In vitro biofilm formation and antimicrobial susceptibility patterns of bacteria isolated from suspected external ocular infected patients attending Jimma University Medical Center eye clinic, southwest Ethiopia

Background: Ocular disease with its complications is a major public health problem which significantly impacts on quality of life in developing countries. An ocular infection due to microbial agents, can lead to reduced vision and blindness. This study was aimed to assess the antimicrobial susceptibility pattern and biofilm forming potential of bacteria isolated from suspected external ocular infected patients attending Jimma University Medical Center (JUMC). Method: A cross sectional facility based study was conducted on 319 suspect patients with external ocular infections from March 2017 to June 2017 at JUMC in Southwest Ethiopia. External ocular specimens were collected and standard operating procedures were followed to handle and culture throughout the study period. Antimicrobial susceptibility pattern of the isolates was determined by disk diffusion method according to CLSI 2015. Microtiter (96 wells) plate method was used to screen biofilm formation by measuring optical density at 570nm. Result: Out of 319 study participants with external ocular infection, the prevalence of bacterial pathogens was 46.1%. The predominant bacterial isolates were Coagulase negative staphylococcus (CoNS) (27.7%) followed by Staphylococcus aureus (19.7%). Among Gram negatives, Pseudomonas aeroginosa (6.8%) was the leading isolate. Increased antimicrobial resistance was observed for tetracycline (64%), erthromycin (66.7%) and penicillin (77.1%). Amoxicillin-clavulanic acid, ciprofloxacin and gentamicin were the most effective drugs for both Gram negative and Gram positive ranging from about 70 to 100% with the later two drugs for external ocular infections. Methicillin resistant S. aureus (MRSA) accounted for 13.8% of S. aureus isolates.. Multidrug resistance (MDR) accounted for 68.7%. The overall biofilm formation rate of bacterial ocular pathogens was 66.1%; with P. aeruginosa (40%), CoNS (34.1%) and S. aureus (31%) formed strong biofilm phenotype. Conclusion: The prevalence of bacterial isolates among external ocular infection was high. Almost


Introduction
The human eye, which is constantly exposed to the external environment, is a unique organ considered to be the window of our body. Ocular disease with its complications, which happen due to microorganisms, is a significant health problem worldwide; particularly it further impacts quality of life in least income countries. Ocular infection can damage the structure of the eye which can lead to reduced vision or even blindness if it is inappropriately diagnosed and treated. The most frequently affected parts of the eye are the conjunctiva, eyelid and cornea as a result of pathogenic microorganisms (1). Conjunctivitis, blepharitis, and dacryocystitis are considered the most common manifestations of external ocular infections (2). The pathogenic micro-organisms in external ocular infections include bacteria, fungi, viruses and parasites (3).
Pathogenic bacteria are the major causative agents of ocular surface infections with possible loss of vision (4). Control of eye infections may involve the use of broad spectrum antimicrobial agents.
Nevertheless the emerging and increasing antimicrobial resistance is a problem worldwide (5). In this regard, inappropriate and irrational use of antimicrobial medicines provide favorable conditions for resistant microbes to emerge, spread and persist (6). The development of bacterial biofilms is presently recognized as one of the most relevant drivers of persistent infections. It constitutes a serious challenge for clinical microbiologists and physicians being 100 to 1000 fold more resistant to antimicrobial agents than their counter parts in planktonic forms (7). Phenotypic and physiological changes in biofilm platform restrict penetration of antibiotics into biofilm forming bacteria. This expression of biofilm provides a higher resistance to antimicrobial treatments (8).
In most parts of Ethiopia, geting antibiotics without prescription order is easy, and this can lead to underuse, overuse or misuse of antibiotics (9). These conditions contribute to the emergence and spread of antimicrobial resistant strains. Moreover, poor sanitary and infection control practice in the anatomic area (face) may play a serious part in an increased prevalence of resistant bacteria. This rising antimicrobial resistance increases the risk of treatment failure with potentially serious consequences (10,11).
Even though a study on ocular infection was conducted in 2012 in Jimma area (4), the bacterial profile and antimicrobial susceptibility pattern can vary over time and place as indicated in different studies (3,12). Therefore, the changing spectrum of microorganisms involved in external ocular infections and the emergence of acquired microbial resistance to antibiotics needs continuous surveillance to guide empirical therapy. As a result of this, updated knowledge of bacterial aetiologic agents in ocular infections and their antibiogram are crucial. On the other hand, bacterial biofilm development was not addressed in bacterial isolates from external ocular infections in Jimma area. Hence, the present study was intended to update profile of bacteria present in external ocular infection. Moreover, this study aimed to assess the antimicrobial susceptibility pattern along with biofilm forming potential of the isolates at Jimma University Medical Center (JUMC) eye clinic, Southwest Ethiopia.

