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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 193-202

Antibiotic Susceptibility Profile and Plasmid Analysis of Micro-Organisms Associated with Ocular Infections


1 Department of Biological Sciences, Spiritan University, Nneochi, Abia State, Nigeria
2 Department of Microbiology and Brewing, Nnamdi Azikiwe University Awka, Nigeria

Date of Submission18-Oct-2021
Date of Acceptance17-Dec-2021
Date of Web Publication17-Jun-2022

Correspondence Address:
Valentine Nnachetam Unegbu
Department of Biological Sciences, Spiritan University, Nneochi, Abia State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_274_21

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  Abstract 


Background: This study was undertaken to determine the antimicrobial susceptibility profile and plasmid analysis of micro-organisms associated with ocular infections. Methods: Ocular specimens were collected from 500 patients. Subsequent identification was done based on morphology and biochemical tests. Susceptibility pattern of the isolates was done using the disk diffusion method. The presence of plasmids was determined using the agar gel electrophoresis technique. Plasmid curing was carried out by treating the MAR isolates with sodium dodecyl sulfate at concentration of 10%. Results: Conjunctivitis was the most prevalent ocular infection of 105 (39.8%). S. aureus was the most prevalent pathogen 63 (23.9%) followed by CoNS 36 (13.6%). S. aureus was 100% sensitive to vancomycin and chloramphenicol. CoNS was also 100% sensitive to ciprofloxacin, vancomycin, and chloramphenicol. K pneumoniae was 100% sensitive to gentamicin and amoxicillin-clavulanic acid while N. gonorrhoeae was 100% sensitive to gentamicin, ciprofloxacin, ceftriaxone, amoxicillin-clavulanic acid, and cefotaxime. The overall MAR bacteria was 38 (16.2%). Plasmid analysis revealed the presence of 1–3 plasmid bands of sizes 6.21–16.22 Kbp from the MAR isolates. Plasmid curing revealed that the gene coding for resistant seen in this study was plasmid mediated. Conclusions: The prevalence of ocular infection was high with conjunctivitis being the dominant. The dominant bacteria species were S. aureus and CoNS. The overall MAR bacteria proportion was relatively high. The presence of plasmids was responsible for the resistance of the isolates to antibiotics.

Keywords: Bacteria, conjunctivitis, drug susceptibility profile, ocular pathogens, plasmid analysis


How to cite this article:
Unegbu VN, Anyamene C, Odibo F. Antibiotic Susceptibility Profile and Plasmid Analysis of Micro-Organisms Associated with Ocular Infections. Biomed Biotechnol Res J 2022;6:193-202

How to cite this URL:
Unegbu VN, Anyamene C, Odibo F. Antibiotic Susceptibility Profile and Plasmid Analysis of Micro-Organisms Associated with Ocular Infections. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Oct 5];6:193-202. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/193/347713




  Introduction Top


The eye is a unique organ that is almost impermeable to almost all external agents.[1] The defense mechanisms of the eye are the tears which contain several substances (e.g., lysozymes and interferon), the eyelids, and eye lashes. Pathogenic microorganisms cause diseases to the eyes due to their virulence and host's reduced resistance from many factors such as personal hygiene, living conditions, socioeconomic status, nutrition, genetics, physiology, fever, and age.[1] The areas in the eye that are frequently infected are the conjunctiva, lid, and cornea.[2]

Ocular infections in man are the contamination and invasion of ocular tissues by micro-organisms leading to the breakdown of the natural defense mechanisms of the eyes (i.e., the bony orbits, eyelids, eyelashes, and tears). This situation results in various ocular disorders including conjunctivitis, keratitis, blepharitis, lid abscess, external hordeolum, dacryocystitis, and blepharo-conjuctivitis.[3] The effects of these ocular infections are enormous as they cause both physical, emotional stress including psychological trauma if it leads to blindness or severe ocular distortions.[3]

In Nigeria, conjunctivitis is one of the most common eye problems which causes “red eye” that affects all age groups. Infective keratitis is a major cause of vision loss and blindness second to cataracts.[4] Blepharitis is an inflammation of the eyelid margins which can result in patient discomfort and decline in visual function while lid abscess may cause vision-threatening ocular complications.[5] Dacryocystitis is an inflammation of the lacrimal sac and duct.[5] Finally, eyelid infection causes redness of the eyelids and the skin around the eyes.[5]

