Available online at International Journal of Research in Pure and Applied Microbiology

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1 Available online at International Journal of Research in Pure and Applied Microbiology Universal Research Publications. All rights reserved ISSN Original Article Analysis of biofilm formation and antibiotic resistance of microbial isolates from intraocular lens following conventional extracapsular cataract surgery Ravish Katiyar, Amreesh Vishwakarma, Shilpa Deshpande Kaistha Department of Microbiology, Institute of Biosciences & Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur , UP, India Corresponding Received 08 May 2012; accepted 11 June 2012 Abstract This study reports antibiogram and biofilm formation abilities of isolates from intraocular lens following conventional extra capsular cataract extraction (ECCE) around Kanpur district. A total of 95 isolates from 36 intraocular lenses obtained following ECCE were studied. Antibiotic resistance was determined using Kirby Bauer disk diffusion assay as per CLSI guidelines. Biofilm formation assay was carried out by recommended techniques % of the isolates were biofilm formers. A total of 88.2% of pan drug resistant were found to be high biofilm formers while 94.4% of multiple drug resistant organisms were high biofilm formers. Antibiotic resistance to gentamycin, tetracycline, co-trimoxazole was positively correlated in biofilm formers. Amongst the pan drug resistant high biofilm forming isolates, least resistance was found to imipenem (4.4%) followed by levofloxacin (33.3%), vancomycin (46.6%) and moxifloxacin (57.7%). This study shows a significant association of biofilm formation with multiple and pan drug resistance of isolates from intraocular lenses Universal Research Publications. All rights reserved Key words: pan drug resistance, biofilm formation, intraocular lenses, ECCE 1. Introduction Cataract surgery has provided relief from blindness to a number of individuals. However, approximately 70% of cataract surgery may lead to certain complications such as post operative endophthalmitis if proper sanitation facilities are not maintained [1, 2]. Infectious endophthalmitis is a rare but vision threatening condition which may be caused due to microbial contamination following traumatic laceration of the eye ball as a consequence of post operative surgery [2]. Although percentage of infectious endophthalmitis is low in western world, in a developing country like India, rates have known to be higher particularly when cataract surgery are performed in rural settings using conventional techniques such as extra capsular cataract extraction (ECCE) [3]. In most cases, infectious endophthalmitis occurring due to post cataract surgery is due to microbial contamination either from the patient s microbiota or from contaminated surgical equipments and surrounding operating environment [3, 4]. The role of the normal micro flora of the conjunctiva has been studied previously to understand its contribution to endophthalmitis [2, 5, 6]. In most such studies conjunctival swabs are taken prior to the surgery [5, 6]. In this report, we have attempted to study the micro flora associated with the excised intra-ocular cataract lens. The rationale of the study being that the micro flora represented on the cataract lens during the excision process would represent the microbiological load to which prosthetic lens being replaced is being exposed. Often eye surgeons utilize pre operative methods to reduce occurrence of endophthalmitis such as administration of topical antibiotic drops [1, 2]. The antibiotic resistance of the isolates from such excised cataract lens is studied in order to determine the effect of any prophylactic antibiotic treatment undertaken to mitigate the normal microbiota [7, 8]. Understanding the antibiotic resistogram of the frequently isolated ocular flora may help in an effective antibiotic strategy for bacterial endophthalmitis prophylaxis. 20

2 The ability to form biofilms is considered one of the important virulence factors in the pathogenecity of ocular microbial pathogens [9, 10, 11]. Biofilms confer on its residence a higher resistance to antimicrobial agents and the immune response as compared to the free living bacteria. Hence the ability of the isolates from extracted intraocular lenses to form biofilms would also determine the virulence of the microbiota. In this study, a snapshot of the microbial load of the intraocular lens/ lens capsule excised during cataract surgery using the conventional extra capsular cataract extraction (ECCE) around Kanpur and Unnao district has been studied with aim of determining the antimicrobial sensitivities and biofilm formation abilities. The antibiograms and biofilm forming abilities of the isolates are determined and its relevance discussed. 