Study Design and Population
A cross-sectional health facility based study was conducted on a total of 319 patients at JUMC, Department of Ophthalmology in Ethiopia from March to June 2017. All patients with external ocular infection that fulfill the eligibility criteria during the study period were recruited prospectively by ophthalmic nurse and confirmed based on clinical examination by ophthalmologists. Patients examined and diagnosed with slit lamp biomicroscope and had an external ocular infection with red eye, discharge, mucoid or mucopurulent secretion, thickening of the conjunctiva, in one or both eyes, and agreed to participate were included in this study. Patients on antibiotics within the last 5 days prior to sample collection date were excluded from the study since bacteria is less frequently detected in culture based tests collected after antibiotic use (13) .

Data Collection Procedures and Process
Socio Demographic and Clinical Characteristics Socio-demographic data (age, sex, monthly income, educational level, occupation and address), clinical data (history of repeated infections, duration of stay in hospital, use of contact lenses, surgery, previous antibacterial therapy, systemic diseases and use of traditional medicine) and others like source of light and use of fire wood at home were collected from each study participants by using structured questionnaire. Ophthalmologists diagnosed patients with clinical picture of external ocular infections.

Specimen Collection, Handling and Transport
All consecutive patients examined clinically with the slit-lamp bio-microscope were set apart for suspected bacterial infection. Specimens from external ocular structures were collected from consented patients. Briefly, patients were requested to look up while lowering the eye lid down, and samples were collected from one or both eyes based on the nature of the infection. The discharge from the surface of the eye was collected gently using sterile cotton swab that had been premoistened with sterile physiological saline. The swab was rubbed over the lower conjunctival sac from medial to lateral side and back again. Purulent material was collected in the cases of dacryocystitis by everted puncta then applying pressure over the lacrimal sac area from the infected eye (12,14). In the cases of ulcerative blepharitis, lashes deposit, tear film foaming content, and corneal punctuate erosions were swabbed. From each patient, two swabs were collected: one for Gram staining immediately after collection and the other one for bacterial culture. The collected swab were inserted into Amies transport media, placed in a cold box and transported to Jimma University Medical Microbiology Laboratory. Standard operating procedures were followed to handle specimens collected from patients throughout the study period.

Isolation and Identification of Bacterial Pathogens
All specimens were streaked onto MacConkey agar, Mannitol salt agar, Blood agar and Chocolate agar plates (all media used from Oxoid, Hampshire, UK). The plates were incubated aerobically at 37⁰C for 24 to 48 hours. The Chocolate agar was kept at 37⁰C for 24 to 48 hours in a 5 to 10% CO2 atmosphere. All plates were initially examined after 24 hours and cultures with no growth were further incubated for another 24 hours. Bacteria were identified on the basis of phenotypic and a series of biochemical tests. For Hemophilus spp., satelitism test was also performed (11).