Studies by investigators from ethiopia and Pakistan indicated that Gram-positive and Gram-negative bacteria are the most commonly isolated pathogens in patients with blepharitis, dacryocystitis, and conjunctivitis, but variations exist in etiologies, drug susceptibilities of pathogens, and antibiotic resistance mechanisms.[6],[7] Gram-positive bacteria such as Staphylococcus aureus, noncoagulase-positive Staphylococci, Bacillus sp, Corynebacterium sp., Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus viridans have been implicated as etiologies of most ocular infections in patients.[5] In Gram negative-mediated ocular infections, pathogens such as Pseudomonas aeruginosa, Escherichia coli, Proteus sp., Moraxella sp., and Neisseria gonorrhoeae have been isolated as etiologic agents.[5]

Plasmids, which are extra-chromosomal double-stranded DNA materials, have been found to be useful for pathogens' genetic diversity and prowess as infectious agents.[8] Profiling pathogens for their harbored plasmids have been found to be very useful in epidemiological studies, diagnosis, and elucidation of mechanisms of drug resistance.[8] Plasmids have also been found useful in knowing whether two or more strains of a pathogen evolve from the same microorganism, thereby providing a reliable insight into the genetic relatedness of pathogens in an environment.[8] Plasmid resistance curing is normally done to determine whether the gene coding for resistance is carried in the chromosomes or plasmids. Plasmid being an extrachromosomal DNA molecule is eliminated from host bacteria after exposure to sublethal concentrations of intercalating agents such as Acridine orange, ethidium bromide, and detergents such as sodium dodecyl sulfate (SDS).[9]

The management of bacterial eye infections may involve treatment with broad-spectrum antibiotics. The indiscriminate use of antibiotics led to the development of resistance to many commonly used antimicrobial medications. The emergence of bacterial resistance toward topical antimicrobial agents may increase the risk of treatment failure with potentially serious consequences.[6] Therefore, up-to-date information is essential for appropriate antimicrobial therapy and management of ocular infections.[7]

The number of people attending various health institutions for eye-related problems in Nigeria is currently in the increase. This has resulted in the establishment of optometry clinics in various hospitals across the country. Therefore, this study was carried out to assess the drug susceptibility pattern and plasmid profile of microorganisms isolated from ocular infections of patients in Abia State, Nigeria.


  Methods Top


Study area

Eye-patients attending the Optometry clinic at Abia State University Teaching Hospital Aba, Federal Medical Centre Umuahia, Abia State Diagnostic Hospital Umuahia, and General Hospital Ugwunagbo were the target population.

Study design

This is a cross-sectional study that included patients with clinically diagnosed bacterial conjunctivitis, keratoconjunctivitis, keratitis, blepharo-conjunctivitis, blepharitis, dacryo-cystitis, and lid abscess. All patients were diagnosed by a number of ophthalmologists using standard protocols.

Sample size/study techniques

A total of 500 ocular specimens, consisting of 125 ocular specimens (swabs), each from the four hospitals were used for this study. All individuals examined and diagnosed using the silt-lamp bio-microscope by ophthalmologists as ocular infection patients were included in this study.

Ethical statement

Ethical permission (ABSUTH/CS/56/VOL 2/48) was obtained from the hospital authorities and the consent of the patients was also obtained before specimen collection.

Eligibility criteria

Inclusion criteria

  • Clinically diagnosed patients suspected with ocular infections
  • Patients who gave their informed consent.


Exclusion criteria

  • Patients on topical antibiotics treatment
  • Patients with trachoma, peripheral ulcerative keratitis, viral keratitis, allergic and viral conjunctivitis, severe ocular trauma, and patients who had recent ocular surgery.


Specimen collection

Specimens were collected with the help of an ophthalmologists. Specimens from the eyes (eye, conjunctiva, lacrimal sac, and cornea) were collected using sterile swab sticks following routine clinical management of the patients.[10]

Culture, isolation and identification of bacteria

The obtained swabs were examined in the laboratory within 20 min to 1 h of collection using two methods, direct wet mount, and culture technique. The swabs were cultured on the appropriate media (chocolate agar, MacConkey agar, and blood agar) for microbial growth.

Specimens were cultured by the streak plate methods using wire loop on chocolate agar, MacConkey agar, and two blood agar plates (Oxoid Basingstoke, UK). MacConkey agar and one blood agar plates were incubated at 37°C aerobically and the other blood agar and chocolate agar plates were incubated at 37°C within a candle jar to enhance the growth of bacterial species that need 5%–10% CO2 (microaerophilic organisms).

