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 ~  Abstract
 ~ Introduction
 ~ Methodology
 ~ Results
 ~ Discussion
 ~ Conclusion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 397-400
 

Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa


1 Department of Microbiology, Assam University, Silcahr, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silcahr, Assam, India
3 Department of Microbiology, Institute of Medical Sciences, Benaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission21-Mar-2020
Date of Decision02-Aug-2020
Date of Acceptance09-Sep-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Amitabha Bhattacharjee
Assistant Professor, Department of Microbiology, Assam University, Silchar, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_118

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 ~ Abstract 


Purpose: Pseudomonas aeruginosa is an opportunistic pathogen with biofilm-forming ability, by the virtue of which they can evade the immune response and antimicrobial chemotherapy. Several methods have been designed for the detection of biofilms but require sophisticated instrumentation and expertise. The present study, therefore, used an improvised device, 'fluorescence foldscope' which is an origami-based fluorescence microscope as an easy and effective tool to detect biofilm formation. Methodology: Three representatives of P. aeruginosa of clinical origin were taken for the study along with two reference strains PA01 and ATCC27853. The strains were cultured in Luria Bertani (LB) broth with and without carbapenem (imipenem and meropenem) and cephalosporin (ceftazidime, cefotaxime and ceftriaxone) pressure, respectively. The cultures were diluted to 1:100 in LB; seeded with sterile glass slides at 90° angle and incubated for 5 consecutive days. The slides were observed with fluorescence foldscope. Results: Fluorescence emission was observed in two test isolates CD1 and CD2 at 48 and 72 h, respectively, whereas no fluorescence was observed in CD3. The fluorescence observed in the isolates was not affected by 2 μg/ml carbapenem pressure, while with 2 μg/ml ceftazidime stress, a change in fluorescence was observed in CD2 in comparison to the fluorescence observed under normal growth condition. Conclusion: Fluorescence foldscopy is an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa under different laboratory conditions. Biofilm-forming P. aeruginosa worsens the medical condition and is difficult to eradicate. The present study came up with an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa.


Keywords: Biofilm, carbapenem, cephalosporin, fluorescence, foldscope, Pseudomonas aeruginosa


How to cite this article:
Deshamukhya C, Das BJ, Chetri S, Paul D, Chanda DD, Banerjee T, Bhattacharjee A. Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa. Indian J Med Microbiol 2020;38:397-400

How to cite this URL:
Deshamukhya C, Das BJ, Chetri S, Paul D, Chanda DD, Banerjee T, Bhattacharjee A. Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 28];38:397-400. Available from: https://www.ijmm.org/text.asp?2020/38/3/397/299812





 ~ Introduction Top


Bacterial biofilms are aggregates of bacterial cells enclosed by an exopolymeric matrix which can remain attached to solid surfaces. The sessile bacteria here are more tolerant to antimicrobials than corresponding planktonic cells.[1] The management of biofilm-associated health issues is increasingly being problematic. Both Gram-positive and Gram-negative bacteria can actively form biofilm.[2]Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, has a remarkable ability to form biofilm. This ability makes it capable of evading immune response and antibiotic therapy thus increasing the complications manifold in medical conditions.[3],[4] There are severalin vitro techniques available for early detection of biofilm formation which includes tissue culture plate technique, Congo red agar (CRA) assay, tube adherence assay,[2] confocal laser scanning[5] and other microscopic techniques.[6] All these require sophisticated instrumentation and expertise which is often not within the scope of many resource-limited settings. Hence, the use of a cost-effective, simple and easy to use device for quick detection of biofilm formation is the need of the hour. In this context, an origami-based microscope called foldscope, designed by Cybulski et al.,[7] was used in this study to determine whether this could be an effective tool for the detection of biofilms in P. aeruginosa.


 ~ Methodology Top


Bacterial strains, media and culture conditions

Three clinical isolates of P. aeruginosa were obtained on the basis of carbapenem susceptibility from Silchar Medical College and Hospital, India, for the present study. All the three isolates were carbapenem non-susceptible. The test isolates were labelled CD1, CD2 and CD3. CD2 and CD3 were blaCTX-M-15 and blaNDM-1 harbouring isolates. Reference strains PA01 and ATCC 27853 which are biofilm producers were used as control in the present study. The isolates were subjected to fluorescence foldscopy for the detection of biofilm formation. Conventional biofilm detection methods such as CRA method and tube method were also performed.

