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Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 48--52

Biofilm formation capability of enterococcal strains causing urinary tract infection vis-a-vis colonisation and correlation with enterococcal surface protein gene

Shubha Garg1, Balvinder Mohan2, Neelam Taneja2,  
1 Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Neelam Taneja
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India

Abstract

Background: Data regarding differences in biofilm formation among urinary isolates of enterococci causing nosocomial infection versus asymptomatic colonisation is lacking. Conflicting data are available for the role of enterococcal surface protein (esp) gene in the development of enterococcal biofilms. Materials and Methods: A total of 50 (25 each of asymptomatic bacteriuria and urinary tract infection (UTI) isolates were collected from admitted patients who had nosocomial acquisition of enterococci in urine culture (≥105 cfu/ml). Biofilm assay was done by the quantitative adherence assay. Screening for esp gene was carried out by polymerase chain reaction, and confocal laser scanning microscopy was used to examine biofilms. Results: Out of 25 enterococcal isolates from asymptomatic patients, 9 (36%) isolates were found to be biofilm producers (6 weak [optical densities [OD]595 < 0.2] and three medium [OD595≥0.2 to<0.5]). Twelve (48%) out of 25 enterococcal isolates from UTI cases, produced biofilms (7 weak, 4 medium, and 1 strong [OD595>0.5]). The esp gene was present in 30 (12 biofilm+, 18 biofilm−) isolates. Seventeen esp positive isolates were from asymptomatic cases whereas 13 were from UTI. However, we found that 100% of medium and strong biofilm producers were esp positive (P < 0.001). On comparing Enterococcus faecalis (n = 10) and E. faecium (n = 40) isolates, 70% of E. faecalis isolates were biofilm producers as compared to only 35% of E. faecium isolates (P > 0.05). The esp positivity was observed more in E. faecium isolates (65%) as compared to 40% in E. faecalis. Vancomycin-sensitive enterococcal and vancomycin-resistant enterococcal isolates and catheter-related and unrelated isolates showed similarity in biofilm production and esp positivity. Conclusion: The esp gene is not compulsorily required to produce biofilm but when present may enhance the biofilm formation. We did not find any correlation between biofilm formation and the ability of the strain to cause symptomatic UTI be associated with catheters or vancomycin resistance.

How to cite this article:
Garg S, Mohan B, Taneja N. Biofilm formation capability of enterococcal strains causing urinary tract infection vis-a-vis colonisation and correlation with enterococcal surface protein gene.Indian J Med Microbiol 2017;35:48-52

How to cite this URL:
Garg S, Mohan B, Taneja N. Biofilm formation capability of enterococcal strains causing urinary tract infection vis-a-vis colonisation and correlation with enterococcal surface protein gene. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 29 ];35:48-52
Available from: http://www.ijmm.org/text.asp?2017/35/1/48/202330

Full Text



 Introduction



Enterococci are established nosocomial pathogens causing urinary tract infections (UTIs), bacteremia, surgical site infections, intra-abdominal, pelvic site wound infections, infections in indwelling devices, etc.[1] Biofilm formation in enterococci has been implicated as an important virulence factor.[2] The prevalence of biofilm production varies widely worldwide from 0% to 100%[3],[4] and is dependent on the enterococcal species, host and environmental factors.[2] Conflicting data are available for the role of enterococcal surface protein (esp) gene in the development of enterococcal biofilms.[5] The previous studies have implicated esp encoded surface proteins in adherence and colonisation of enterococcus to uroepithelial tissues.[6] Enterococci are ascendant organisms causing catheter-associated bacteriuria and catheter-associated UTI isolated from 6% to 7% of all urine samples submitted for culture at our centre. There are hardly any data available on the biofilm forming abilities of urinary isolates causing nosocomial infection versus asymptomatic colonisation and the comparative role of esp gene in formation of biofilms in these groups. We also explored the relationship between biofilm formation and the presence of esp gene with catheterisation and vancomycin resistance in nosocomially acquired enterococci.

Bacterial strains

This prospective analytic study was done in Enteric Laboratory, Department of Medical Microbiology in our tertiary care centre. A total of 50 (25 in each group) urinary isolates were collected from admitted patients of all age groups and both sexes who had nosocomial acquisition of enterococci in urine culture (≥105 cfu/ml). Twenty-five isolates were taken from the clinically asymptomatic patients with no pus cells in direct urine microscopy, and another 25 enterococcal isolates were taken from the symptomatic UTI (SUTI) cases. Centers for Disease Control and Prevention definition for nosocomial UTIs were followed for defining SUTI.[7] Identification of isolates was done by colony morphology, Gram staining, and standard biochemical tests and confirmed by matrix-assisted laser desorption ionisation-time of flight.