After 24 h incubation, plates were examined for microbial growth. Specimens taken from the eyelid, conjunctiva, or lacrimal sac were considered as culture positive according to CLSI.[10] In the case of microbial keratitis, a culture was considered positive when there was growth of the same organism on two or more media or confluent growth of a known ocular pathogen at the site of inoculation on one solid medium.[10] Plates which did not show any growth were further incubated for additional 24 h. All observed colonies were identified by their characteristic appearance on their respective media. Furthermore, it was confirmed by the pattern of biochemical reactions using the standard method according to Clinical Laboratory Standard Institute.[10]

Chromogenic agar test

A loopful of the isolates was aseptically inoculated onto the surface of plates containing chromogenic agar medium. The inocula were spread all over the agar medium by streaking for bacteria isolates. The plates were incubated for 48 h. Color change was observed after incubation.[10]

Molecular characterization

Background

Some bacterial isolates cannot be taxonomically identified from phenotypic characteristics. Bacterial isolates can be characterized by sequencing the 16S Rdna. The universal primers 27F and 1492R are used to amplify the 16S target region.[9]

Extraction and sequencing of bacterial isolates

Using the ZR Fungal/Bacterial DNA Kit™ (Zymo Research), DNA was obtained from the cultures received. The 16S target region was amplified using DreamTaq™ DNA polymerase (Thymo Scientific™) and the primers shown in [Table 1]. PCR products were gel extracted (Zymo Research, Zymoclean™ Gel DNA Recovery Kit) and sequenced in the forward and reverse directions on the ABI PRISM™ 3500xl Genetic Analyser. Purified sequencing products (Zymo Research, ZR-96DNA Sequencing Clean-up Kit™) were analyzed using CLC Main Workbench 7 followed by a BLAST search (NCBI).
Table 1: 16S primers sequence

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Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed for all bacterial isolates (307 isolates) using disk diffusion method on Mueller–Hinton agar (Oxoid Basingstoke, UK) according to the direction of the Clinical and Laboratory Standards Institute.[10] Briefly, 3–5 colonies of the test organism were emulsified in 5 ml of nutrient broth and mixed gently. The suspension was incubated at 37°C and the turbidity of the suspension becomes adjusted to 0.5 McFarland standards. The suspension was uniformly rapped onto Mueller-Hinton agar. The antimicrobial impregnated disks were placed using sterile forceps on the agar surface and the plates were incubated at 37°C for 24 h and the zone of inhibition was determined. The antimicrobial agents on the disks and their concentrations are as follows: ofloxacin (5 μg), gentamicin (GEN, 10 μg), ciprofloxacin (CIP, 5 μg), vancomycin (VA, 30 μg), chloramphenicol (CHL, 10 μg), tetracycline (TET, 10 μg), ceftriaxone (CTR, 30 μg), and ampicillin (AMP, 10 μg). The rest are amoxicillin-clavulanic acid (AMC, 30 μg) and cefotaxime (CEF, 30 μg). The zones of inhibition were measured to the nearest millimeter using a transparent foot ruler. The results obtained were interpreted as sensitive or resistant according to the direction of the Clinical and Laboratory Standards Institute. (Resistance 0–16 mm and sensitive >16 mm).[10]

Identification of Multiple antibiotic resistance (MAR) bacteria

The multiantimicrobial resistance bacteria in this study were identified by observing the resistance pattern of the isolates to at least one antimicrobial drug in three or more antimicrobial categories used in this study.[10]

Determination of multiple antibiotic resistance (MAR) index

Multiple antibiotic resistance (MAR) index was determined for each of the selected bacterial isolates by dividing the number of antibiotics to which the isolate showed resistance by the total number of antibiotics to which the isolates were exposed. Thus, MAR index = a/b, where (a) represents the number of antibiotics to which the isolates were resistant and (b) the number of antibiotics to which the isolates were exposed.[10]

Plasmid profile studies using agarose gel electrophoresis

Culture purification

Purity of bacterial cultures to be used for the test was ascertained by subculturing the isolates on Nutrient and MacConkey agars.