Since the isolates were collected from a secondary source and no patient data were involved in this study, no ethical approval was required.

Fluorescence foldscope method

Foldscope, an origami-based paper microscope, was used as a tool in the present study for the detection of biofilm formation. As described by Cybulski et al., the foldscope was converted to a fluorescence foldscope with the help of blue light and filters (excitation and emission).[7] All the isolates (including the reference strains) were cultured in Luria Bertani (LB) broth (HiMedia, India) (pH maintained at 7) up to stationary phase which contained 3 × 109 CFU/mL. The stationary phase cultures were then diluted to 1:100 in LB. Each diluted culture was seeded with sterile, clean, grease-free glass slides at a 90° angle and incubated for 5 consecutive days in static condition. Biofilm formed by bacteria adherent to the slides was observed each day under fluorescence foldscope on the basis of the amount of fluorescence emitted by the cells adherent to the slides at 12, 24, 48, 72, 96 and 120 h of incubation, respectively. The test isolates CD1, CD2 and CD3 were further grown in LB broth supplemented with 2 μg/ml imipenem (Merck, France), meropenem (AstraZeneca, UK) and cephalosporin (ceftazidime, ceftriaxone, cefotaxime; Alkem, India) pressure, respectively. The stationary phase cultures were then diluted to 1:100 in LB and each diluted culture was seeded with sterile, clean, grease-free glass slides at a 90° angle and incubated for 5 consecutive days in static condition. The slides were then observed under fluorescence foldscope for the detection of changes if any in biofilm formation under antibiotic stress. The experiment was performed in triplicate and the scoring was done in accordance with the fluorescence emitted by the reference strains PA01 and ATCC 27853 which are biofilm producers. CRA assay and tube method were also performed for further confirmation.

Device details

P. aeruginosa being pigment-producing microorganisms have the capacity to fluoresce.[8] Hence, on excitation with light of specific wavelength, the pigments emit fluorescence.[9] This property was utilised in the present study for the detection of biofilm formation in P. aeruginosa. The foldscope was converted to a fluorescence foldscope with the help of excitation and emission filters and also a source of illumination. The assembly was done according to the instructions mentioned in the paper.[7]

Congo red agar method

CRA method is a simple qualitative method for the detection of biofilm production using CRA medium.[10] CRA was prepared by mixing sucrose (HiMedia, India), agar (HiMedia, India) and Congo red indicator (HiMedia, India) in brain heart infusion (BHI) broth (HiMedia, India). The Congo red indicator was prepared as a concentrated aqueous solution and autoclaved separately from the other constituents. The solution was then added to autoclaved BHI agar with sucrose at 55°C. The clinical isolates as well as the reference strains were then streaked on CRA plates and then incubated for 48 h at 37°C. Black, dry crystalline colonies indicated biofilm production.[10]

Tube method

Tube method is also a qualitative method for the detection of biofilm formation.[11] The isolates were seeded in 10 mL of LB broth with 1% glucose in test tubes and kept for incubation for 48 h at 37°C. After 48 h of incubation, the tubes were decanted and washed with phosphate-buffered saline (HiMedia, India) (pH 7.3) and dried. 0.1% aqueous solution of crystal violet stain (HiMedia, India) was prepared and the dried tubes were stained with the solution. De-ionised water was used to wash off the surplus stain and then the tubes were allowed to dry in an upturned position. A noticeable film coating the wall and the bottom of the tube indicated biofilm formation.[11]


