Ocular bacterial infections at Quiha Ophthalmic Hospital, Northern Ethiopia: an evaluation according to the risk factors and the antimicrobial susceptibility of bacterial isolates

Background External and intraocular infections can lead to visual impairments, which is a major public health problem. Bacteria are the most frequent pathogens affecting ocular structures; the increasing rate of antimicrobial drug resistance is a worldwide concern. The aim of this study was to determine the occurrence of bacteria in ocular infections, their antimicrobial susceptibility patterns, and risk factors in bacterial ocular infection. Methods A hospital based cross-sectional study was conducted from September 2015 to December 2015 at Quiha Ophthalmic Hospital, Tigray, northern Ethiopia. Ocular specimens from blepharitis, blepharoconjunctivitis, conjunctivitis, keratitis, endophthalmitis, periorbital cellulitis and dacrocystitis were collected from 270 individuals with suspected ocular infection. Data on sociodemographic and risk factors were also collected using a structured questionnaire. Data analysis was performed using SPSS version 21 and 0.05 with a corresponding 95% confidence interval (CI) was considered statistically significant. Results Among 270 study subjects, 180 (66.7%) were culture positive for different bacterial isolates. The predominant bacterial isolates were Staphylococcus aureus (40, 22.2%), coagulase negative staphylococci (31, 17.2%) and Pseudomonas aeruginosa (21, 11.7%). Ocular surface disease, ocular trauma, hospitalization and cosmetic application practices were significantly associated with the occurrence of bacterial infection. Concerning antimicrobial susceptibility, most isolates were susceptible to amikacin (137, 93.2%), gentamicin (131, 89.1%) and ciprofloxacin (141, 89.2%). Overall, 40 (22.5%), 34 (19.1%) and 62 (34.8%) isolates were resistant to one, two, and three or more antimicrobials, respectively. Conclusion Bacteria were isolated from the majority of the study subjects. More than half of the bacterial isolates were resistant at least to one drug and a significant rate of multidrug resistance was detected. Therefore, identification of the etiologic agent and antimicrobial susceptibility testing should be practiced to select the appropriate antimicrobial agent to treat eye infections and prevent the emergence of drug resistant bacteria.

Bacteria are the most common agents causing external ocular infections, including blepharitis, keratitis, dacryocystitis, and orbital cellulitis. Bacteria are responsible for 70-80% of conjunctival morbidity which poses a huge socioeconomic burdens to the general public [16][17][18][19]. Blepharitis, an inflammation of the eyelid, can cause complete loss of the eyelashes if not diagnosed early [20]. Bacterial keratitis is the leading cause of corneal blindness [18,21,22].
External ocular infections may remain localized, or progress to adjacent tissues. For instance, external and internal hordeolum can result from the spread of eyelid infection to the respective glands [23]. Dacryocystitis, inflammation of the nasolacrimal duct, is the result of nasolacrimal duct obstruction. The accumulation of fluid and edema in the eye secondary to dacryocystitis, is a potential danger to ocular tissues such as the cornea and conjunctiva [24,25]. External ocular infections that result from systemic spread, surgery or are secondary to ocular trauma, can lead to sight threatening intraocular infections such as endophthalmitis [1,22,26,27]. Left untreated, bacterial ocular infections, bacterial products and vigorous inflammation following infection, can irreversibly damage ocular structures [28,29]. Inflammation and scarring, once present, may not be easily resolved and can result in visual impairment or permanent loss of vision [30,31].
Additionally, bacterial ocular infections have been complicated by multidrug resistance;, a problem that is intensifying over time [32,33]. This poses a challenge in the clinical management of bacterial ocular infections [34][35][36].
Eye infections are remarkably common in northern Ethiopia; however, no studies have been conducted on this topic in this region. Similarly, the antimicrobial susceptibility patterns of infecting bacteria in this region are not known. Therefore, we sought to determine the ocular bacterial infection, the risk factors and the antimicrobial susceptibility of bacterial isolates in ocular infections at Quiha Ophthalmic Hospital, Tigray, northern Ethiopia.

