Bacterial isolates and their antimicrobial susceptibility pattern among patients with ocular infections at the University of Gondar Hospital, Northwest Ethiopia: A 10-years retrospective study

Abstract Objective: The aim of this study was to identify potential bacterial isolates and their antimicrobial susceptibility pattern among patients who visited eye clinic of Gondar university hospital. Results: From year of January 2009 to January 2019 a total of 319 eye discharge specimens were submitted for microbiological analysis, of which 133(41.7%) were culture positive. A total of 42(31.6%) Gram negatives and 91(68.4%) Gram-positive bacteria were isolated. Among isolated Gram-negative bacterial species, Klebsiella spp was the most predominant, 42.9% followed by E. coli 26.2%. Among isolated Gram-positive pathogenic bacterial species, S. aureus was the most prevalent, 59.5% followed by S. pyogenes , 8% and S. pneumoniae , 5.5%. Most of the bacterial isolates were resistant to cotrimoxazole, 81.6%, amoxicillin, 78.7%, tetracycline,76.2%, and ampicillin 75.9%. About 47.4 % of bacterial isolates showed multi drug resistance to three or more classes of antimicrobials. Antimicrobials like: ampicillin, amoxicillin, ceftriaxone, cipro�oxacin, and nor�oxacin exhibited year to year increment of resistance (p<0.0001).


Introduction
The eye is one of the sense organs which is very important throughout our life.Ocular infection is a challenge for all age groups and can cause damage to structures of the eye, which can lead to loss of vision and even blindness if left untreated [1].Dust, high temperature, microorganisms, and other factors can lead to various eye diseases which can lead to blindness [2].However, bacteria are the major contributor to ocular infections throughout the world [3].
Ocular disease and its complications are a signi cant health problem worldwide that impacts greatly on quality of life.World Health Organization (WHO) estimates that about 285 million peoples are visually impaired worldwide and 90% of these individuals live in low-resource countries [4].
In the case of sub-Saharan Africa, an estimated 26 million individuals live with visual impairment, of whom 5.9 million individuals are classi ed blind [5].The costs of conjunctivitis are substantial.Bacterial conjunctivitis, which comprises about 50 percent of all cases of conjunctivitis, costs an estimated $377 million to $857 million annually in the U.S [6].
Internationally, there is a growing concern over antimicrobial resistance (AMR) which is currently estimated to account for more than 700,000 deaths per year worldwide [7].In this study, we will review the microbiology and antimicrobial susceptibility pattern of ocular bacterial infection in Gondar University Comprehensive Referral Hospital over the past 10 years.

Study Area, Study Design and Period
The study was conducted at University of Gondar Hospital, North Gondar, based on the 2005 census, the total population of Gondar is 194,773 (97,625 males and 97,148 females) [8].Hospital based crosssectional retrospective study was conducted at the University of Gondar Hospital among patients with ocular infection from the year January,2009 to January,2019.

Data collection
A standard data extraction sheet was prepared by using different literature and the data were collected from the registration book.

Laboratory methods
The eye discharge specimens were collected aseptically and streaked on blood agar, MacConkey agar and chocolate agar (Oxoid, UK).The inoculated plates were incubated at 37 0 C for 24-48hrs.The growth on the plates were identi ed by standard microbiological techniques.AST was done by the modi ed Kirby-Bauer disk diffusion method on Mueller Hinton agar (Oxoid, UK).For fastidious isolates, Mueller Hinton agar supplemented with 5% sheep blood was used.Diameters of a zone of inhibition around the disk were measured with the ruler and interpreted according to the Clinical Laboratory Standard Institute guidelines.Bacteria resisting for more than two drugs of the different classes are considered as "multidrug resistant" [9].

Data Analysis and Interpretation
Data was entered to SPSS version 20 statistical packages for analysis.Descriptive statistics were done to calculate frequencies of categorical variables.Chi-square test was done to see trend of drug resistance among bacterial isolates.Results were presented in the form of tables, texts, and graphs.P value<0.05 were considered as statistically signi cant.

Ethical Consideration
Ethical clearance was obtained from Research and Ethics Committee of the School of Biomedical and Laboratory Science, Collage of Medicine and Health Science.