 ~ Results Top


The fluorescence emitted by PA01 and ATCC 27853 was observed. A negative control (sterile LB broth) was also used and as expected, no fluorescence was observed in case of negative control during the period of incubation [Figure 1]. In case of the test isolates CD1 and CD2, the fluorescence emitted was observed at 48 and 72 h, respectively [Figure 1]. In case of CD3, whereas, no significant fluorescence was observed during the period of incubation [Figure 1]. Treatment of CD1 and CD3 with 2 μg/ml imipenem and meropenem had no significant effect on biofilm formation. There was no significant difference in fluorescence emission by these isolates even at antibiotic stress. In case of treatment of CD2 with 2 μg/ml ceftazidime, significant fluorescence could not be observed in comparison to the normal condition [Figure 1]. This indicates that in case of CD2, ceftazidime treatment may have affected biofilm formation, whereas no significant change in biofilm formation was observed in CD2 with 2 μg/ml ceftriaxone and cefotaxime stress. On CRA, ATCC 27853 and PA01 formed black, dry crystalline colonies which are indicative of biofilm production. Similar colonies were formed by CD1 and CD2 thus characterising them as biofilm producers, whereas CD3 produced translucent pink colonies. Tube assay result revealed CD1 and CD2 as biofilm producers and CD3 as a non-biofilm producer.
Figure 1: Observation of fluorescence of ATCC, PA01 and test isolates CD1, CD2 and CD3, respectively, under normal as well as 2μg/ml antibiotic pressure

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 ~ Discussion Top


Bacteria that produce biofilm are the reason behind causing many refractory infections which are extremely difficult to eliminate. Biofilm producing bacteria are more tolerant to antibiotics by exhibiting resistance by different methods such as decreased growth rate and expression of resistance genes.[12] Currently, a limited number of cost-effective methods are available to detect biofilm formation by bacteria. Hence, in the present study, fluorescence foldscope was tested to be used as a tool for detecting biofilm formation in the study isolates. The fluorescence foldscope device allowed the detection of biofilm formation in three clinical strains of P. aeruginosa as well as two standard strains (ATCC 27853 and PA01) on the basis of their fluorescence emission. A previous study by Ritenberg et al.[6] in 2016 reported the detection of biofilm formation in P. aeruginosa by fluorescence microscopy utilising amphiphilic fluorescent carbon dots. Another previous study by Cerca et al.[5] in 2012 analysed Staphylococcus epidermidis biofilms by confocal laser scanning microscopy. However, in our present study, the fluorescing ability of pigment-producing P. aeruginosa was taken into consideration and hence, no stain was used. Instead, a blue light was used to excite the pigment. Two of the three test strains (CD1 and CD2) and both the standard strains emitted significant fluorescence along the period of incubation, thus indicating biofilm formation. CRA assay and tube adherence assay for the test isolates also showed correlation with the results of fluorescence foldscopy in the present study. As several earlier studies have reported that treatment with the subinhibitory concentration of antibiotics affects biofilm formation in P. aeruginosa,[13],[14],[15] so in the present study, the subinhibitory concentration of carbapenem and cephalosporins were used for the test isolates to check their effects on biofilm formation. However, after analysing the results, it was revealed that subinhibitory concentration of imipenem and meropenem had no significant effect on biofilm formation by CD1 and CD2 when compared to the untreated strains. This result was in concordance to a previous study by Yamasaki et al.[16] in 2001 which also reported that imipenem did not affect the number of viable cells of Staphylococcus aureus as compared to the control. However, Chen et al.[17] in 2014 revealed in their study that imipenem treatment did not wipe out Klebsiella pneumonia biofilm, but higher concentrations of imipenem significantly reduced viable cells in K. pneumonia biofilm. Treatment of CD3 with sub-minimum inhibitory concentrations (MIC) ceftazidime had an effect on biofilm formation in the present study as there was no significant fluorescence observed as compared to the untreated strain. Similar results were reported by Otani et al.[18] in 2018 where they revealed a major reduction in the biofilm formation in sub-MIC ceftazidime treated P. aeruginosa cells as compared to the untreated cells, thus suggesting that sub-MIC of ceftazidime affects biofilm formation.

The present study has limitation and one obvious limitation in the study was that it involved only isolates of P. aeruginosa which has the ability to fluoresce and entirely this property was taken into consideration in the present study. Although, biofilm formation is not just confined to P. aeruginosa, and many clinically significant bacteria also possess this ability to cause biofilm-associated infections. Therefore, further comprehensive studies are required to include modifications such that the device can be utilised to detect biofilm formation in all clinically significant genera of bacteria as a whole.


 ~ Conclusion Top


Biofilm-forming bacteria presently worsen medical conditions and have become a challenge to detect and eradicate. Early detection of these biofilms can assist in early intervention and proper care. Hence, the use of a simple and reliable device in the form of a fluorescence foldscope can suffice to detect biofilms even in resource-limited settings. Therefore, from the present study, it can be concluded that fluorescence foldscope can be used as an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa.