2.0 Material and Methods 2.1. Sampling: Intraocular lenses following cataract surgery were collected from local hospitals in sterile saline. Samples were vortexed vigorously as well sterile loop was used to scrap the lens surface prior to inoculating Brain Heart Infusion Agar, Trypticase Soya Agar, Mannitol Salt Agar, Nutrient Agar and Sabouraud s Agar (Hi Media). Plates were incubated at 37 C for 24 hrs to 48 hrs. Microbiological and biochemical characterization of the isolates was performed as per Bergey s determinative bacteriology [12] Antibiotic susceptibilities: Antibiotic susceptibility tests for each isolates were performed by disk diffusion method as per CLSI guidelines [13]. Mueller Hinton Agar plates were spread with log phase cultures with OD corresponding to 0.5 McFarland standards. Antibiotics disks (Hi Media) were placed on the spread culture, and plates incubated at 37 C. The inhibition zones were measured after h. Isolates were characterized as resistant, moderate and susceptible following the standard antibiotic disc sensitivity as per CLSI guideliness. The antibiotics tested include ampicillin (10µg), co-trimoxazole (25µg), colistin sulphate (10µg), ciprofloxacin (5µg), gentamicin (10µg), imipenem (10µg), levofloxacin (5µg), moxifloxacin (5µg), streptomycin (10µg), tetracycline (30µg) and vancomycin (30µg). Standard American Type Culture Collection (ATCC) bacteria Staphylococcus aureus ATCC and Pseudomonas aeruginosa ATCC were used for quality control. 2.3 Biofilm formation Assay: Static biofilm formation assay was used as per O Toole et al with some modifications [14]. Isolates were grown in 96 well micro titer plates containing 200 µl of TSB for 24h at 37 C. Planktonic cultures were removed and the static surface with biofilms was washed with sterile saline. Adherent bacteria were stained with 1% w/v crystal violet for 20 min. Excess stain was removed by washing the plate with saline. Stained adherent bacteria were detached using 200 µl of dimethyl sulfoxide and solubilized biofilms measured using microplate reader (Thermo Fisher Scientific, USA) at A630. Isolates were characterized as high biofilm former: O.D> 0.5; moderate O.D and poor Figure 1. Correlation between biofilm formation and resistance to number of different classes of antibiotic in the isolates. Antibiotics tested include ampicillin (10 µg), cotrimoxazole (25 µg), colistin sulphate (10 µg), ciprofloxacin (5 µg), gentamicin (10 µg), imipenem (10 µg), levofloxacin (5µg), moxifloxacin (5µg), streptomycin (10 µg), tetracycline (30 µg) and vancomycin (30 µg) as per CLSI guidelines [13]. biofilm former OD< Statistical Analysis Statistical analysis was performed wherein categorical variables were compared using Chi Square test. Pearson s Correlation was performed between biofilm formation and antibiotic resistance. Differences were considered statistically significant if p value < Results A total of 95 bacterial isolates were characterized from 36 excised intra ocular lenses following conventional extra capsular cataract extraction (ECCE) surgery from local hospitals and clinics. Gram positive rods (30.7%) were the predominant isolates followed by Gram negative rods (28%), Gram negative cocci (26.3%) and Gram positive cocci (15%). Most cataract lens showed multiple bacterial species. Microbiological and biochemical characterization showed that there was no predominant isolate with wide coverage ranging from, Pseudomonas spp. (9%) with Ps. aeruginosa (5%), Staphylococcus aureus (15%), Staphylococcus epidermis (5%), Micrococcus luteus (3%), Serratia marcescens (2%), Neisseria spp. (7%), Moraxella spp. (7%), Bacillus spp. (20%), members of the Enterobacteriaceae family (27%) including the following: E. coli (3%), Proteus mirabilis (2%), Enterobacter agglomerans (3%), Klebsiella spp. (4%) and others (15%). All the isolates were further screened for biofilm formation using the static biofilm formation assay. A total of 85.2% (81/95) of the isolates were biofilm formers. Isolates showing resistance to five or more antibiotic classes were characterized as pan drug resistant (PDR) and those showing 21

3 Table 1: Correlation between biofilm formation and antibiotic susceptibility in isolates Biofilm formation Antibiotic resistance Biofilm Former Non Formers of Isolates Total (OD: ) (OD<0.15) Pan Drug Resistant (PDR 5 classes) Multiple Drug Resistant (MDR 3 classes) Low DR (<2 classes) Sensitive TOTAL Chi square test: ; p= Table 2: Antibiotic Susceptibility of isolates Antibiotics Ampicillin ß lactam (90.1) 5 (9.8) 51 (53.6) Co-trimoxazole Analogue (87.2) 6 (12.7) 47 (49.4) Colistin Sulphate Antimicrobial peptide (90.6) 4(9.3) 43 (45.2) Ciprofloxacin Quinolone (83.3) 6(16.6) 36 (37.8) Gentamicin Aminoglycoside (78.3) 8(21.6) 37 (38.9) Streptomycin Aminoglycoside (75.6) 10 (24.3) 41(43.1) Tetracycline Macrolide (82.8) 6(17.1) 35(36.8) * Value within the parentheses in the entire column is percentage of isolates resistant to antibiotic over total resistance to the antibiotic. Value within the parentheses in the entire column is the percentage of antibiotic resistance to total number of isolates. Table 3: Antibiotic susceptibility of biofilm forming pan drug resistant isolates to third and fourth generation antibiotics Antibiotic Family Concen- -tration (µg) Concentration ( CLSI nomenclature) µg No of Isolates* (N=45) 22 Percentage (%) Moxifloxacin Levofloxacin Vancomycin Imipenem * Pan Drug Resistant biofilm forming isolates Table 4: Resistance to fluoroquinolones amongst