Extraction of plasmid DNA

Selected resistant isolates were grown in a 5 ml double-strength Mueller–Hinton broth for 72 h at 37°C. The 72 h grown cultures were harvested into Eppendorf tubes which have already been labeled with the name of each isolate and centrifuged in a microcentrifuge for 10 min at 10,000 rpm to obtain pellets. The supernatant was gently decanted and the cell pellets were vortexed for 5 min. Thereafter, 300 μl of Tris EDTA (TE) buffer and 150 μL of 3.0 M sodium aqueous acetate was added at pH 5.2 and was vortexed for 3 mins to lyse the bacteria cell pellet. The samples were centrifuged again for 10 min in a microcentrifuge (Biofuge, Biotra Bio-trade Hecrus Sepatech Co. Ltd USA), and the supernatant was transferred to a fresh Eppendorf tube, mixed well with 0.9 ml of 70% ethanol which had been precooled to −20°C (in a refrigerator) to precipitate the plasmid DNA. It was centrifuged again for 10 min and the supernatant was discarded. The pellet was rinsed twice with 1 ml of 70% ethanol and was air dried for 10 min (excess alcohol was wiped away from the sides of the tube surrounding the pellet very carefully using an absorbant paper), after which it was resuspended in 20–40 μL of TE buffer for further use. It was stored at 4°C or frozen until needed.[9]

Preparation of Gel

The gel casting apparatus was set up as instructed in the product manual. The comb was straight, and there were few millimeters of clearance between the bottom of the comb and the bottom of the gel tray. A 0.5 g of agarose was dissolved in 50 ml of Tris Borate EDTA buffer (TBE), thus forming 1.0% gel. The agarose was completely melted and the agarose solution was gently swirled while looking out for translucent “flecks” of nonmelted agarose. Heat was later applied until all flecks are gone. The agarose solution was allowed to cool to a temperature of about 50°C–55°C before pouring. Thereafter ethidium bromide (2 μl of a 10 mg/ml EtBr solution [per 50 ml gel]) was added, swirled to mix and the mixture poured into a gel tray. Hand glove was worn when handling ethidium bromide as ethidium bromide is a well-known mutagen. This was allowed for 20 min to solidify (the solution turned from translucent to opaque when solidified) and the comb was carefully removed from the gel. The gel carrier was removed from the pouring tray and was placed in the gel electrophoresis tank. A 250 ml of 1 × TBE was used to fill the electrophoresis tank until the gel was submerged. Air bubbles left in the sample well were carefully dislodged with a pipette because the presence of air bubbles will exclude the buffer and make the wells difficult to fill with the sample.[9]

Electrophoresis of the DNA samples

Using micropipette, a 50 μL sample of DNA and 3 μL of loading dye were added together and carefully mixed by pipetting the solutions up and down.[9] Each sample was loaded carefully into the gel wells, one sample per well, and this was placed on the gel box at the negative charge end of the electrophoresis machine. Buffered water was added which sealed the agarose containing the sample DNA and acted as electrolyte by moving the current as well as the sample DNA towards the positive end for 2 h with a voltage of 63 V. The agarose containing the sample DNA was removed and allowed to drain off. With the aid of ultraviolet (UV) light, UV certified safety glasses and camera, a picture showing size and movement of the sample DNA was taken to determine the mobility in millimeter using a known sample standard.[9]

Determination of molecular weight of plasmids of the isolates

Using the distance of migration of the bands (plasmid unit) in each isolate and matching the value of the marker in the standard with it, the plasmid sizes of the isolates were determined.[9]

Plasmid curing using sodium dodecyl sulfate

Plasmid curing was carried out on organisms that were resistant to minimum of 3 antibiotics using subinhibitory concentration of 10% SDS as described by Coba et al.[9] Overnight broth culture was inoculated into 4.5 ml nutrient broth. About 1.0 ml of sodium dodecyl sulfate (10% concentration) was added and incubated for 48 h at 37°C. 5.5 ml of the broth was added into a freshly prepared 4.5 ml nutrient broth, it was incubated for another 24 h at 37°C, after which another plasmid antibiotic susceptibility test was carried out on each of the isolates. The suspension was uniformly spread onto Mueller-Hinton agar. The antimicrobial impregnated disks were placed using sterile forceps on the agar surface, and the plates were incubated at 37°C for 24 h.

Reference strains

Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATTC 27853) were used as reference strains for culture and sensitivity testing.

Controls

Prior to actual data collection, comprehensiveness, reliability, and validity of questionnaires were pretested on ten patients each from the four aforementioned hospitals. All specimens were collected following standard operating procedure for ophthalmic specimen collection. The sterility of culture media was ensured by incubating 5% of each batch of the prepared media at 37°C for 24 h. Performances of all prepared media were also checked by inoculating standard-strains such as Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853) obtained from Nigerian Institute of Medical Research Yaba, Lagos State. The qualities of biochemical testing procedures were checked by these reference strains.