Study design, data and specimens collection
A hospital based cross-sectional study was conducted from September-December 2015 in Quiha Ophthalmic Hospital, Quiha, Tigray, Ethiopia. A total of 270 patients who presented with suspected ocular infection were consecutively included in the study. Sociodemographic, clinical and risk-factor related data were collected using a pretested structured questionnaire using intervieweradministered questionnaires. Study subjects were examined using a slit-lamp biomicroscope to screen for the presence of ocular infection. Ocular specimens were collected from periorbital cellulitis (N = 8), dacryocystitis (N = 13), blepharitis (N = 26), blepharoconjunctivitis (N = 16), conjunctivitis (N = 123), keratitis (N = 57) and endophthalmitis (N = 27) diagnoses. An ophthalmic surgeon collected specimens from all patients except those patients who had endophthalmitis who had their specimemens collected by an ophthalmologist [37][38][39].

Specimen transportation
The respective specimens were then transferred into 2 ml of brain-heart infusion broth (Oxoid, Hampshire, UK). Tubes were tightly capped, gently mixed, labeled and placed in cold chain. Finally, samples were transported to the Microbiology Laboratory, Mekelle University, for microbiological analysis [38,39].

Cultivation and identification of isolates
The broth was gently mixed to homogeneity; 100 μl of inoculum was then dispensed and streaked onto blood agar, chocolate agar, MacConkey agar, Mannitol salt agar and modified Thayer-Martin agar (MTM) (Oxoid, Hampshire, UK). To maintain the presence of CO 2 , chocolate agar and MTM agar plates were placed in candle jars. All media were incubated overnight at 37°C. After 24 h of incubation, each plate was inspected for any growth and negative plates were incubated for an additional 24 h. For eyelid and conjunctival swabs, culture positivity was determined based on a threshold criteria. Corneal specimens were considered as positive if there was a confluent growth at the site of inoculation [39]. All bacterial isolates were identified using standard clinical laboratory methods [40,41].

Quality assurance
Standard operational procedures were strictly followed from the pre-analytical to the post-analytical phase. The questionnaire was pretested on 16 patients with ocular infection. Data collectors were trained on data collection procedures. Completed questionnaires were proofread immediately after completion by the research team to clarify any labelling errors or illegibility. Standard Operating procedures were followed during Specimen collection, handling, transportation, microbiological analysis and interpretation of results. Reagents, media and antimicrobial disks were checked for the expiry date, damage and storage problems. Media sterility was strictly monitored by random sampling and incubation to test for any growth. In addition, reference strains of Staphylococcus aureus ATCC25923, E. coli ATCC25922 and P. aeruginosa ATCC27853 were used to monitor the quality of this work [42].

Statistical analysis
Data was entered into SPSS version 21, and descriptive statistics, bivariate and multivariate logistic regression analyses were performed. Bivariate logistic regression was employed to look for association between the outcome variable and independent variables. Variables with P-values <0.05 were reanalyzed in multivariate logistic regression to identify risk factors for bacterial occurrence. The corresponding P-value <0.05 and the confidence interval (CI) of 95% were considered statistically significant.

