Biofilm Formation Among Enterococci Species

 

Tandra Das. T ¹ , Dr. Gopinath. P²

¹2 ^nd  Year BDS, Saveetha Dental College and Hospitals, Chennai, India.

² Senior Lecturer, Department of Microbiology, Saveetha Dental College and Hospitals ,Chennai, India.

 

ABSTRACT:

Enterococci are an important member of human gastrointestinal flora, and are also an important cause of opportunistic nosocomial infections. The ability of enterococci to form biofilms may confer an ecological advantage in certain situations. For example, clinical strains of E. faecalis isolated from infective endocarditis patients were significantly associated with the greater biofilm formation than nonendocarditis clinical isolates. A sum of 20 clinical isolates of Enterococcus spp were subjected to antibiotic susceptibility pattern followed by detection of biofilm formation by TCP method. We found varied resistance pattern by disc diffusion method and none of the isolates was found to be vancomycin resistance. We have very little percentage of biofilm formers this indicates the less prevalence of biofilm in enterococci in our isolates. However in order to detect the same, more number of isolates to be included from different specimens. This TCP method is a much reliable and convenient method to detect the biofilm load in even a simple lab setup.

 

 

INTRODUCTION:

Enterococci are an important member of human gastrointestinal flora, and are also an important cause of opportunistic nosocomial infections ^[1]. These organisms are capable of causing different infections such as bacteremia, intra-abdominal infections, endocarditis, and urinary tract infections^[2]. Enterococcus faecalis and Enterococcus faecium are the most common enterococci species, and are responsible for up to 95% of human enterococcal infections.^[3] Biofilms are defined as microbially derived sessile communities characterized by the cells that are irreversibly attached to a substratum or to each other. They are embedded in a matrix of extracellular polymeric substances (EPS) they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription ^[4]. Within a biofilm, they communicate with each other their chemotactic factors or pheromones, this phenomenon is termed as quorum sensing ^[5].

 

Availability of key nutrients, chemotaxis towards surface, motility of bacteria, surface adhesins and presence of surfactants are some factors which associate with the biofilm formation .The ability of enterococci to form biofilms may confer an ecological advantage in certain situations. For example, clinical strains of E. faecalis isolated from infective endocarditis patients were significantly associated with the greater biofilm formation than nonendocarditis clinical isolates^[6]; this may be attributable in part to specific virulence factors in enterococci ^[7].

 

MATERIALS AND METHODS:

Clinical Isolates:

A total of 20 different non-repetitive clinic isolates of Enterococci were collected from different clinical specimens were included in this study. These isolates were identified by standard biochemical parameters as described by elsewhere. Isolates were preserved in semi-solid brain heart infusion medium and stored at 4ºC until further use.

 

Antimicrobial susceptibility test:

Antibiotic susceptibility test was determined for these strains to routinely used antibiotics such as ampicillin (10µ), vancomycin (30µ), teicoplanin (30µ), erythromycin (15µ), ciprofloxacin (5µ), amikacin (200µ), gentamycin (10µ), tetracycline (30µ) and linezolid (30µ) (Hi Media, Mumbai) by kirby-bauer disc diffusion method ^[8].

 

Biofilm formation:

Overnight grown cultures of Enterococcal isolates from agar plates were inoculated in 0.5 % of glucose and incubated at 37˚C overnight. Individual wells of sterile polystyrene 96 well flat bottom tissue culture plates were filled with 200µl of culture suspension from afore mentioned broth. Uninoculated broth served as negative control. The plates were incubated at 37˚C for overnight. After incubation, content of each well was gently discarded by tapping the plates downwards. The wells were washed three times with 200 µl of PBS (pH 7.2) in order to remove planktonic bacteria. Biofilms are formed by adherent sessile isolates in plates were fixed with 2 % sodium acetate and stained with 0.1% W/V crystal violet. Excess stain was removed by washing the wells with distilled water and plates were kept for drying at an inverted position. Optical density of stained adherent bacteria was determined with an ELISA reader (BioRad)at wavelength of 570nm. These OD values were taken as index of bacteria adhering the surface and formed biofilm. Experiments were carried in triplicate and their mean was taken for the analysis.^[9]

The below mentioned interpretation charge was applied to categorize the ability of Enterococci spp to form biofilm.