Statistical analysis

Statistical analysis was carried out using the SPSS 21.0 (IBM SPSS, Chicsgo, Illinois, USA) window-based program. The proportion of isolated bacteria with patient demographic information and susceptibility to commonly used antibiotics was compared using the Chi-square test. A value of P < 0.05 was considered to be statistically significant.


  Results Top


[Table 2] shows the prevalence of bacteria pathogens across the different clinical features of ocular infections. S. aureus was the most prevalent pathogen 63 (23.9%) followed by CoNS 36 (13.6%) and S. pneumoniae 35 (13.3%). The least pathogen was Neisseria gonorrhoeae 3 (1.1%). Pathogens isolated from conjunctivitis includes S. aureus 25 (23.8%), CoNS 16 (15.2%), E. coli 14 (13.3%), P. aeruginosa 6 (5.7%), S. pneumoniae 24 (22.9%), Moraxella catarrhalis 3 (2.9%), and Neisseria gonorrhoeae 3 (2.9%). Pathogens isolated from keratitis include S. aureus 11 (30.6%), P. aeruginosa 8 (22.2%), and S. pneumoniae 10 (27.8%). Other pathogens isolated from clinical features are found in [Table 2].
Table 2: Prevalence of bacteria pathogens across the different clinical features of ocular infections at ASTHA, GHU, FMCU, and ASDHU

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[Table 3] shows the antimicrobial susceptibility pattern of bacteria isolated from the hospitals. S. aureus was 100% sensitive to vancomycin and chloramphenicol. CoNS was also 100% sensitive to ciprofloxacin, vancomycin, and chloramphenicol. K pneumoniae was 100% sensitive to gentamicin and amoxicillin-clavulanic acid while N. gonorrhoeae was 100% sensitive to gentamicin, ciprofloxacin, ceftriaxone, amoxicillin-clavulanic acid, and cefotaxime.
Table 3: Antimicrobial susceptibility pattern of bacteria isolated from the selected hospital

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[Table 4] shows the multiple antimicrobial resistance (MAR) bacteria. The overall MAR bacteria was 38 (16.2%). They include S. aureus 11 (28.9%), E. coli 12 (31.6%), P. aeruginosa 8 (21.1%), and K. pneumoniae 7 (18.4%).
Table 4: Multiple antimicrobial resistance bacteria

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[Table 5] shows the relationship between the distance moved by the standard and the log of molecular weight of the standard. When the graph of the distance moved by the standard is plotted against Log of molecular weight of standard, the result is a linear equation [Figure 1].
Table 5: Determination of molecular weight of the plasmids

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Figure 1: Distance moved by standard against log of molecular weight of standard

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[Table 6] shows the resistant pattern of some selected antibiotic mar bacteria before and after plasmid curing at 10% SDS concentration. The SDS was lethal to isolate 8. It had no effect on isolates 7 and 9–11 but it partially cured isolates 3–6 and 12–15.
Table 6: Resistant pattern of some antibiotic resistant bacteria isolated from ASTHA, FMCU, GHU, and ASDHU before and after plasmid curing at 10% sodium dodecyl sulfate

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[Figure 2] shows the Plasmid profile of selected 13 MAR isolates before plasmid curing. Plasmid profile shows the presence of plasmids in the isolates with bands between 1 to 3 in each of the isolates ranging from 6.21kbp to 16.22kbp. Comparison of plasmid sizes and numbers showed that some of the isolates have plasmid band of the same number and sizes. [Figure 3] shows the Plasmid profile of selected MAR isolates after curing with sodium dodecyl sulphate (SDS) at 10% concentration. Of these 13 isolates, the curing was lethal to one of the isolates in lane 8. Furthermore, the isolates in lane 7, 9 11 wasn't affected by the curing agent while the curing was partially efficient in some isolates (lane 3-6, 12-14).
Figure 2: Plasmid profile of selected 13 MAR isolates. KEY: Lane 1 = Molecular Weight Standard, Lane 2 = Empty well, Lane 3 = S. aureus3, Lane 4 = S. aureus4, Lane 5 = S. aureus5, Lane 6 = S. aureus6, Lane 7 = E. coli7, Lane 8 = E. coli8, Lane 9 = E. coli9, Lane 10 = P. aeruginosa10, Lane 11 = P. aeruginosa11, Lane 12 = P. aeruginosa12, Lane 13 = K. pneumoniae13, Lane 14 = K. pneumoniae14, Lane 15 = K. pneumoniae15

