|Year : 2020 | Volume
| Issue : 1 | Page : 23-28
Study of biofilm formation in bacterial isolates from contact lens wearers
L Raksha1, Nagaraju Gangashettappa2, GB Shantala1, Bhavna R Nandan2, Deepa Sinha1
1 Department of Microbiology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
2 Department of Ophthalmology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
|Date of Submission||20-May-2019|
|Date of Acceptance||07-Aug-2019|
|Date of Web Publication||19-Dec-2019|
Dr. Nagaraju Gangashettappa
Minto Eye Hospital, Fort, Krishna Rajendra Road, Bengaluru - 560 002, Karnataka
Source of Support: None, Conflict of Interest: None
Purpose: To detect biofilm forming capacity of bacterial isolates obtained from the conjunctiva, contact lens and accessories of contact lens wearers using phenotypic and genotypic methods. Methods: Bacterial strains were collected from the conjunctiva, contact lens and lens storage cases of contact lens wearers. The phenotypic detection of biofilm production was done using the tube method and congo red agar method. The biofilm-forming related genes, icaA, of Coagulase negative Staphylococcus (CONS) and Staphylococcus aureus, and pslA, of P. aeruginosa, were detected using PCR. Results: A total of 265 bacterial isolates which included S. aureus, CONS, Pseudomonas, Nil-fermenter Gram-negative bacilli (NFGNB), Bacillus spp, Diphtheroids, Micrococci, Klebsiella pneumonia, Klebsiella oxytoca, E. coli, Proteus mirabilis, Proteus vulgaris, Citrobacter koseri, Citrobacter freundii, Enterobacter cloacae, Moraxella were obtained. Of the 265 isolates, 53.5% were moderately positive, 33.2% strongly positive and 13.2% negative for biofilm production by tube method and 36.6% were moderately positive, 40% strongly positive and 23.3% negative for biofilm production by congo red agar method. Of the four S. aureus isolates, two (50%) showed the presence of icaA gene. Of the 23 CONS isolates, three (13%) showed the presence of icaA gene. All the Pseudomonas isolates were negative for presence pslA (1119 bp) gene though most of them were phenotypically positive for biofilm formation. Conclusion: Most of the bacterial isolates obtained from contact lens wearers had the potential to produce biofilms. Tube method and Congo red agar method exhibited significant statistical correlation (P-value = 0.006) and picked up a good number of biofilm-forming isolates, hence may be used for detection of biofilm production. The absence of biofilm-forming gene did not rule out the possibility for phenotypic biofilm production by bacteria.
Keywords: Biofilm, congo red agar method, contact lens, lens storage case, polymerase chain reaction, tube method
|How to cite this article:|
Raksha L, Gangashettappa N, Shantala G B, Nandan BR, Sinha D. Study of biofilm formation in bacterial isolates from contact lens wearers. Indian J Ophthalmol 2020;68:23-8
|How to cite this URL:|
Raksha L, Gangashettappa N, Shantala G B, Nandan BR, Sinha D. Study of biofilm formation in bacterial isolates from contact lens wearers. Indian J Ophthalmol [serial online] 2020 [cited 2020 Sep 30];68:23-8. Available from: http://www.ijo.in/text.asp?2020/68/1/23/273277
Contact lens (CL) use has been increasing for cosmetic or therapeutic purposes and is preferred because of their optical and cosmetic benefits over spectacles. Microbial contamination and eye infections are proved to be present in cases where there is a lack of compliance and poor hygiene towards lens care., Microbial keratitis is a rare but feared complication of contact lens use as this may result in permanent loss of vision as a consequence of corneal scarring or perforation. A biofilm has been defined as a “functional consortia of micro-organisms, organized at interfaces, within exopolymer matrices”. Biofilm protects microbes against antibiotics, phagocytes and bacteriophages and hence help in their survival. Both Gram-positive and Gram-negative bacteria possess the ability to form biofilm such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Escherichia More Details coli, Enterococcus faecalis, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa. Bacterial biofilms are thought to play a major role in more than 80% of bacterial infection., Biofilms were observed on CLs, IOLs, glaucoma tubes, stents, punctual plugs, corneal sutures, scleral buckle, or other ocular prostheses., Extracellular polymeric substances (EPS) are secreted by bacteria that hold together heterogeneous mixtures of bacteria and therefore are an important component of biofilm production. The main components of EPS in S. aureus and S. epidermidis are Polysaccharide intercellular adhesion (PIA) and capsular polysaccharide/adhesin (PS/A). Several studies have shown that the intercellular adhesion (ica) locus, particularly the icaA gene, encodes the production of both PS/A and PIA., The pslA gene performs an essential function in biofilm formation of P. aeruginosa., To our knowledge, biofilm formation ability of clinical strains of bacteria obtained from the conjunctiva, CL and its accessories have not been analyzed using both genotypic and phenotypic methods. Therefore, the aim of the present study was to: (a) Detect biofilm forming capacity of bacterial isolates obtained from conjunctiva, CL and accessories of CL wearers, (b) Comparison of biofilm detection by two methods: Tube method and Congo red agar (CRA) method, and (c) Corroboration of biofilm formation with detection of gene for biofilm formation by PCR (Staphylococcus and Pseudomonas isolates).