Socio-demographic characteristics
Within the last ten years (January 2009-January 2019), a total of three hundred nineteen (319) ocular infection suspected patients visited University of Gondar Comprehensive specialized referral hospital.Among this, a total of 133(41.7%patients were culture positive.About 81(60.3%)patients were males and 52(39.7%)were females.However, the age of most patients was in the age group of 1-5 years [Table 1].

Prevalence of bacterial isolates from ocular specimens
In this study, the overall bacterial isolation rate was 133(41.7%).Both Gram-negative and Gram-positive bacteria were isolated from the positive cases.A total of 42(31.6%)Gram negatives and 91(68.4%)Grampositive bacteria were isolated.Among isolated Gram-negative bacterial species, Klebsiella spp was the most predominant, 42.9% followed by E. coli 26.2%.Among isolated Gram-positive bacterial species, S. aureus was the most prevalent, 59.5% followed by CONS 18.7% [ Table 2].

Multi drug resistance property of isolates
The overall multidrug resistance rate of bacterial isolates was 47.4% [Table S3].

Discussion
Ocular infection is a major public health problem especially in developing countries like Ethiopia.The external ocular infections are responsible for increased incidence of morbidity and blindness worldwide, their morbidity vary from self-limiting light infection to sight threatening infection [10,11].In this study, the overall, prevalence of isolated bacteria was 133/319(41.7%).This nding is in agreement with the previous studies carried out in Hawassa [12], in which, about 48.8% of the study participants were culture positive.However, our result was signi cantly lower than ndings reported in Sudan [13], Tigray [14] and Jimma [10], about 63.7%, 66.7% and 74.7% positivity rate respectively.This difference may result from difference in geography of the study setting and types of study design.However, compared to study ndings in China [15], 20.43% and India [16], 34.5%, this nding is higher.This difference may be resulted from difference in self-hygiene, socioeconomic status of the study population and disease prevention practices.
Different scholars reported that both Gram-negative and Gram-positive bacterial species involved in ocular infection [13,15,17,18] and this nding is in line with our ndings.Among isolated Gram-negative bacterial species, Klebsiella spp was the most predominant bacterial isolate recovered from eye discharge.Similarly, this bacterial species was isolated from eye discharge specimens at University of Gondar in 2016 [ 19].In contrast to our nding, the study done in Sudan [13], Hawassa [12], Tigray [14] and Jimma [10] reported that P. aeruginosa was the most isolated bacteria.Geographical location and acquisition of hospital acquired infection in the study participants may be the possible reason for this difference.E. coli was another Gram-negative bacterial isolate recovered in this study.Similarly, this bacterium was also reported in different studies [ 15,16,19].
In this study, the dominant bacterial isolates from eye discharge specimens were Gram positive cocci.This nding is in agreement with other several previous studies done in many parts of Ethiopia [10,12,14], and countries like China [15], India [17] and Sudan [13].In the present study, S. aureus was the predominant Gram-positive bacterial isolate concordant with previous study ndings reported in Hawassa [12], Tigray [14], Jimma [10] and India [17].
On the contrary, other study ndings from Saudi Arabia [18] and Uganda [20] stated that CoNS was the most prevalent Gram-positive bacterial species.This difference may result from the immune status of patients and specimen collection procedures.
In this study, Gram-positive bacterial species like S. pyogenes and S. pneumoniae were also signi cantly isolated.This nding is supported by ndings reported in Sudan [13] and Hawassa [12].However, bacterial species like N. gonorrheae, H. in uenzae and Pseudomonas were not isolated in this study unlike to other studies [12,13,17].
Speci cally, antimicrobial resistance by Gram-negative bacterial species become a big threat for the world, especially for developing countries [21].Ampicillin, amoxicillin, cotrimoxazole, gentamycin, chloramphenicol, nor oxacin and ceftriaxone were among tested antimicrobials resisted by K.
pneumoniae, one of the isolated Gram-negative bacteria with the resistance rate of 100%, 100%,84.6%,73.3%,66.7%, 66.7% and 60% respectively.In case of ampicillin, this nding is comparatively in agreement with results reported in India [16] in which, K. pneumoniae was 100% resistant to ampicillin.In a reverse, unlike to our ndings, results reported in Gondar [22] showed that complete sensitivity was observed to gentamycin (100%), chloramphenicol (100%), cipro oxacin (100%) and cotrimoxazole (100%).In the study setting, an irrational use of antimicrobials without prescription, improper dosage regimen, misuse of antimicrobials for viral and other non-bacterial infections and extended duration of therapy could result in increased antimicrobial resistance.
E. coli also showed a signi cant level of resistance to ampicillin (77.8%), cotrimoxazole (77.8%), tetracycline (72.7%) and amoxicillin (71.4%).Similarly, a high level of resistance was observed in the study done by Yared A et al., 2016 [22], in which, E. coli showed 100% resistance to amoxicillin and tetracycline.In the study setting, however, some tested antibiotics like nalidixic acid (100%), erythromycin (100%) and nor oxacin (75%) are still effective antimicrobial agents against E. coli.This nding was consistent with study done in southern parts of Ethiopia [12], gram negative isolate was more susceptible to cipro oxacin and gentamicin.
In this study, antimicrobials susceptibility pattern of Gram-positive bacterial species was also determined.S. aureus, one of the most predominantly isolated Gram-positive bacteria, had great extent of resistance to ampicillin (100%), penicillin G (80.8%), tetracycline (77.8%) and amoxicillin (75%).Different literatures supported our ndings.For instance, a prospective study done in India [16] showed that S. aureus was resistant to penicillin (100%), erythromycin (100%), tetracycline (87.5%) and amoxicillin (62.5%).However, rate of resistance in our study by S. aureus was higher than ndings reported in Uganda [20], about 31% and 55.2% of S. aureus resisted against erythromycin and tetracycline, respectively.Similar study done in Sudan [13] also showed that about 100% and 72% of S. aureus was resistant to penicillin and tetracycline, which is almost comparable to the present study.
The overall prevalence of multi-drug resistance (MDR) to three or more classes of commonly prescribed antimicrobials was 47.4%.this is lower than results reported in Hawassa [12], 69.9% and Gondar [19], 87%.But, MDR rate in the present study is higher than nding reported in Tigray [14], only 34.8% MDR rate was observed.
Investigation on antimicrobial resistance trend is indispensable for empirical treatment of infections, implementing resistance control measures, and preventing the spread of antimicrobial-resistant microorganisms.In the present study, the antimicrobial resistances of bacteria isolated from eye discharge was analyzed.In this study, increased trend of resistance to many tested antimicrobials was observed in the past 10 years.Increased trend of resistance to ampicillin was observed over a period of