Click here to view
Figure 3: Plasmid profile of selected MAR isolates after curing with SDS at 10% concentration. Lane 1 = Molecular Weight Standard, Lane 2 = Empty well, Lane 3 = S. aureus3, Lane 4 = S. aureus4, Lane 5 = S. aureus5, Lane 6 = S. aureus6, Lane 7 = E. coli7, Lane 8 = E. coli8, Lane 9 = E. coli9, Lane 10 = P. aeruginosa10, Lane 11 = P. aeruginosa11, Lane 12 = P. aeruginosa12, Lane 13 = K. pneumoniae13, Lane 14 = K. pneumoniae14, Lane 15 = K. pneumoniae15

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  Discussion Top


Out of 500 ocular specimens, a total of 79.5% isolates of bacterial pathogens were isolated. This finding is in agreement with the previous studies carried out in ethiopia where 54.9% and 59.4%, respectively, were recorded.[3],[4],[5],[6],[7],[8],[9],[10],[11] Higher prevalence was reported in Jimma 74.7% and in India 64%.[12],[13] The varying rate of bacterial pathogen isolation may be due to geographical location or study period when this study was carried out.

Conjunctivitis was the most common eye infection seen in this study (39.8%) as was found in previous studies.[14],[15] This was followed by blepharitis (28.8%), keratitis (13.6%), lid abscess (7.6%), blepharo-conjuctivitis (7.2%), and dacryocystitis (1.9%). This was in agreement to other similar studies.[16],[17] The causes of bacterial conjunctivitis were due to the alteration in the normal flora which can occur by external contamination, by infection spread from adjacent sites or through blood-born pathway and disruption of epithelial layer covering the conjunctiva.[17]

The predominant bacterial isolates were Staphylococcus aureus (23.9%) followed by coagulase-negative staphylococci (13.6%), which was the normal conjuctival flora.[18] This finding is in agreement with previous study by Anagaw et al. who reported 37.4% and 12.3% as the isolation rates for S. aureus and coagulase-negative Staphylococci, respectively, in the same environment.[3] In southeast Nigeria, Ubani also recovered S. aureus (23.7%) and Staphylococcus albus (19.3%) as the predominant Gram-positive pathogens of ocular infections in patients.[19] In India, isolation rates of 25% and 18.3% have been reported in S. aureus and noncoagulase Staphylococci as ocular pathogens, respectively.[20] In general, the predominance of Staphylococcus aureus, CoNS, and S. pneumoniae as major ocular pathogens might be due to the fact that these organisms represent the major flora of the eye lid and the conjunctiva and under normal conditions, their clinical manifestations are averted by eye innate immune defense system constituted by tear flow, secretory immunoglobulin, and the presence of cidal agents such as lysozyme and lactoferrin.[20] In other study, the predominant isolates were coagulase-negative staphylococcus (22%) followed by Streptococcus pneumoniae (19.3%).[21] The little difference may be due to the difference in climate and geographical location between Nigeria and India reconfirming previous reports that ocular pathogens vary in etiology in different countries and different locations within a country.[19],[20]

With regard to Gram-negative pathogens in ocular infections in this study, Pseudomonas aeruginosa was found to be the predominating species with an isolation rate of 9.8%, whereas in a previous study in the same environment, Klebsiella pneumoniae was the most commonly isolated pathogen (10.3%) followed by P. aeruginosa (8.7%).[19] Findings in this study, therefore, indicate a changing trend in the Gram-negative etiology of ocular infections in Abia State. In other parts of the country, K. pneumoniae was also most commonly isolated, followed by E. coli coupled with the involvement of other Gram-negative pathogens such as N. gonorrhoeae and Neisseria meningitides.[2] This again corroborates the influence of locations on the etiology of ocular infections.

The high prevalence of Gram-negative enteric bacteria in this study could be due to ineffective personal hygiene, as the most important mode of transmission for enteric pathogens is faeco-ocular contamination.[2] During data collection, we noticed that the surrounding communities near the hospitals lack proper waste and sewage disposal system.