| Methods|| |
The study was an observational study conducted in the Department of Microbiology and Ophthalmology of a tertiary care hospital attached to a medical college. Institution ethical clearance was obtained. Ethical committee approval was obtained. Informed written consent was obtained from those who volunteered to participate. A total of 40 CL wearers in the age group 18-35 years which consisted of undergraduate and postgraduate medical students were included in the study. All the 40 CL users were lens wearers for a period of more than 4-5 years.
The study subjects were silicone hydrogel soft contact lens users (SiHy). Frequency of change of CL wear is as follows:
- Monthly wear CLs: 37 participants
- Daily wear CLs: 1 participant
- Quarterly wear CLs: 2 participants.
The CLs storage case was used for duration of 4-6 months by the study population.
All the participants were examined by an ophthalmologist using a slitlamp. Individuals with ocular infections, co-existing ocular diseases, antibiotic use within 1 month and systemic diseases were excluded from the study.
Samples were collected from conjunctiva, CL and lens storage cases of both the eyes of CL wearers. Thus, a total of six samples each was collected from 40 CL wearers (N = 240). The samples were obtained by swabbing the lower conjunctival sacs, lens storage cases using sterile cotton swabs and CLs were collected (aseptically) from people just as they were to be discarded. All the samples were incubated for 24 hours at 37°C in Brain heart infusion broth and then sub-cultured onto blood agar, MacConkey agar and Sabouraud's dextrose agar (SDA). The blood agar and MacConkey agar was incubated at 37°C whereas SDA was incubated at 25°C. Organisms grown were identified using standard microbiological technique. Of the 240 samples obtained, 6 samples had sterile growth and 27 of them exhibited polymicrobial growth; hence the total number of bacterial isolates obtained was 265.
Detection of biofilm production
The bacterial isolates obtained were subjected to two tests to detect biofilm production:
The bacterial isolates (loopful of bacteria) obtained were inoculated into Trypticase soy broth supplemented with 1% glucose (TSBglu) and incubated for 24 hours at 37°C. Tubes were decanted and washed with PBS (pH 7.3) and dried. Dried tubes were stained with crystal violet (0.1%). Excess stain was removed, and tubes were washed with deionized water. Tubes were then dried in an inverted position and observed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. Based on the intensity of the color formed, they were characterized as moderately positive and strongly positive [Figure 1]. Lab-confirmed biofilm producer strain was used as a positive control.
|Figure 1: Bacterial biofilm detection by the tube method, showing strongly positive (a), moderately positive (b) and negative (c) results|
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The bacterial strains obtained were inoculated into CRA and was incubated for 24-48 hours at 37°C.
CRA was prepared as follows: BHI broth was supplemented with 5% sucrose and Congo red stain. Medium composed of BHI (37 g/L), Sucrose (50 g/L), Agar (10 g/L) and Congo red stain (0.8 g/L). Congo red was prepared as a concentrated aqueous solution and autoclaved at 121°C for 15 minutes separately from other medium constituents and was added when the agar was cooled to 55°C. Isolates were considered as strongly positive when there was the presence of black colonies with a dry crystalline consistency. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated a moderately positive biofilm producer. Colonies that remained pink were designated as non-biofilm producer [Figure 2]. Lab-confirmed biofilm producer strain was used as a positive control.
|Figure 2: Biofilm detection by Congo red agar method, showing strongly positive (a), moderately positive (b) and negative (c) results|
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Polymerase chain reaction (PCR)
Colony PCR method was used in order to detect genes related to biofilm formation in Staphylococcus species and P. aeruginosa. Bacterial cultures were lysed, DNA extracted, and gene-specific primers were used to amplify DNA fragments using PCR. The DNA template was obtained using the crude method: Loop full of culture were picked up using sterile pipet tip, suspended in 50 μl nuclease free water, boiled at 95°C for 10 minutes and centrifuged at 14,000 rpm speed for 1 minute. The supernatant (2 μl) obtained was used as the template. The Primer Sequences and Product Length for icaA of Staphylococcus spp. and pslA of P. aeruginosa (common organisms implicated in biofilm formation):
PCR sequences product length
IcaA F: 5′-TCTCTTGCAGGAGCAATCAA-3′ 188 bp
PslA F: 5′-CACTGGACGTCTACTCCGACGATAT-3′ 1119 bp
Reaction mix (20 μl)
2.0 μl of template suspension, 1.0 μl of 10 μM FP (Forward primer), 1.0 μl of 10 μM RP (Reverse primer), 6.0 μl of nuclease-free water, 10 μl of Master mix which consisted of: Taq DNA polymerase, dNTPs, Magnesium chloride and reaction buffers at optimal concentrations.