Bacterial Biofilm Tests
Bacteria isolated from fresh agar plates were inoculated into tube filled with sterile trypton soya broth (TSB) with 1% glucose and incubated at 37⁰C for 24 hours. This culture was diluted 1:100 into fresh media. Then, 200 μL of the suspension was added into sterile 96 wells flat-bottom microtiter plate and incubated at 37⁰C for 48 hours. The bacterial suspension of each well was gently removed and washed three times with phosphate buffer saline (pH 7.2). Plates were fixed with methanol (99%), and then stained with 220 μL of crystal violet (CV, 0.1% w/v) at room temperature for 15min. Each well was washed three times with PBS to remove unbound CV dye. After drying, 220 μL of ethanol (95%) was added to each well. Finally, the solubilized CV was transferred to new microtiter plate. The optical density (OD) of the biofilm was determined by a microplate reader (HumaReader HS, German) at a wavelength of 570 nm. The experiment was performed in triplicate separately for each strain and the average values was calculated (15).
The cutoff optical density (ODc) was proof of the biofilm formation and was defined as the sum of the arithmetic mean of negative controls and a triple value of its standard deviation (ODc = ẍ+3σ). The liquid medium without bacterial suspension incubated in microtiter plate was used as negative control. Biofilm formations of the isolates were classified into four categories as stated in previous study (16): non-adherent (OD < ODc), weakly-adherent (ODc < OD < 2xODc), moderately-adherent (2xODc < OD < 4xODc), and strongly-adherent (4xODc < OD).

Data quality assurance
All ophthalmic specimens were collected following standard operating procedure by ophthalmologists.
Cross-checking and data cleaning were done on daily basis. All laboratory and clinical data were recorded during the study period as back up. The sterility of culture media was ensured by incubating five percent of each batch of the prepared media at 37⁰C for 24 hours. For better results, any physical change like cracks, excess moisture or dehydration, color change, hemolysis, contamination, deterioration and expiration dates were checked before using culture media. The quality and performance of culture media, biochemical tests and antimicrobial susceptibility discs were checked Frequency count and percentage were used to present the finding. Prevalence figures were calculated for the total study population and separately by clinical feature of the disease. Bivariate and multivariate logistic regression was used to asses associated variable with bacterial prevalence. Pvalues less than 0.05 were considered statistically significant.

Socio-demographic and clinical feature of study participants
A total of 319 study participants clinically diagnosed with external ocular infection were included in this study; out of which 172(53.9%) were males and the remaining 147(46.1%) were females. The age of the study participants ranged from 1 month to 95 years of age with median of 21 years old. The majority of study subjects were children below the age of two years which accounts for 103(32.3%) followed by ages of above 45 years groups accounting for 74(23.2%). Bivariate analysis (COR) did not show significant association between sociodemographic characteristics and bacterial isolation pattern.

Correlation of antimicrobial resistance and biofilm formation
From the total bacterial isolates, higher biofilm formers were observed among multidrug resistant bacteria. Non-biofilm formers predominanted among those bacterial isolates with resistance to one or two antimicrobial agents. But in bacterial isolates where there was MDR features, higher numbers of biofilm formers were the principal ones. Significant rate of strong biofilm formers (48.4%) were seen in bacterial isolates that were resistant to five or more antimicrobial agents. In this study, Chi-square analysis revealed a significant relationship between multidrug resistant and biofilm former bacterial isolates, p<0.05, despite that the correlation coefficient (0.491) did not show strength (Figure 1). Figure 1, place here.