Among the clinical features, significant association of culture positivity was observed among study subjects with blepharitis (28.8%) with S. aureus (25%), CoNS (18.4%), P. aeruginosa (6.6%), and K. pneumoniae (26.3%) being the major etiological agent. This is in agreement with the study done in ethiopia.[4]

Streptococcus pneumoniae (27.8%), Staphylococcus aureus (30.6%), and Pseudomonas aeruginosa (22.2%) were found to be the predominant isolates in the cases of microbial keratitis (13.6%). This was also in agreement with previous works in ethiopia.[11] In contrast, other studies reported P. aeruginosa as the major isolate.[12],[21] This may be due to inter-population variations and environmental dissimilarities in different countries.[12] Microbial keratitis is often related to contact lens wear, especially improper contact lens use or storage; and wearing of contact lens overnight (i.e., extended wear). Bacterial keratitis cannot be spread from person to person.[22]

More so, Staphylococcus aureus (23.8%), CoNS (15.2%), S. pneumoniae (22.9%), E. coli (13.3%), P. aeruginosa (5.7%), Moraxella catarrhalis (2.9%), and Neisseria gonorrhoeae (2.9%) were also found to be predominant in the cases of conjunctivitis. This was also in agreement with the study done by Anteneh et al.[4] Bacterial conjunctivitis is highly contagious; most bacteria that cause conjunctivitis are spread through direct hand-to-eye contact from contaminated hands or improper lens hygiene.[4]

Staphylococcus aureus (10.5%), Coagulase-negative staphylococcus (31.6%), E. coli (26.3%), and K. pneumoniae (10.5%) were the predominant bacterial isolates observed in blepharo-conjuctivitis cases. This was in agreement with the study conducted in Nigeria.[2] The reason for the high rate of S. aureus and CoNS among blepharitis and blepharo-conjuctivitis may be virulence factor such as exo-enzymes and a surface slime that may play a role in the pathogenesis.[2]

Moraxella catarrhalis was implicated in conjunctivitis (2.9%) and lid abscess (10.0%) in this study. This contrasted with previous study in ethiopia which implicated it in dacryocystitis and keratitis.[4] Moraxella catarrhalis being an opportunistic pulmonary invader and which causes harm, especially in immune-compromised individuals have been reported to be an emerging bacterial pathogen of ocular infection.[23]

The Neisseria gonorrheae seen in conjunctivitis cases were likely from the age group 0–15 years. Susceptibility to infection is increased in babies due to low immunity at such ages.[24] In addition to this, the air facilitates the transfer of bacteria to hospital delivery rooms, especially when opening the doors and windows.[24]

In this study, different bacterial species showed high level of resistance pattern to different antimicrobial agents. For example, most of the bacterial isolates have shown high resistance to tetracycline and ampicillin. This is in agreement with the studies done by Musa et al.[25] Reduced efficacy to the above-mentioned antibiotics could possibly be due to frequent usage of these drugs by patients with or without prescription as they are readily available and easy to purchase in any chemist store around.[25]

S. aureus was 100% sensitive to vancomycin and chloramphenicol. CoNS was also 100% sensitive to ciprofloxacin, vancomycin, and chloramphenicol. K pneumoniae was 100% sensitive to gentamicin and amoxicillin-clavulanic acid while N. gonorrhoeae was 100% sensitive to gentamicin, ciprofloxacin, ceftriaxone, amoxicillin-clavulanic acid, and cefotaxime. This observation is consistent with those studies conducted in Libya and India.[21],[26] Yet, it is contradictive to the findings obtained from a recent study in ethiopia which reported high resistance to ciprofloxacin and ceftriaxone.[3] The high susceptibility shown by vancomycin, ciprofloxacin, and chloramphenicol to all Gram-positive isolates in this study implies that such drugs can be used to treat ocular infections caused by Gram-positive bacteria in such environment. Furthermore, the high susceptibility shown by gentamicin, amoxicillin-clavulanic acid, and ceftriaxone to Gram-negative bacteria also implies that such drugs can be used to treat ocular infections caused by Gram-negative bacteria in the study environment.

The prevalence of multiantimicrobial-resistant (MAR) bacteria to at least one antimicrobial drug in three or more antimicrobial categories used in this study was 16.2%; and this is similar to results from previous studies.[27],[28] In Nigeria, antimicrobial drugs can be purchased at any pharmaceutical store without prescription, which may contribute to the emergence and spread of antimicrobial resistance.[12] Other factors may include improper dosage regimen and substandard antimicrobial drugs which is sold all over Nigeria.[29]

All the 38 (16.2%) isolates with multiantibiotic resistant bacteria had MAR indices >0.2. This was similar to the work of Ugwu et al.[30] who recorded a high level of MAR index of 46% within Nsukka metropolis, Enugu State. When the MAR index is >0.2, it shows that the organisms were isolated in an environment where antibiotics are widely abused,[31] which was the case in this study.