Sociodemographic characteristics
A total of 270 study subjects with clinically diagnosed ocular infections were included in the study, of whom 151 (55.9%) females, 119 (44.1%) were males and 158 (58.5%) were rural dwellers ( Table 1). The mean (SD) age of the study subjects was 37.8 (22.9) years.
As compared to published literature from other areas in Ethiopia, Nigeria, UK, Australia and India, most streptococcal isolates were from conjunctivitis diagnoses, with some isolates from blepharoconjunctivitis and keratitis. In the literature, Staphylococci and streptococci are known to be the main agents of post-traumatic periorbital cellulitis [54]; in our study, S. aureus (50%), S. pneumoniae (12.5%) and S. pyogenes (12.5%) were the main Gram-positive isolates from periorbital cellulitis. As has been seen in Gondar [19], Borumeda [44], and India [47], S. pneumoniae and S. pyogenes were the predominant species among patients with dacryocystitis.
The most common isolates among the Gram-negative bacteria were P. aeruginosa (11.3%), E. coli (8.1%) and Klebsiella spp. (6.4%) as has been seen in Hawassa; however, the proportions of P. aeruginosa (4.9%) and E. coli (4.9%) observed in Hawassa were lower than in our study [23]. This may be due to the additional isolates from endophthalmitis diagnoses we identified.
P. aeruginosa was the predominant isolate in keratitis (23.4% of diagnoses) similar to findings reported in Nigeria (23.8%) [23], the UK (24.3%) [7], Australia (21%)      [8], and Iran (24.7%) [15]. Pseudomonal keratitis is a progressive infection with large infiltrate and scarring [22,53]; this therefore means majority of patients in our population are at risk of blindness. Our assessment illustrated that the presence of ocular surface disease, ocular trauma, hospitalization and cosmetic application practices were significantly associated with the occurrence of bacterial infection. Risk factors for bacterial ocular infections that have been described include a history of hospital stay in Columbia and Portugal [48,55], ocular trauma in Australia, Florida, Iran, China and Mexicao [1,8,15,22,50] and ocular disease in Florida and Colombia [22,48] Cosmetic application practices were considered only for female participants. Limited information exists on the relationship between cosmetic application practices and bacterial ocular infections.
Almost one quarter of the bacterial isolates in were susceptible to all of the tested antimicrobials. Resistance to two or more antimicrobials was seen in 53.9% of the isolates, which is lower than that reported in other regions in Ethiopia (69.9-87.1%) [10,11]. S. pneumoniae (87.5%), CoNS (87%) and S. aureus (82.5%) were the most resistant among Gram-positive isolates. Acinetobacter spp. (100%),   [56,57].
Here, relatively, amikacin (93.2%), gentamicin (89.1%) and ciprofloxacin (89.2%) were revealed high efficacy towards Gram-positive and Gram-negative isolates. Studies in Gondar have reported lower susceptibility to gentamicin (54.8%) and ciprofloxacin (74.2%) [10,19]; differences in the variety of isolates tested may partly explain this. Outside Ethiopia, comparable susceptibility patterns to amikacin, ciprofloxacin and chloramphenicol were reported in Iran [15] and India [47]. However, the gentamicin susceptibility in this study is much higher than that in the study conducted in India (58.6%) [47]; regional variations in antibiotic prescription practices could explain this [38]. The resistance we observed to tetracycline and erythromycin was high, and this paralleled a study from India [3]. High resistance is usually due to the over use and empirical treatment of patients that ultimately leads to the emergence of drug resistant strains [58,59].
Methicillin resistance was seen in 17.5% of the isolated S. aureus, which is in line with a study conducted in India (14%) [60], but lower than the rates in Taiwan (52.8%) [57] and Uganda (31.9%) [49]. In addition, 45.2% of CoNS were methicillin resistant, which was higher than detected in Uganda (27.6%) [49]. To date, other than a study conducted in Gondar, most studies in Ethiopia did not determine methicillin resistance rates for ocular staphylococcal isolates [19]. Our study was not without limitation; we did not isolate anaerobic bacteria or Chlamydia trachomatis due to unavailability of the prerequisite facilities. We also did not determine the antimicrobial susceptibility of Moraxella species or Aeromonas species due to a lack of antimicrobials for agar dilution.

Conclusions
In conclusion, a high prevalence of bacteria was found in patients with ocular infection. "Ocular surface disease, ocular trauma, hospitalisation and cosmetic application practices were significantly associated with the occurrence of bacterial infection. Overall, S. aureus, CoNS, and P. aeruginosa were the predominant isolates. The majority of the bacterial isolates were multidrug resistant. Methicillin resistance was higher in CoNS than in S. aureus. S. pneumoniae, CoNS of which S. aureus was the most resistant species among Gram-positive isolates; Klebsiella species and E. coli were most resistant among Gram-negative isolates. Identification of the specific etiologic agent and antimicrobial susceptibility testing should be practiced during the management of ocular infections to reduce the further emergence of multidrug-resistant bacteria".