in Fig.2 and Staphylococcus epidermis, the preferred antibiotic would be rifampicin and linezolid respectively.
Following PCR to determine mecA gene in 11 cefoxitin-resistant staphylococci sample, it was found that despite resistance to disc cefoxitin, none of the resistant strains had mecA gene (Fig.3).

Discussion
Considering the wide variety of microorganisms that can be involved in the development of conjunctivitis [1], studying the bacterial causes of this disease may help us better understand its epidemiology and devise appropriate therapeutic strategies accordingly. Many bacterial and viral agents can contribute to the development of ocular infections [5,17,18] In the present study, Pseudomonas aeruginosa was most resistant to ampicillin. In the case of In the present study, 100% of MRSA strains had PCR negative. This result could be related to the presence of a mutation in the methicillin resistance gene in these strains or the presence of physiological differences such as alteration in the bacterial wall [12]. Despite the high resistance to cefoxitine in these strains, there may be some difficulty regulating mecA gene expression. In future studies, it is recommended to conduct research in this area.
This report is one of the most comprehensive studies ever conducted on the relative frequency of bacterial and adenoviral causes of conjunctivitis in the population of Kashan and their antibiotic resistance patterns. These results can contribute to the planning and adjustment of therapeutic strategies, and especially antibiotic medication, for patients with conjunctivitis in this region.

Conclusion
In this study, Staphylococcus aureus and Staphylococcus epidermidis is the most common cause of conjunctivitis in all age groups, however, this condition decreases with age and is also influenced by other factors such as season and weather conditions. The results of this study can be helpful in planning more prudent treatment strategies for patients with conjunctivitis in Kashan.

Consent for Publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this published article.

Competing interests
No competing financial interests exist.

Funding
This research was supported by the Kashan University of Medical Sciences (Grant no. 9631). The funding agency had no role in design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Authors' contributions
HF, MM and AA conceptualized, acquired funding, collected the data, analyzed and wrote the first draft of paper. AN and HF validated the data, and analyzed the data. HA and HF assisted in analyzing data and writing the draft manuscript. HF, MS, AN and RM were involved in project administration, supervision of data collection team, revision of draft paper and its finalization. All authors read and approved the final manuscript.    The antibiotic sensitivity of organisms isolated from conjunctivitis patients (A: Pseudomonas, B: Staphylococcus epidermidis, C: Staphylococcus aureus)