Validity Of Congo Red Agar And Modified Congo Red Agar To Detect Biofilm Of Enterococcus Faecalis

Purpose: Enterococcus faecalis causes nosocomial infections such as bacteremia, urinary tract infections, intraabdominal infections, and endocarditis. These infection is associated with biofilm and intrinsically resistant to many antibiotics. This study aims to determine the validity of the CRA and MCRA for detecting biofilms of Enterococcus faecalis Method: This is a laboratory observational study with 30 sample of Enterococcus faecalis. We performed biofilm examination for Enterococcus faecalis by using Congo red Agar, Modified Congo red Agar and Microtitter Plate Assay as gold standard. Result: Both MCRA and CRA were compared MPA as a gold standard was obtained p value is 0.309 (p> 0.05), with a Kappa agreement coefficient is 0.067, which indicates there is no significant agreement to detect biofilm of Enterococcus faecalis. MCRA and CRA have almost no compatibility with MPA for biofilm forming of Enterococcus faecalis. Conclusion: Both MCRA and CRA has a very high sensitivity (100%), but the specificity is very low 6.67% for detecting the biofilms of Enterococcus faecalis. MCRA and CRA can not determine negativity well and it have a high false positive rate, so to increase specificity of biofilm forming, we must combine these method with the others.


INTRODUCTION
The infections that caused by biofilm-producing bacteria has become an urgent in clinical setting. Biofilms producing bacteria is associated with a number of persistent infections that respond very poorly to antibiotic therapy, change ability of the bacteria to survive against immune responses and help spreading of antibiotic resistant nosocomial infection (Gaca et al., 2012, Ponnusamy andNagappan, 2013). One of the agents that commonly causes of severe nosocomial infections, such as bacteremia, urinary tract infections, intra-abdominal infections, and endocarditis, is Enterococcus faecalis (Richard et al, 2000).
The ability of Enterococcus faecalis forming biofilms shows their virulence. This ability enables colonization of inert and biological surfaces while protect itself against antibiotic substances and mediate adhesion to host cells (Hashem et al, 2017). The structure of biofilms provides an optimal micro-environment for growth and facilitates the transmission of cellular genetic elements between bacteria (Sienko et al, 2015). Biofilms play an important role in bacterial virulence, because they protect bacteria, especially uro-pathogen from the antibactericidal activity of antibiotic in various ways. This bacterial defense mechanism is due to the changing the of bacterial cells characteristics in environmental adaptations, such as slowing growth of microorganisms or undergoing metabolic dormant, which leads to increase antibiotics tolerance and resistance (Fujiwara et al., 1998;Donlan, 2003;Trautner et al., 2004).
Strains of bacteria that produce biofilms, including Enterococcus faecalis, show higher resistance to antibiotics compared to those that do not produce biofilms. Levy's (2002) shows that 100% of enterococci strains produce biofilms that are resistant to 2 (two) or more antibiotics and even to the phenotypic MDR. Komiyama et al (2017) said that Enterococcus faecalis often forms biofilms in stents and other devices, which require long-term antibiotic administration when removal of the devices are not possible. Chen and Wen (2011) and Atray and Atray (2015) show that biofilm production is often associated with organisms that are in the urinary tract for longtime and dramatically increases antibiotic resistance.
Ch 'ng et al. (2018) revealed that Enterococci cause 25% of all catheter-related urinary tract infections. These bacteria are also often isolated in wounds and are increasingly found in infective endocarditis. All of these infections are associated with biofilms. Enterococcal biofilms are intrinsically resistant to antibiotics so it pose a serious obstacle in the treatment of infections. Christopher (2003), revealed that Enterococcus faecalis can form biofilms in vitro, so these organisms are often isolated from biofilms on the surface of various medical devices. However, the molecular mechanisms that regulate biofilm formation in clinical isolates are largely unknown. The formation of biofilms as pathogenesis of disease can be detected using several methods, including: Microtiter Plate Assay (MPA), Congo Red Agar (CRA) and Tube Method (TM) (Hasan et al., 2011).
The Microtiter Plate Assay (MPA) method was considered as the gold standard for biofilm examinations (Christensen et al., 1985).
According to Kaiser et al. (2012), the CRA is considered to have several advantages over other methods, cheap, fast, and easy to do even for small laboratories, and does not require technical expertise. However, this method also has disadvantages because it needs subjective evaluation. Triveda et al (2016) considered that sensitivity and specificity of the CRA to detect biofilm Enterococcus were 25% and 46.67% respectively. Melo et al (2013), considered that the sensitivity and specificity of the MCRA method using additions of glucose to detect the biofilms production of Staphylococcus aureus were 90.63% and 90.6% (compared with MPA) as the gold Normanita R. et. al./ SM Vol.16 No.1 June 2020 Page 55-65 57 standard). The sensitivity and specificity of the MCRA (compared with PCR) were 89% and 100%.
There has been no research comparing the sensitivity and specificity of CRA to compare with MCRA to detect biofilms producing Enterococcus faecalis. This study aims to determine the sensitivity and specificity of CRA and MCRA for detecting biofilms producing Enterococcus faecalis from clinical isolates at Dr. Soetomo Hospital, Surabaya.