Table 2 :
Number and percentage of bacteria isolates from eye discharge specimens at University of Gondar specialized and referral hospital (January 2009 -January 2019), Gondar, Northwest, Ethiopia.

Table 3 :
Overall antimicrobial susceptibility pro les of bacterial isolates from eye discharge at University of Gondar referral hospital (January 2009 -January 2019), Gondar, Northwest, Ethiopia

Table S1 .
Antimicrobial susceptibility patterns of Gram-negative bacterial isolates from eye discharge at University of Gondar referral hospital laboratory (2009-2018), Gondar, Northwest, Ethiopia.This table shows the antimicrobial susceptibility pattern of some gram-negative bacterial species isolated from eye discharge.This table should be put just after Table3(see above the "Discussion").

Table S2 .
Antimicrobial susceptibility patterns of Gram-positive bacterial isolates from eye discharge at University of Gondar referral hospital (2009-2018), Gondar, Northwest, Ethiopia.This table shows the antimicrobial susceptibility pattern of some gram-positive bacterial species isolated from eye discharge.This table should be put just after TableS1(see above the "Discussion").

Table S3 .
Multidrug Resistance Patterns of Bacteria Isolates from eye discharge at University of Gondar referral hospital laboratory (2009-2018), Gondar, Northwest, Ethiopia.This table shows the Multidrug resistance pattern of bacterial isolates from eye discharge.This table should be put just after Table S2 (see above the "Discussion").
Table S4.Chi-square test for trend applied to bacterial resistance rates to various antimicrobials at University of Gondar comprehensive specialized hospital laboratory (2009 -2018), Gondar, Northwest,