PCR reaction condition for gene 1 (icaA)
A thermal step program was used, including the following parameters: Incubation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 45 seconds (denaturation), 55°C for 30 seconds (annealing), 72°C for 20 seconds (elongation) and 72°C for 10 minutes after conclusion of the 30 cycles. Amplification products were analyzed using 2% agarose gel electrophoresis.
PCR reaction condition for gene 2 (pslA of P. aeruginosa)
A thermal step program was used, including the following parameters: Incubation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 45 seconds (denaturation), 55 C for 30 seconds (annealing), 72°C for 1 minute 20 seconds (elongation) and 72°C for 10 minutes after conclusion of the 30 cycles. Amplification products were analyzed using 1% agarose gel electrophoresis.
The data obtained were in the form of percentages and were analyzed using appropriate statistical tests and represented using tables and bar graphs.
| Results|| |
The bacterial isolates obtained from the conjunctiva, CL (soft CLs-monthly disposable) and lens storage cases of contact lens wearers were: S. aureus, Coagulase negative Staphylococcus (CONS), Pseudomonas, Nil-fermenter Gram-negative bacilli (NFGNB), Bacillus spp, Diphtheroids, Micrococci, Klebsiella pneumonia, Klebsiella oxytoca, E. coli, Proteus mirabilis, Proteus vulgaris, Citrobacter koseri, Citrobacter freundii, Enterobacter cloacae ont class="spl"> Moraxella More Details. The distribution of the bacterial isolates obtained is depicted in [Figure 3].
|Figure 3: Frequency of bacterial isolates obtained from the conjunctiva, Contact lens (soft Contact lenses –monthly disposable) and lens storage cases of contact lens wearers|
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Detection of biofilm formation
Total bacterial isolates (n = 265) were assessed by both tube method and CRA method to look for phenotypic production of biofilm. Of the 265 isolates, 53.5% were moderately positive, 33.2% strongly positive and 13.2% negative by the tube method. Of the 265 isolates, 36.6% were moderately positive, 40% strongly positive and 23.3% negative by the CRA method. The consistency between the CRA method and the tube method was 75.8% (P-value = 0.006). The results of tube method and CRA are depicted in [Table 1].
|Table 1: Analysis of results obtained in tube method and congo red agar method|
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Microbial isolates and biofilm formation
Among 57 Bacillus sp obtained, 44 (77.1%) of them were biofilm positive in tube method and in CRA method. Of the 35 Diphtheroids isolates obtained, 22 (62.8%) of them were biofilm positive in tube method and 21 (60%) in CRA. Among 75 Micrococci isolates obtained, 63 (84%) of them were biofilm positive in tube method and 58 (77.3%) in CRA method. 86.9% of CONS isolates were biofilm producers. In other bacterial organisms, most of them were biofilm producers. The results are depicted in [Table 2].
|Table 2: Detection of biofilm production of bacterial isolated obtained from contact lens wearers|
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PCR-based confirmation of bacterial biofilm formation from CLs wearers
Of the four S. aureus isolates, two (50%) showed the presence of icaA gene. Among 23 CONS isolates, three (13%) showed the presence of icaA gene [Figure 4]. The isolates which showed the presence of icaA gene were phenotypically positive for biofilm formation by both the methods (tube method and CRA method). The isolates which were phenotypically negative for biofilm formation did not show the presence of icaA gene. All the Pseudomonas isolates were negative for presence pslA (1119 bp) gene though most of them were phenotypically positive for biofilm formation.