MATERIALS AND METHODS
The observational analytic study using the clinical isolate of Enterococcus faecalis was   with an average age of 43.8 years. The most specimens of Enterococcus faecalis from urine were 18 isolates (60%), followed blood were 11 isolates (36.7%), pus was 1 isolate (3.3%) (   There is no significant difference between MCRA and MPA (p=0.309 (p> 0.05) ). Kappa agreement coefficient of 0.067 (away from 1) can be meant there is suitability between MPA and MCRA to detect biofilms formation in Enterococcus faecalis isolate is very low. Validity of MCRA compared with MPA as gold standard is sensitivity of 100% and specificity of 6.67%. The positive predictive value is 51.72% and negative prediction value is 100%. Congo red stain will interact with polysaccharides which are secondary metabolic products of bacteria and growth media so that they form complexes with stain that cause the colonies to appear dark in color (Jain and Agarwal, 2009). Bacteria fermented sugars or polysaccharides needed to produce certain metabolites, that combine with Congo red so as give a black colony color that indicates the formation of biofilms (Mack et al, 1992).

Enterococcus faecalis is a predominant pathogen in urinary tract infections after
Phenotypic evaluation of CRA in order to show the formation of black color associated with the presence of sucrose in CRA media. The concentration of sucrose is contained in the medium affects the production of EPS. When the concentration of sucrose is low, the diffusion of black pigment from the colony is also reduced. Sucrose in CRA acts as a carbon source. As a comparison in this study using MCRA. According to Kaiser et al. (2012), MCRA to be a modification of the way of inoculation of Enterococcus faecalis so that the interpretation of colony and pigmentation is more easily evaluated. The difference in MCRA compared to CRA, is changing the streaking method to spot inoculation. MCRA method is considered to be more economical for the media used to identify biofilms. This method is fast, easy, sensitive, and reproducible and colonies growing on this medium can still be used for further analysis. The initial study proposed MCRA is an alternative MPA for screening biofilm forming (Knobloch, 2002) because it is easy to do, time-consuming, sensitive and specific, while MCRA and CRA have high sensitivity but very low specificity. MCRA and CRA can not determine negativity well and it will have a high false positive rate. So both of tool are good for catching actual case of Enterococcus faecalis but they also come with fairly high false positive rate. To increase specificity of detection of biofilm forming, we must combine MCRA or CRA method with the others. Actually we may use these method to detect biofilm forming, because MCRA and CRA have some advantages, i.e easy and simple implementation, cheap and simple production of media, and simple assessment of result.
MCRA and CRA is not fully able to show how the bacteria formed biofilm, because the mechanism of biofilm formation can go through several pathway. MCRA also has a difference compared to CRA, in which it is given glucose addition of 10g/l. Based on research Rossi et al  MCRA is a better method than CRA for the same thing. (Panda, et al. 2016) This study resulted several findings, are: (1) Urine is the most common specimen of Enterococcus faecalis isolate compared to other specimens; (2) Sensitivity and specificity of the CRA and MCRA is same to MPA so that CRA and MCRA can be used as an initial screening alternative to detect biofilm producing Enterococcus faecalis but cannot be used as a standardized microbiological examination as a determinant of biofilm formation; (3)  negativity well and it will have a high false positive rate. To increase specificity of detection of biofilm forming, we must combine MCRA or CRA method with the others.

LIMITATION
We have some limitation in this study : sample size and limitation of financial resources and timing of study that might influence the interpretation of result