Analysis of conjunctival flora in diabetic and non-diabetic individuals and their antibiotic sensitivity pattern

Antibiotic resistance in ocular microorganisms

An experimental study was conducted on rabbits and it was found that endophthalmitis caused by resistant S. epidermidis caused more ocular tissue destruction and inflammation than non-resistant strains.^[8] A nationwide US-based multicenter surveillance program, TRUST, was established in 1996, in which isolates are sent, from over 200 clinical laboratories to an independent central laboratory for in vitro susceptibility testing. A sub study was initiated in 2005, which was ocular-specific (Ocular TRUST1), looking to gather prospective data each year as well as to retrospectively analyze ophthalmic samples from previous years. The TRUST study specifically looks at three microorganisms — Haemophilus influenza, Staphylococcus aureus, Streptococcus pneumoniae S. aureus further being divided as methicillin-susceptible (MSSA) or methicillin-resistant (MRSA). A similar surveillance program, ARMOR^[9] study, was specifically set up to monitor ocular pathogens across the United States. Initial results were published in 2011 (ARMOR 2009) and the study was based on isolates which were collected from 34 institutions in 2009, and subsequent data from 2009 through 2013 (ARMOR 2013) were published this year. The ARMOR study extends data collected for TRUST studies with analysis of Pseudomonas aeruginosa and CoNS.3237 isolates were analyzed in the ARMO R 2013 study and is the largest study of its kind till date.

Adam M et al^[10] investigated aerobic bacteria in the conjunctival flora of diabetic patients in comparison with that of non-diabetic individuals and found that in the diabetic group there were 34 (64.15%) women and 19 (35.85%) men; in the control group there were 26 (60.45%) women and 17 (39.55%) men. The average age of the patients in the diabetic group was 53.94±9.24 years (range, 38-70 years) and in the control group was 55.23±10.97 years (range, 40-71 years). No noteable differences were observed between the two groups in terms of demographic characteristics (gender, p=0.71; age, p=0.89). Suresha K et al^[11] compared the normal conjunctival flora of non diabetics with that of diabetics and antibiotic sensitivity pattern of the organisms identified was carried out. It was found that age demographics of the patients varied from 17-85 years. There were 49 male patients and 51 female patients. Suto C et al^[12] study compared the isolates from the conjunctival sac before cataract surgery and the differences of isolates and resistance to antimicrobial agents was studied. It was found positive in 234 males (40.4%) and 345 females (59.6%). The range in age was from 51 to 100 years (mean ± standard deviation: 71.7 ± 9.5 years).

Hayashi Y et al^[13] retrospective study compared isolates from the conjunctival sac bacterial flora prior to cataract surgery and identified differences of isolates and resistance to antimicrobial agents. They found 90 patients (3.3%; 83 eyes of 30 females and 42 eyes of 30 males) in a total of 3754 eyes of 2384 patients. The mean age of the subjects with duct obstruction was 79 ± 8.5 years.

In our study, the mean fasting blood sugar level of patients in Group A (Diabetics) and Group B (Non-Diabetics) was 100.48 ± 20.68mg/dl and 86.93 ± 4.72mg/ dl respectively. The mean difference was statistically significant as per Student t-test (p<0.05). The mean glycocylated haemoglobin (HbA1c) level of patients in Group A (Diabetics) and Group B (Non-Diabetics) was 5.40 ± 0.92% and 4.85 ± 0.22% respectively. The mean difference, as per Student t-test, was statistically significant (p<0.05). The duration of diabetes mellitus in majority of patients in Group A (Diabetics) (78.3%) was <5 years while 7 (15.2%) and 3 (6.5%) patients had diabetes was 5-10 years and >10 years respectively. 40 (86.9%) patients in Group A (Diabetics) were on oral hypoglycaemics while 6 (13.1%) patients were on insulin therapy for treatment of diabetes mellitus. Adam M et al^[10] study investigating the aerobic bacterial conjunctival flora in diabetic patients and comparing it to non-diabetics reported that in the diabetic group,^[3] 83 % individuals were on an oral anti-diabetic and 17% were on insulin. The mean duration of diabetes was 7.81±5.77 years (range, 1-20 years). Diabetic retinopathy (DR) was seen in 12 patients (22.6%).