Acknowledgement

The authors would like to thank Biotech Hub, Assam University, Silchar, India, for providing the infrastructure and the Department of Biotechnology, Government of India, for financial support vide no (BT/IN/INDO-US/Foldscope/39/2015).

Financial support and sponsorship

The study was financially supported by the Department of Biotechnology, Government of India programme (BT/IN/INDO-US/Foldscope/39/2015).

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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Olsen I. Biofilm-specific antibiotic tolerance and resistance. Clin Microbiol Infect2015;34:877-86.  Back to cited text no. 1
    
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Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011;15:305-11.  Back to cited text no. 2
    
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Anastasiadis P, Mojica KD, Allen JS, Matter ML. Detection and quantification of bacterial biofilms combining high-frequency acoustic microscopy and targeted lipid microparticles. J Nanobiotechnology 2014;12:24.  Back to cited text no. 3
    
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Rasamiravaka T, Labtani Q, Duez P, El Jaziri M. The formation of biofilms by Pseudomonas aeruginosa: A review of the natural and synthetic compounds interfering with control mechanisms. Biomed Res Int 2015;2015:1-17.  Back to cited text no. 4
    
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Cerca N, Gomes F, Pereira S, Teixeira P, Oliveira R. Confocal laser scanning microscopy analysis of Staphylococcus epidermidis biofilms exposed to farnesol, vancomycin and rifampicin. BMC Res Notes 2012;5:244.  Back to cited text no. 5
    
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Ritenberg M, Nandi S, Kolusheva S, Dandela R, Meijler MM, Jelinek R. Imaging Pseudomonas aeruginosa biofilm extracellular polymer scaffolds with amphiphilic carbon dots. ACS Chem Biol 2016;11:1265-70.  Back to cited text no. 6
    
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Cybulski JS, Clements J, Prakash M. Foldscope: Origami-based paper microscope. PLoS One 2014;9:e98781.  Back to cited text no. 7
    
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Wasserman AE. Absorption and fluorescence of water-soluble pigments produced by four species of Pseudomonas. Appl Microbiol 1965;13:175-80.  Back to cited text no. 8
    
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Xiao R, Kisaalita WS. Purification of pyoverdines of Pseudomonas fluorescens 2-79 by copper-chelate chromatography. Appl Environ Microbiol 1995;61:3769-74.  Back to cited text no. 9
    
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Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982;37:318-26.  Back to cited text no. 10
    
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Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 1989;42:872-4.  Back to cited text no. 11
    
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Brauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol 2016;14:320-30.  Back to cited text no. 12
    
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Balaji K, Thenmozhi R, Pandian SK. Effect of subinhibitory concentrations of fluoroquinolones on biofilm production by clinical isolates of Streptococcus pyogenes. Indian J Med Res 2013;137:963-71.  Back to cited text no. 13
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Jiang H, Li J. Effects of subinhibitory concentrations of antibiotics on the biofilm formation of Pseudomonas aeruginosa. Eur Respir J 2011;38:2539.  Back to cited text no. 14
    
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Narasanna R, Chavadi M, Oli A, Chandrakanth K. Effect of subinhibitory concentration of cefetoxime on biofilm formation. J Microbiol Infect Dis 2017;7:67-75.  Back to cited text no. 15
    
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Yamasaki O, Akiyama H, Toi Y, Arata J. A combination of roxithromycin and imipenem as an antimicrobial strategy against biofilms formed by Staphylococcus aureus. J Antimicrob Chemother 2001;48:573-7.  Back to cited text no. 16
    
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Chen P, Seth AK, Abercrombie JJ, Mustoe TA, Leung KP. Activity of imipenem against Klebsiella pneumoniae biofilmsin vitro and in vivo. Antimicrob Agents Chemother 2014;58:1208-13.  Back to cited text no. 17
    
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Otani S, Hiramatsu K, Hashinaga K, Komiya K, Umeki K, Kishi K, et al. Sub-minimum inhibitory concentrations of ceftazidime inhibit Pseudomonas aeruginosa biofilm formation. J Infect Chemother 2018;24:428-33.  Back to cited text no. 18
    


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