Biofilm assay on polystyrene plates

A modified protocol as described by Alejandro Toledo-Arana et al.[8] was followed. Briefly, enterococcal strains were grown overnight in brain heart infusion (BHI) with 0.25% glucose at 37°C. Cultures were diluted in fresh BHI–0.25% glucose, and the optical densities (OD) of the bacterial suspensions were measured using a U-2900 ultraviolet–visible spectroscopy spectrophotometer (Hitachi, Tokyo, Japan) and normalised to optical density of 0.2 at 595 nm (OD595 = 0.2). A 200 µl of this cell suspension was added to sterile 96-well polystyrene microtitre plate. After 24 h incubation at 37°C, wells were gently washed three times with 200 µl of phosphate-buffered saline (PBS); dried in an inverted position, and stained with 200 µl 1% crystal violet (CV) for 15 min followed by washing with PBS. Afterwards, ethanol-acetone (80: 20, v/v) was added, and the absorbance of extracted CV was determined at 595 nm (OD595) with Multiskan FC Microplate photometer (Thermoscientific Inc., USA). As a control, CV binding to the wells was measured for wells exposed only to the medium with no bacteria. The mean optical density of blank wells plus two standard deviations (SDs) was subtracted from individual readings for each experiment to give final corrected optical density results. Any optical density value >0 after correction was considered positive. Strains were classified as biofilm non-producers (OD595 ≤0), weak (OD595 595 ≥0.2 to <0.5), strong (OD595 ≥0.5) biofilm producers according to Seno et al.[9] Each assay was repeated three times on different days with three replicates for each strain per assay from which mean values were calculated.

Screening for enterococcal surface protein gene

All strains of enterococci included in the study were screened for esp gene by the polymerase chain reaction (PCR). PCR was performed using primers (Sigma, Bangalore, India) for amplifying 407 bp fragments of the esp variants (5'-GGTCAC AAA GCC CAA CTT GT-3' and 5'-ACG TCG AAA GTT CGA TTTCC-3') from Enterococcus faecalis or E. faecium.[10] Amplification conditions have been described earlier.[11] PCR was carried out with the following thermal cycling prof1itial activation step at 95°C for 15 min; 30 cycles of denaturation at 90°C for 30 s, annealing at 58°C for 1 min and extension at 72°C for 1 min; final extension at 72°C for 10 min. The PCR product was run on 1.5% agarose gel stained with ethidium bromide. Specific bands 407 bp for esp were visualised using a digital gel documentation system (Alpha Innotech, SanLeandro, CA, USA). The isolate was considered positive for PCR if a specific band was seen. A 100-bp ladder was used as a molecular marker.

Confocal laser scanning microscopy [12]

Confocal laser scanning microscopy (CLSM) was used to examine biofilms formed using two strains: one esp-positive strain which formed strong biofilm [Figure 1], and the other esp-negative strain which failed to form the biofilm [Figure 2]. These strains were grown at 37°C for 24 h in BHI with 0.25% glucose and on next day, growth was suspended in the fresh medium. The optical density of final suspension was normalised to optical density of 0.2 at 595 nm (OD595 = 0.2) and used to grow bacterial biofilms in Labtek ® two-chambered #1.0 borosilicate cover glass system (Hatfield, PA). After overnight incubation, for confocal imaging, supernatant media was gently aspirated from them. The wells were washed twice with sterile PBS, and biofilms were stained with 100 µl of 1% aqueous auramine solution for 10 min. Then, subsequently, washing was done twice with sterile PBS. Fully hydrated biofilms with 2 ml PBS were visualised through CSLM. Confocal images were captured using Nikon A1 R confocal, and an argon laser (488 nm) was used for sample excitation. The images were viewed using the Nis-Elements AR 4.1 software (Nikon Corporation, Tokyo, Japan).{Figure 1}{Figure 2}

 Results



Biofilm formation by enterococcal isolates from symptomatic and asymptomatic patients

OD595 values following CV staining ranged from 0.241 to 1.355 [Figure 3]. Out of 25 enterococcal isolates from asymptomatic patients, 9 (36%) isolates were found to be biofilm producers (6 weak [mean ± 2SD OD595 595 ≥0.2 to <0.5]). Twelve (48%) out of 25 enterococcal isolates from SUTI cases, produced biofilms [7 weak, 4 medium and 1 strong (mean ± 2SD OD595 >0.5) biofilm formers]. However, the difference was not statistically significant between asymptomatic and symptomatic isolates. Other characteristics of all 50 strains screened for biofilm production are listed in [Table 1].{Figure 3}{Table 1}