It was observed in our present study that the rate of positive culture in Group A (Diabetics) was significantly higher as compared to Group B (Non -Diabetics) (21.7% vs. 4.3% respectively). The difference was statistically significant as per Student t-test (p<0.05). This is comparable to the studies of Adam M et al,^[10] Suresha K et al,^[11] Suto C et al^[12] and Hayashi Y et al.^[13] Suresha K et al^[11] comparative study comparing the normal conjunctival flora of non-diabetics with that of diabetics and identifying the organisms from the conjunctival flora and their antibiotic sensitivity pattern reported total positive cultures in 148 from 100 patients (diabetic=72 and non-diabetics=74). 20 patients had negative cultures. Adam M et al^[10] study investigating the aerobic bacterial conjunctival flora in diabetic patients and comparing it to non-diabetics reported growth in 38.5% of diabetics and 34.9% of non-diabetic individuals. Suto C et al^[12] study comparing isolates from the conjunctival sac bacterial flora prior to cataract surgery and identifying differences of isolates and resistance to antimicrobial agents reported bacteria were isolated from 227 (39.2%) of the 579 eyes. Out of these, one isolate was found in 179 eyes (78.9%), 2 isolates were found in 39 eyes (17.2%), 3 isolates were found in 8 eyes (3.5%), and 4 isolates were found in one eye (0.4%). Hayashi Y et al^[13] retrospective study comparing isolates from the conjunctival sac bacterial flora prior to cataract surgery and identifying differences of isolates and resistance to antimicrobial agents reported microbial growth from the swab samples in 56 of 125 subjects from 90 patients at the initial bacterial culturing (44.8%).

The most common bacteria isolated in Group A (Diabetics) of our study was Staphylococcus epidermidis (13.2%) followed by Staphylococcus aureus (6.6%) and Klebsiella (2.2%) and in Group B (Non-Diabetics) was Staphylococcus epidermidis (2.2%) and Staphylococcus aureus (2.2%). There was significantly higher incidence of Staphylococcus epidermidis in Group A (Diabetics) as compared to Group B (Non-Diabetics) (p<0.05). This is concordant to the studies of Suto C et al^[12] and Hayashi Y et al.^[13] Suto C et al^[12] study comparing isolates from the conjunctival sac bacterial flora prior to cataract surgery and identifying differences of isolates and resistance to antimicrobial agents reported Gram-positive cocci (191 strains), accounting for 67% of all isolates, (44.5%) of methicillin-sensitive coagulase negative staphylococci (127 strains), (12.7%) of methicillin resistant coagulase-negative staphylococci (37 strains), (2.9%) of methicillin-sensitive Staphylococcus aureus (8 strains), (1%) of methicillin-resistant S. Aureus (3 strains). Out of 76 isolates of Gram-positive bacilli (26.7%), all belonged to genus Corynebacterium. Only one Gram-negative coccus (0.4%), Moraxella catarrhalis was isolated. 16 Gram-negative bacillary isolates were seen (5.9%), including three isolates of Escherichia coli (1.0%); two isolates (0.7%) each of Pseudomonas aeruginosa, Acinetobacter spp, and nonglucose -fermenting bacteria; and one isolate (0.4%) each of Proteus vulgaris, Proteus mirabilis, Enterobacter aerogenes, Serratiamarcescens, Klebsiellaoxytoca, and Alcaligenes spp. Hayashi Y et al^[13] retrospective study comparing isolates from the conjunctival sac bacterial flora prior to cataract surgery and identifying differences of isolates and resistance to antimicrobial agents reported reported percentages as follows: Gram-positive cocci (64%), Gram-positive rods(24%), Gram-negative bacilli (11%), and fungi (1%). Among 125 eyes with positive results for bacteria, coagulase-negative Staphylococcus (CNS) was found in 28 eyes, Corynebacterium species was found in 17 eyes, and S. aureus was found in 7 eyes. Methicillin-resistant S. aureus (MRSA) was found in two of seven eyes with S. aureus.

In our study, the association of Bacterial Isolates and Age of patients in Group A (Diabetics) showed that the incidence of Staphylococcus epidermidis was higher in the age group of 61-70 years (8.8%) as compared to 40-50 years (2.2%) and 51-60 years (2.2%). Also the incidence of Staphylococcus aureus was higher in the age group of 61-70 years (4.4%) as compared to 51-60 years (2.2%). The incidence of Klebsiella was seen in patient in the age group of 51-60 years. There was no significant association of bacterial isolates and age of patients in Group A (Diabetics) as per Chi-Square test (p>0.05). The association of Bacterial Isolates and Sex of patients in Group A (Diabetics) showed that the incidence of Staphylococcus epidermidis was present equally in male and female patients (6.6%). The incidence of Staphylococcus aureus was higher in male patients as compared to female patients (4.4% vs. 2.2%). The incidence of Klebsiella was seen in a female patient. There was no significant association of bacterial isolates and sex of patients in Group A (Diabetics) as per Chi-Square test (p>0.05). The association of Bacterial Isolates and Duration of Diabetes Mellitus (DM) of patients in Group A (Diabetics) showed that the incidence of Staphylococcus epidermidis was present equally in patients with diabetes mellitus for 5-10 years and >10 years (6.6%). The incidence of Staphylococcus aureus and Klebsiella was seen in patients with diabetes mellitus for 5-10 years (6.6% and 2.2% respectively). No bacterial isolate was seen in patients with diabetes mellitus for <5 years. There was no significant association of bacterial isolates and duration of diabetes mellitus of patients in Group A (Diabetics) as per Chi-Square test (p>0.05).