Discussion
In this study, a total of 319 patients suffering from external ocular infections were included in four months period in 2017. The overall prevalence of bacterial external ocular infections rate was 46.1%.
This result is comparable with previous study conducted in Southern Ethiopia (18). However, the result is lower than the prevalence reported from elsewhere ranging between 74% and 88% (4,19,20).
But it is higher than the study conducted in Bangalore (11). The different rate of bacterial isolation from one place to another or within the same place might be due to different factors including geographic variation, sample collection and transportation method, culture media used, differences in study period, and varied cases in inclusion criteria.
Conjunctivitis was the predominant external ocular infection accounting for 51.7% followed by blepharoconjunctivitis (23.2%), blepharitis (16.3%), dacryocystitis (4.1%), and other eye infection accounting for the remaining 4.7%. This is similar with previous studies conducted in Ethiopia (18) and India (19) where conjunctivitis was the leading causes of external ocular infections. In contrast to this study; however, one finding in southwest Ethiopia (4) reported blepharoconjunctivitis as the predominant types of external ocular infection. The differences within the study might be due to differences in study period, and smaller sample size.
In this study, the most common isolates observed were Gram positive cocci (65.3%). The finding is indicative of Gram positive cocci as primary cause of external ocular infections in Jimma area and it is comparable with other previous studies (9,21,22). From the Gram positives, CoNS (27.9%) was the most predominant isolates followed by S. aureus (19.7%) and S. pneumoniae (8.8%). Similar studies conducted in Ethiopia (23,24) and India (25)  The prevalence of Gram negative bacterial isolate among patients suffering from external ocular infection was 34.7%, with P. aeruginosa (6.8%) as the leading Gram negative agent followed by K. pneumoniae (6.1%) in this study. Similar studies in different geographic locations (4,32-34) also reported that P. aeruginosa is the most frequent isolate. Transient contamination of patient hand may be the source of infection of external eye structures.
Among the clinical features of external ocular infections, the predominant bacterial isolates identified among cases of bacterial conjunctivitis and blepharitis were CoNS followed by S. aureus. In the case of dacryocystitis, in addition to CoNS and S aureus, S. pneumoniae was also the predominant strains recovered. This is in agreement with several studies conducted in Ethiopia and elsewhere (18,20,21,22,24,(35)(36)(37)(38)(39)(40). Hence, most ocular cases may be managed by considering these members of bacterial etiologies.
This study showed high rate of antimicrobial resistance to different agents in both Gram positive and Gram negative bacteria, which is consistent with findings from Ethiopia (4,23) and Uganda (36). In this in vitro study, high frequency of resistance to ampicillin, penicillin, erythromycin, trimethoprim sulphamethoxazole, tobramycin and tetracycline has been observed. From these antimicrobial categories, medicinal preprations usable in ophthalmic purposes, including tetracycline and tobramycin, needs particular concern at the study location. The observed resistance of the bacteria might be due to accessibility of the antimicrobial drugs over the counter in Ethiopia. In addition, indiscriminate use of antimicrobial drugs and empirical treatment by the health professionals without susceptibility testing results for severe external ocular infections, and shortage of microbiological services for culture and susceptibility testing or unavailability of updated guideline regarding the selection of drugs are some of the factors which can lead to the development of the increased resistance rate.
In this study, the overall resistance rate of isolated Staphylococcus spp. to commonly prescribed antibiotics such as to ampicillin, tetracycline, erythromycin and penicillin was between 73% and 85%.
Among these antimicrobials, tetracycline prepration is avialble in external ophthalmic treatments which may warrant prudent use of it as most S. aureus and CoNS were resistant. Consistent results were reported from different studies elsewhere (4,9,18,39). P. aeruginosa, on the otherhand, was highly resistant to ceftazidime (90%) and tetracycline (80%). This is consistent with a study done in Ethiopia (4) where P. aeruginosa was highly resistant to tetracycline. Other Gram negative isolates were highly resistant to tetracycline and tobramycin in the range between 40% and 80%.
Epidemiological factors, study period and geographic location may be among the factors contributing to highly variable resistance rates. The necessity of bacterial culture and scesuptibility for suspected cases of external ocular infected patienst may be compulsory to select the most effective ophthalmic antimicrobial preparations.
All Gram positive bacterial isolates were susceptible to amoxicillin-clavulanic acid, ciprofloxacin and gentamicin within the susceptibility range of 69%-100%. This is comparable with previous studies done in Ethiopia (18,23,40) and India (41). On the otherhand, most Gram negative bacteria from our study were susceptible to Amoxicillin-clavulanic acid, ciprofloxacin, ceftriaxone and gentamicin within the range of 66%-100%. This is in agreement with previous studies done in different locations (18,23).