isolates Levofloxacin Moxifloxacin Isolates* Sensitive Resistant Sensitive Resistant Ciprofloxacin Sensitive (50) 45(94%) 5 (10%) 43(86%) 7 (14%) Ciprofloxacin Resistant (30) 12(40%) 18 (60%) 8 (26.6%) 22(73.4%) TOTAL (80) *Isolates tested for ciprofloxacin resistance or susceptibility were analyzed for resistance to levofloxacin and moxifloxacin using disk diffusion assay as per CLSI norms. resistance to three or more drugs were characterized as multiple drug resistant organisms (MDR). Antibiotic resistance was determined to a range of antibiotic classes which included ampicillin (10 µg), co-trimoxazole (25 µg), colistin sulphate (10 µg), ciprofloxacin (5 µg), gentamicin (10iµg), streptomycin (10iµg), tetracycline (30iµg) vancomycin (30iµg). The correlation between number of classes of antibiotic resistance and biofilm formation for all Biofilm formers (OD 0.3) N=81* Number of Resistant Non (OD 0.15) N=14* Total (95) the 95 isolates was found to be positive (pearson s correlation r=0.3) (Figure 1). 88.2% (45/51) of pan drug resistant were found to be high biofilm formers while 94.4% (17/18) of multiple drug resistant organisms were high biofilm formers (Table 1). Chi square analysis suggests high correlation for biofilm formation and pan drug resistance (P = ). An analysis was performed to determine the total percentage resistance to individual antibiotics which were tested (Table

4 2). The highest percentage of resistance is seen to ampicillin (53.6%) followed by co-trimoxazole (49.4%). In order to correlate the role of biofilm formation in resistance to the antibiotics, isolates were further classified on the basis of their ability to form biofilms. Hence, for the antibiotics colistin sulphate (90.6%), ampicillin (90.1%), co-trimoxazole (87.2%), ciprofloxacin (83.3%) and tetracycline (82.5%),, a high correlation is found for antibiotic resistance and biofilm formation (p 0.05). Fluoroquinolones, carbepenems and vancomycin are the choice of third and fourth generation drugs for treatment of severe ocular infections [15, 16, 17]. Isolates found to be pan drug resistant were tested for their susceptibility to moxifloxacin, levofloxacin, vancomycin and imipenem. 57.7% of the isolates were moxifloxacin resistant, 46.6 % vancomycin resistant, 33.3 % levofloxacin resistant and only 4.4 % of the isolates were resistant to imipenem (Table 3). Fluoroquinolone resistance analysis was furthered by determining susceptibility of ciprofloxacin resistant (second generation fluoroquinolone) and sensitive isolates to moxifloxacin (fourth generation fluoroquinolone) and levofloxacin (third generation fluoroquinolone) for 80 random isolates. Table 4 indicates that 10% of ciprofloxacin sensitive isolates were resistant to Levofloxacin and 14% were resistant to moxifloxacin. Also, 60% of the isolates were ciprofloxacin and levofloxacin resistant while 73.4% were ciprofloxacin and moxifloxacin resistant. 4. Discussion In the present study, we report a high correlation between biofilm formation and antibiotic resistance amongst ocular isolates following ECCE procedures. Hence, biofilm formation is one of the mechanisms of drug resistance; while many other factors may be responsible for drug resistant amongst the poor biofilm forming isolates [15, 16]. Mechanisms of pan antibiotic resistance and biofilm formation are found to be multifactorial. Delayed antimicrobial penetration due to polysaccharide matrix, multiple efflux pumps, differential growth and metabolic rates within the biofilm, quorum sensing systems and increased incidence of persister cells within biofilms are some mechanism responsible for increased biofilm resistance [15, 16]. Biofilm formation in itself promotes colonization, increased resistance to antibiotics and avenues for horizontal gene transfer. The current findings are agreement with earlier report of endophthalmitis associated microbiota shows that wide microbiological spectrum of isolates is highly region specific and depends on the experiences of individuals [2, 4, 5, 7]. The reported incidence for fluoroquinolone resistance amongst ocular isolates has been steadily increasing since its introduction [17, 18]. A study of antibiotic susceptibilities in the asian landscape shows that moxifloxacin shows activity against pan Gram positive and Gram negative ocular bacterial infections [8]. However, in a recent study of in vitro antibiotic susceptibilities of ocular infections with Gram positive cocci, ciprofloxacin, ofloxacin and gatifloxacin were found to be more effective than Moxifloxacin [19]. However, the third and fourth generation fluoroquinolones when introduced showed higher activity against Gram positive pathogens as well single step topoisomerase mutants [18]. Our study indicates an increase in the number of isolates which show resistance to fourth generation quinolones. Also, the presence of ciprofloxacin sensitive but levofloxacin and moxifloxacin resistant isolates indicate a rise in multi step topoisomerase mutants. Biofilm growth has been shown to produce extensive genetic diversity in the presence as well absence of external pressure [20]. 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