Correlation between presence of enterococcal surface protein and biofilm formation in enterococcal isolates

Isolates were screened for the presence of esp gene and categorised based on their capacity to form biofilm and presence of esp gene [Table 2]. The gene was present in 30 (60%) of 50 isolates and 57% of biofilm producers. Eighteen (62.07%) of 29 non-biofilm producers were also esp positive. Seventeen esp positive isolates were from asymptomatic cases, whereas 13 were from SUTI. However, we found that 100% of medium and strong biofilm producers were esp positive. The esp negative isolates were only weak biofilm producers (P < 0.01 by Chi-square test).{Table 2}

Subgroup analysis

When subgroup analysis was done between catheterised and non-catheterised patients, there was no statistically significant difference in above four categories of isolates described [Table 2]. Biofilm formation was observed in 9 out of 23 (39.13%) catheter-related isolates and 12 of 27 (44.44%) catheter unrelated isolates. However, esp was detected in 13 (56.52%) and 17 (62.96%) catheter-related and unrelated isolates, respectively.

On comparing E. faecalis (n = 10) and E. faecium (n = 40) isolates, 70% of E. faecalis isolates were biofilm producers as compared to only 35% of E. faecium isolates (P > 0.05 by fisher's exact test). The esp positivity was observed more in E. faecium isolates (65%) in comparison to E. faecalis strains (40%) though this difference was also not statistically significant.

Vancomycin-sensitive enterococcal (VSE) and vancomycin-resistant enterococcal (VRE) enterococcal isolates showed similarity biofilm production and esp positivity. Out of 33 VSE isolates 14 (42.42%) showed biofilm production, and of the 17 VRE isolates, 7 (41.18%) were biofilm formers. The esp gene was present in 20 (60.61%) VSE and 10 (58.82%) VRE strains. [Table 3] shows antimicrobial resistance profile of various antibiotics in biofilm producer and non- producer enterococcal isolates in symptomatic and asymptomatic cases.{Table 3}

 Discussion



Enterococci are common nosocomial uropathogens isolated in significant numbers (≥105/ml) from 6.8% of urine samples at our tertiary care referral centre (unpublished data). Biofilm formation in enterococci enhances their tolerance to antibiotics and also contributes in genetic exchange and dissemination of resistance properties.[1] In the present study, we tried to explore the relationship between biofilm formation and the presence of esp gene with SUTI, catheterisation and vancomycin resistance in nosocomially acquired enterococci. Although earlier studies by Upadhyaya et al.[13] and Di Rosa et al.[14] observed biofilm production to be more commonly associated with clinical infection isolates than commensals or environmental isolates, we did not find statistically significant difference in biofilm formation in isolates from symptomatic patients and asymptomatic patients. However, in symptomatic patients, biofilm production might enhance the establishment of the infectious process and emergence of symptoms. To identify genetic determinants of enterococcal biofilms, we tried to establish correlation between esp and biofilm formation. The unique morphology of esp with multiple repeat motifs helps enterococci to adhere to ligands on the host uroepithelium. It also has a role in colonisation and persistence of UTI.[6] Sequencing of esp region shows sequence similarity with bap (biofilm-associated protein) of Staphylococcus aureus which is critical for biofilm formation by this organism.[15] In the present study, 40% esp positive isolates produced biofilm and 45% esp negative isolates also produced biofilm. However, all medium and strong biofilm producing enterococcal strains were esp positive. Unlike the studies done by Seno et al.,[9] Toledo-Arana et al.[8] and Tendolkar et al.[12] and in concordance with studies by Dworniczek et al.[16] and Ramadhan and Hegedus,[11] our study suggests that esp is not a compulsory genetic component to produce biofilm by enterococcal strains but more than one components may function synergistically to express biofilm formation. Other virulence factors that have been implicated in biofilm formation include gelatinase-serine protease (gel E-Spr E) operon whose expression is under positive control of the quorum sensing system encoded by the fsr locus; agg surface protein which helps in facilitating the transfer of plasmids between donor and recipient cells; epa (enterococcal polysaccharide antigen); sal A and B (secretory antigen-like protein), dltA (D-alanine lipoteichoic acid) and sugar-binding transcriptional regulator, bopD etc.[2]

We also looked into whether the strains isolated from Foleys catheter formed more biofilm or were associated with the presence of esp gene. We did not find any correlation between biofilm formation and indwelling catheter similar to the study by Seno et al.[9] Enterococci are known to form biofilm on various types of medical devices.[2] It is possible that enterococci are capable of forming biofilms in response to bacterial factors mediating adherence to the host extracellular matrix. Genetic susceptibility of the host to biofilm formation and the relative significance of host factors versus bacterial virulence determinants in biofilm formation could be an interesting factor to the study.