From all S. aureus strain isolated, 13.8% of them were MRSA strains. The finding is comparable with other study reported in India (37). However, in this study significant proportion of CoNS isolates were also MRSA. The most frequent ocular infections resistant to methicillin were found among conjunctivitis cases. In this study, those MRSA isolates were highly susceptible to chloramphenicol (75%), but resistant to clindamycin, tetracycline and gentamicin. Other similar studies reported chloramphenicol as clinically effective in MRSA (>81%) isolates from conjunctivitis cases and highly resistant to clindamycin, tetracycline and gentamicin (42,43).
About two third of bacterial isolates in this study showed ability to produce biofilm ranged from weak to strong adherence abilities. This feature can contribute to antimicrobial drug resistance development and play a vital role in pathologic processes over a long period by withstanding the effect of immune defence mechanisms. Bacterial biofilm formation feature is little noticed and studied in Ethiopia. Biofilm formation rate found in this study is comparable with study conducted in Chicago (44). However, comparatively lower biofilm formation rate was reported in Saudi Arabia (45). P. aeruginosa (80%), K. pneumoniae (77.8%), CoNS (75.6%), S. aureus (72.4%) and E. coli (60%) were among the leading biofilm former. This biofilm producing features may be responsible for many recalcitrant or refractory infections due to Gram negative bacilli and Gram positive cocci, and are notoriously difficult to eradicate. It is a well-known pathogenic mechanism in most bacterial strains as they exhibit resistance to antibiotics by various means like restricting penetration of antibiotic into biofilms, decreasing growth rate and expressing resistance genes. It is noted that biofilm formation allows microorganisms to survive and thrive in hostile environment, to disperse so that forming new niches and to give them significant advantages in protection against environmental fluctuations. Bacteria in biofilms display increased cell-to-cell communication while becoming less sensitive to chemical and physical stresses, and this may further complicate patient treatment outcome (46, 47).
Multiple drug resistance (MDR) to three or more antimicrobial agents were observed in 101(68.7%) of the tested isolates. This is a significant proportion that can influence patient cure from external ocular infection. Comparable findings is reported in studies conducted from southern and northern Ethiopia (18,24). It has been suggested that indiscriminate and prolonged use of a wide range of antibiotics, and lack of personal hygien might be a major factor leading to emergence of multidrug resistant strains.
In the current study, strains capable of forming biofilms were more frequently observed to be MDR phenotype. Simultaneous occurrence of most MDR and biofilm forming strains show that biofilm phenotype may play a great role in antimicrobial resistance, despite the correlation was not appreciable in this study. Other studies (48,49) reported biofilm formation is higher in MDR bacteria.
Phenotypic changes in the bacterial shape, physiological changes within cells, low diffusion of antibiotics across the biofilm matrix, elevated expression of efflux and quorum-sensing may be some of the reasons for this high MDR property. Most of the Gram positives and Gram negative bacteria isolated in this examination that are biofilm positives were found to be resistant to aminoglycosides, penicillins, fluoroquinolones, folate pathway inhibitors, chloramphenicol and tetracycline. Similar previous studies have also shown biofilm formation is higher in those described antimicrobial categories (50-52). In this study, as biofilm forming features of bacteria were assessed in in vitro set up, the result might be different from the real biofilm formed on external ocular site infection of the patients. Moroever, the absence of routinely performed and standardinezd antibiofilm susceptibility protocols worldwide may undermine the influence of bacterial biofilm in patient morbidity. Anaerobic bacterial culture and Chlamydia trachomatis test were not included in this research due to resource conistraint. In addition, anti-biofilm drug susceptibility test was not done for biofilm former bacterial isolates due to lack of antimicrobial constituents for agar dilution method.

Conclusion
Both Gram positive and Gram negative bacteria were responsible for external ocular infections with the most predominant isolates: CoNS followed by Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeroginosa and Klebsiella pneumoniae. Increased resistance rate was observed to ampicillin, penicillin, erythromycin, trimethoprim-sulphamethoxazole, tobramycin and tetracycline.
Ciprofloxacin and gentamicin were the most effective agents against a good number of isolates from external ocular infections. MDR bacterial isolate was prevalent and methicillin resistance was detected in about a fifth of staphylococci isolates. P. aeruginosa (80%) was the leading biofilm former followed by Klebsiella pneumoniae (77.8%) and CoNS (75.6%). Almost all biofilm formers were MDR.

Declarations
Ethics approval and consent to participate: Ethical clearance was approved and obtained from Institutional Research and Ethical Review Committee Board under the Institute of Health, Jimma University. Permission was also obtained from Jimma University Medical Center clinical director and ophthalmology department. Written informed consent and assent was obtained from the study subjects immediately before data collection. Consent for publication: It was obtained from each participant or guardian as incorporated in the 'participant information sheet' before inclusion in this study.
Availability of data and material: The data sets used and/or analysed during this study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.