The association of Bacterial Isolates and Treatment of Diabetes Mellitus (DM) of patients in Group A (Diabetics) showed that the incidence of Staphylococcus epidermidis was higher in patients on insulin therapy as compared to oral hypoglycaemics (8.8% vs. 4.4%). All the incidence of Staphylococcus aureus was seen in patients on oral hypoglycaemics (6.6%). The incidence of Klebsiella was seen in patient on insulin therapy (2.2%). There was no significant association of bacterial isolates and treatment of diabetes mellitus of patients in Group A (Diabetics) as per Chi-Square test (p>0.05). These findings were consistent with the studies of Suto C et al,^[12] Adam M et al^[10] and Suresha K et al.^[11] Adam M et al^[10] study investigated the aerobic bacterial conjunctival flora in diabetic patients and compared it with that of non-diabetics and it was found that the bacteria found in cultures from the nondiabetic group was Staphylococcus aureus (gram-positive cocci) in 8 cultures (53.3%); coagulase-negative Staphylococci (CONS, gram positive cocci) in 4 cultures (26.7%); Klebsiella pneumonia (gram-negative bacilli) in 1 culture (6.7%) and more than one species of bacterium in 2 cultures (13.3%). In the diabetic group, 6 cultures identified Sta phylococcus aureus (30%); Escherichia coli (gram-negative bacilli) in 4 cultures (20%); CNS in 2 cultures (10%); Klebsiella pneumoniae in 2 cultures (10%); and more than one species of bacterium in 6 cultures (30%). There was no difference between the diabetic and non-diabetic groups in the ratio of positive cultures (chi-square=0.129, p=0.719), but gram-negative bacteria were more common in diabetics than in non-diabetics. Suresha K et al^[11] comparative study comparing the normal conjunctival flora of non-diabetics with that of diabetics and identifying the organisms from the conjunctival flora and their antibiotic sensitivity pattern reported most common organism isolated was Staphylococcus aureus in the diabetic group (40.2%) as well as in the non-diabetic group (58.1%). Diphtheroids were more commonly isolated in the diabetic group (18%). The gram negative bacilli were very few in number, EnterobacterSp (69.9%) being the most common in the diabetic group.

In the present study, the antibiotic sensitivity of bacteria isolated in patients in Group A (Diabetics) showed the following observations - in Staphylococcus epidermidis, maximum sensitivity was seen with Linezolid (100%), Cotrimoxazole (100%), Gentamicin (100%), Vancomycin (100%), and Clindamycin (100%) while resistance was seen with Ciprofloxacin (33.3%), Erythromycin (33.3%) and Oxacillin (33.3%). In Staphylococcus aureus, maximum sensitivity was seen with Linezolid (100%), Cotrimoxazole (100%), Gentamicin (100%), Vancomycin (100%) and Oxacillin (100%) while resistance was seen with Clindamycin (33.3%), Ciprofloxacin (33.3%) and Erythromycin (33.3%). The single incidence of Klebsiella showed sensitivity to Chloramphenicol, Norfloxacin, Amikacin, Gentamicin, Cefoxitin, Ceftazidime, Tazebactem and Imipenem and was resistant to Ampicillin, Cotrimoxazole, Cefotaxime and Clavulinic acid.

In our study, the antibiotic sensitivity of bacteria isolated in patients in Group B (Non-Diabetics) showed the following observations - in Staphylococcus epidermidis and Staphylococcus aureus, maximum sensitivity was seen with Gentamicin (100%), Cotrimoxazole (100%), Clindamycin (100%), Ciprofloxacin (100%), Vancomycin (100%), Linezolid (100%), Erythromycin (100%) and Oxacillin (100%). Similar observations were noted in the studies of Adam M et al,^[10] Suto C et al^[12] and Suresha K et al.^[11] Adam M et al ^[10] study investigating the aerobic bacterial conjunctival flora in diabetic patients and comparing it with non-diabetics reported positive culture ratio (33.3%) in patients having proliferative diabetic retinopathy (PDR) and 55.6% in patients on insulin therapy; however, both groups did not show a significant difference from the control group in the frequency of positive cultures. There was no correlation between diabetes duration and positive cultures. Suresha K et al^[11] comparative study comparing the normal conjunctival flora of non- diabetics with that of diabetics and identifying the organisms from the conjunctival flora and their antibiotic sensitivity pattern reported that among gram positive cocci, Erythromycin (38%) showed maximum resistance among the diabetic group. In the non diabetic group (30%), maximum resistance was seen with Ampicillin. Among the gram negative bacilli, Ampicillin showed maxim um resistance in both groups (56%). Klebsiella showed intrinsic resistance to Ampicillin.