In the current study, among the E. faecalis isolates, biofilm formation was more common (70% vs. 35% in E. faecium isolates); however, the presence of esp was more frequent in E. faecium isolates (65% vs. 40% in E. faecalis isolates) (P > 0.05). Overall, E. faecalis has been associated more frequently with biofilm formation as compared to E. faecium.[14],[17],[18] However, in contrast to our study, Shankar et al.[6] and Di Rosa et al.[14] showed esp gene was more frequent in E. faecalis irrespective of their source of isolation. Woodford et al.[10] found esp gene to be more common in E faecium strains isolated from urine.

In the present study, we did not find any difference in the biofilm formation ability and the presence of esp gene in VSE and VRE strains (41.18% in VRE and 42.42% in VSE). In contrast, Ramadhan and Hegedus [11] showed that acquisition of vancomycin resistance resulted in a lower ability to form biofilm. In the study done by Woodford et al.,[10]esp was present in 61% of VRE and 64% of VSE similar to our study. However, Willems et al.[19] detected the gene only in VRE strains.

 Conclusion



Our study suggests that esp is not a compulsory genetic component to produce biofilm but when present may enhance the biofilm formation. Ability to form biofilm is independent of resistance to vancomycin. We also did not find any significant association of catheterisation with biofilm production. The exact genetic mechanisms of biofilm production in enterococci are still unknown;[20] however, the process appears to be multi-factorial.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990;3:46-65.
2Mohamed JA, Huang DB. Biofilm formation by enterococci. J Med Microbiol 2007;56(Pt 12):1581-8.
3Prakash VP. Clinical Prevalence, Identification and Molecular Characterization of Enterococci. PhD Thesis, Pondicherry University, India; 2005.
4Sandoe JA, Witherden IR, Cove JH, Heritage J, Wilcox MH. Correlation between enterococcal biofilm formation in vitro and medical-device-related infection potential in vivo. J Med Microbiol 2003;52(Pt 7):547-50.
5Mohamed JA, Huang W, Nallapareddy SR, Teng F, Murray BE. Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis. Infect Immun 2004;72:3658-63.
6Shankar N, Lockatell CV, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE. Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect Immun 2001;69:4366-72.
7CDC/NHSN. CDC/NHSN Surveillance Definition of Healthcare-Associated Infection and Criteria for Specific Types of Infections in the Acute Care Setting; 2013. Available from: http://www.cdc.gov/nhsn/. [Last accessed on 2016 Feb 01].
8Toledo-Arana A, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, et al. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol 2001;67:4538-45.
9Seno Y, Kariyama R, Mitsuhata R, Monden K, Kumon H. Clinical implications of biofilm formation by Enterococcus faecalis in the urinary tract. Acta Med Okayama 2005;59:79-87.
10Woodford N, Soltani M, Hardy KJ. Frequency of Esp in Enterococcus faecium isolates. Lancet 2001;358:584.
11Ramadhan AA, Hegedus E. Biofilm formation and Esp gene carriage in enterococci. J Clin Pathol 2005;58:685-6.
12Tendolkar PM, Baghdayan AS, Gilmore MS, Shankar N. Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis. Infect Immun 2004;72:6032-9.
13Upadhyaya GP, Lingadevaru UB, Lingegowda RK. Comparative study among clinical and commensal isolates of Enterococcus faecalis for presence of Esp gene and biofilm production. J Infect Dev Ctries 2011;5:365-9.
14Di Rosa R, Creti R, Venditti M, D'Amelio R, Arciola CR, Montanaro L, et al. Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium. FEMS Microbiol Lett 2006;256:145-50.
15Cucarella C, Solano C, Valle J, Amorena B, Lasa I, Penadés JR. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 2001;183:2888-96.
16Dworniczek E, Wojciech L, Sobieszczanska B, Seniuk A. Virulence of Enterococcus isolates collected in Lower Silesia (Poland). Scand J Infect Dis 2005;37:630-6.
17Baldassarri L, Cecchini R, Bertuccini L, Ammendolia MG, Iosi F, Arciola CR, et al. Enterococcus spp. produces slime and survives in rat peritoneal macrophages. Med Microbiol Immunol 2001;190:113-20.
18Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, et al. Survey for virulence determinants among Enterococcus faecalis isolated from different sources. J Med Microbiol 2004;53(Pt 1):13-20.
19Willems RJ, Homan W, Top J, van Santen-Verheuvel M, Tribe D, Manzioros X, et al. Variant Esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals. Lancet 2001;357:853-5.
20Kristich CJ, Li YH, Cvitkovitch DG, Dunny GM. Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol 2004;186:154-63.