The results from this study revealed that among 13 chosen multidrug resistance isolates from the studied government hospitals in Abia State, plasmid bands (1–3 bands) were detected in all thirteen isolates with size ranging from 6.21 to 16.22 Kbp. Plasmid-mediated resistance to various infections caused by antibiotic-resistant bacteria has been demonstrated by various studies from various states in Nigeria and these researchers have highlighted these diverse plasmid profiles.[32],[33] The implication of this is that most of the patients may not respond positively if they are infected with any of the isolated organisms and treated with any of the antibiotics tested in this study.

Comparison of plasmid sizes and numbers showed that some of the isolates have plasmid band of the same number and sizes which indicates that they are likely of same origin, most probably the same community or close to the hospitals were this study was carried out. The presence of some plasmid DNA in the isolates corresponding to the reference standard DNA fragments suggests that their antimicrobial resistance is possibly plasmid-mediated.[34] The isolated plasmids may be responsible for possibly mediating some or all of the expressed resistances of the microorganisms. Similar findings were also discovered in the work of Sheikh et al.[35] The plasmid profiling also showed that two isolates of S. aureus (one isolated from Abia State Teaching Hospital Aba and the other isolated from Federal Medical Centre Umuahia) had identical plasmid band and sizes, suggesting that they came from the same origin.

Resistance plasmid curing using SDS at concentration of 10% was done to firmly establish the role of the isolated plasmids in the observed resistance patterns of the micro-organisms. Curing agents such as SDS if administered to bacterial populations in sublethal doses can lead to the elimination of plasmid DNA without harming the bacterial chromosome and thus maintaining the ability to reproduce and generate offspring.[36] Population of bacteria containing plasmids that are subjected to agents such as SDS will become more and more dominated by plasmid-free cells with time.[36] Thus, curing by SDS normally involves loss of the whole plasmids. The 13 isolates were consequently subjected to a plasmid curing procedure with SDS at concentration of 10% to cure their respective antibiotic resistance properties. Of these 13 isolates, the curing was lethal to one of the isolates in lane 8 (E. coli8). Furthermore, the isolates in lane 7 and lanes 9-11 (E. coli7, Lane 9 = E. coli9, Lane 10 = P. aeruginosa10, Lane 11 = P. aeruginosa11) still remained resistant to most of the drugs they were resistant to before curing. The curing was partially efficient in some isolates (Lane 3 = S. aureus3, Lane 4 = S. aureus4, Lane 5 = S. aureus5, Lane 6 = S. aureus6, Lane 12 = P. aeruginosa12, Lane 13 = K. pneumoniae13, Lane 14 = K. pneumoniae14, Lane 15 = K. pneumoniae15) as only the lighter plasmids were eliminated but the heavy plasmids remained. This might be due to the fact that the heavy plasmids had higher rate of stability than the lighter plasmids, which made it possible for the lighter plasmid borne multiresistance genes to be removed by the 10% concentrated SDS used as curing agent in sublethal concentration.[36] By this result, it shows that the gene coding for resistant seen in this study was carried plasmid mediated. This is similar to the works of other researchers,[37],[38] who posited that resistance in multidrug-resistant organisms was plasmid mediated.


  Conclusions Top


The prevalence of ocular infection in this study was very high. Conjunctivitis was the dominant ocular infection followed by blepharitis and keratitis. S. aureus was the predominant isolated bacteria followed by CoNS and S. pneumoniae. Gram-positive isolates were highly susceptible to ciprofloxacin, vancomycin, and chloramphenicol while Gram-negative isolates were highly susceptible to ceftriaxone, amoxicillin-clavulanic acid, and gentamicin. The overall MAR bacteria was relatively high. Plasmid analysis revealed the presence of 1–3 plasmid bands of sizes 6.21–16.22 Kbp from the MAR isolates. Plasmid curing revealed that the gene coding for resistant seen in this study was plasmid mediated. Therefore, the identification of potential pathogenic bacteria implicated in these infections through culture and biochemical tests methods as well as conducting drug susceptibility test and plasmid profiling should be practiced as a routine diagnostic procedure to prevent the increasing rate of antimicrobial resistance bacteria seen in this study.

Acknowledgments

The authors wish to thank all the staff at the Optometry clinic of Abia State University Teaching Hospital Aba, Federal Medical Centre Umuahia, Abia State Diagnostic Hospital Umuahia, and General Hospital Ugwunagbo.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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