|Figure 4: Lane 1 – 100 bp DNA ladder; Lane 2-7 – Staph icaA PCR amplicon|
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| Discussion|| |
Biofilms are the survival strategy of bacteria which help them survive harsh environmental conditions. They have the ability to bear a high concentration of antimicrobial agents. To best of our knowledge, there are few large studies analyzing the biofilm-forming capacity of clinically obtained bacterial isolates (conjunctiva, CLs and its accessories). Various gram-positive (200) and gram-negative bacterial (65) isolates were obtained from the conjunctiva, CLs and its accessories of CLs wearers and subjected to detection of biofilm production using tube method and CRA. The percentage of biofilm producers in our study was 86.7% by tube method and 76.7% by CRA method which is higher compared to studies conducted by Mathur et al., Afreenish Hassan et al. and Juárez-Verdayes et al. where the percentage of biofilm formers is 41.4%, 63.6% and 66% respectively. Of the 265 isolates assessed, 53.5% were moderately positive, 33.2% strongly positive and 13.2% negative by tube method. As compared with results of a study conducted by Mathur et al., where tube Method picked up 18 (11.8%) isolates as strong biofilm producers and 45 (29.6%) were moderate biofilm producers, the present study showed a higher proportion of biofilm producers. In a study conducted by Afreenish Hassan et al. – Among 110 isolates, strong biofilm producers were 21, moderate were 33 and weak or non-biofilm producers were 56 which again is lesser compared to results obtained in the present study.
CRA enables for the direct analysis of the colonies. Of the 265 isolates, 36.6% were moderately positive, 40% strongly positive and 23.3% negative which is higher compared to other studies conducted by Mathur et al. Hou et al. and Afreenish Hassan et al. where the percentage of biofilm formers using CRA was 3.4%, 34.38% and 3.6% respectively. Tube method picked up higher positive isolates when compared with CRA method. Based on our experience with the tube method and CRA, the interpretation of results was easier with CRA as tube method was subjective in nature. Consistency between the tube method and CRA method in our study was 75.8%. Though there are differences between the results of the tube method and CRA method, they show significant statistical correlation P value = 0.006). Therefore, we suggest that the phenotypic methods (tube method and CRA method) can be used as a convenient way to detect biofilm-related infections. The unique attempt of the study was to detect biofilm production by commensals. Among the commensals, 77.1% of Bacillus isolates, 62.8% of Diphtheroids, isolates and 84% of micrococci isolates were biofilm producers which show that they are also indeed potential biofilm producers. In all, 86.9% of CONS isolates were positive for biofilm formation which is similar to studies conducted by Catalanotti et al. where 74.1% of S. epidermidis strains were biofilm positive. All the Pseudomonas isolates (13/13 by tube method and 12/13 by CRA method) obtained were phenotypically positive for biofilm formation. Oncel et al. reported that 60% (6/10) of P. aeruginosa isolates from chronic rhinosinusitis produced bacterial biofilms. Coban et al. also reported 33.3% (20/60) of P. aeruginosa samples tested for the biofilm-formation ability of isolates in patients with cystic fibrosis were biofilm-positive.
Major synthetic pathway of biofilm formation in Staphylococcus species are encoded by icaA operon which is comprised four genes namely ica A, D, B and C., Hence, ica A was considered as the representative gene for the icaA operon in our study. S. aureus and CONS where assessed for the presence of icaA gene (188 bp) using PCR. Of 23 CONS isolates three (13%) and two out of four (50%) S. aureus isolates showed the presence of icaA gene. Our results differ from study conducted by Hou et al. where 40.63% of Staphylococcus epidermidis and 11.11% S. aureus strains carried icaA gene. These results indicate that the formation of biofilms requires a complicated network of factors such as icaC, icaD  and the icaA gene is likely to be one of many factors that regulate biofilm formation. The isolates which were genotypically positive for icaA gene were also phenotypically positive for biofilm formation by both the methods (tube method and CRA method) which is similar to study conducted by Takashi et al. The isolates which were phenotypically negative for biofilm formation did not show the presence of icaA gene.
All the Pseudomonas isolates were negative for presence pslA (1119 bp) gene but most of them were phenotypically positive for biofilm formation. The finding in our study is different from the study conducted by Hou et al. where 31.03% of Pseudomonas strains carried pslA gene. Previous reports by Overhage et al. and Colvin et al. suggested an essential role for the psl gene cluster in the initial step of P. aeruginosa biofilm formation, so we focused on the functional assessment of the pslA gene. P. aeruginosa produces at least three polysaccharides (alginate, Pel and Psl) which determine the stability of the biofilm structure., Among the 60 two-components systems found in the genome of P. aeruginosa, the GacS/GacA system acts as a super-regulator of the QS system and is involved in the production of multiple virulence factors as well as in biofilm formation. Hence, further studies are required to genotypically detect biofilm production by Pseudomonas spp as the process of biofilm formation is determined by the interaction of many gene clusters, some of which have not yet been identified.
| Conclusion|| |
In conclusion, most of the bacterial isolates obtained from CLs were potential enough to produce biofilms. Tube method and CRA exhibited significant statistical correlation and picked up a good number of biofilm-forming isolates, hence may be used for detection of biofilm production. The absence of biofilm-forming gene does not rule out the possibility of phenotypic biofilm production by bacteria.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]