 

Table 1: Interpretation chart for biofilm detection by TCP method.

 

RESULTS:

Sample wise distribution of Clinical Isolates of Enterococci:

of the 20 clinical isolates of Enterococci, 12/20 (60%) were  obtained from urine, 4/20 (20%) were from blood, 2/20 (10%) and 2/20 (10%) were from stool samples and wound swabs respectively. Figure 1 depicts sample wise distribution of clinical isolates of Enterococci.

 

Fig 1: Pie chart showing the sample wise distribution of clinical isolates of Enterococcus spp.

Bacterial Isolates:

Out of 20 Enterococci isolates, 14/20 (70%) were found to be E.faecalis, whereas 6/20 (30%) were E. faecium. Figure: 2 , denotes the species wise distribution of Enterococci from clinical samples.

 

Fig 2: Pie chart showing species distribution of Enterococcus spp Antibiotic susceptibility testing

 

We found increased percentage of isolates were shown to be resistant to all the antibiotics used in this study. For ampicillin, amikacin, erythromycin, gentamicin, our isolates were found to resistant between 80-90%. Better sensitivity was observed in linezolid, teicoplanin and vancomycin antibiotics. The detailed results of antibiotic sensitivity patter of Enterococci was given in table 1.

 

Table 2: Results of antibiotic sensitivity patter of Enterococcus spp

 

 

 

Biofilm formation by Enterococcus spp:

All isolates were subjected for biofilm assay by tissue culture plate method. Of 20 Enterococcus spp isolates 6/20 (30%) of isolates were found to be weak biofilm producer. While, 3/20 (15%) were found to be strong biofilm formers.

 

Table3: Results of biofilm formation of Enterococcus spp

Biofilm category	Enterococcus spp isolates (n=20) (%)
Non biofilm	11(55)
Weak	6(30)
Strong	3(15)

 

Fig 3: Picture showing biofilm formers of Enterococcus spp by TCP method.

 

DISCUSSION:

Adherence to body surfaces is considered a major virulence factor responsible for the pathogenicity of clinical isolates of Enterococcus species. Biofilm formation plays a crucial role in nosocomial infections like catheter-associated UTIs and even blood stream infections dueto pacemakers. Investigations to understand the pathogenesis of these infections have focused upon the process of adherence of these microorganisms on these devices and have used various methods to quantify number of organisms adhering to surface ^[10]. In this study we have analyzed 20 clinical isolates of enterococcal species for biofilm production by tissue culture plate method. By this method, we observed 3/20 (15%) of strong and 6/20 (30%)of weak biofilm formers. Similar kind of study conducted by Triveda and coworkers from Chennai in 2016, have reported 21.8% of strong, 17.1% of moderate and 16.4% of weak producers of biofilm in enterococcal spp ^[10] .This is not in agreement with our report, as we found very less percentage of strong followed by weak and non-producers of  biofilm.

 

CONCLUSION:

As enterococci is a troublesome pathogen in terms of showing multitude of resistance to routinely used antibiotics and wide spectrum of infections caused by them as an important nosocomial pathogen, These infections are mainly attributed by biofilm formation and making them inevitable pathogen in hospital setups. However in order to detect the same, more number of isolates to be included from different specimens. This TCP method is a much reliable and convenient method to detect the biofilm load in even a simple lab setup.

 

ACKNOWLEDGEMENT:

We thank Dr. Kalyani, Professor and Head , Department of Microbiology, Saveetha Medical College Chennai for kindly providing clinical samples to carry out research work fruitfully.

 

REFERENCES:

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6.     Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime – producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 37;1982:318–26

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8.     Wayne M. Clinical and laboratory standard institute performance standards for antimicrobial disc susceptibility test; approved standard, CLSI document 10;2015:100-S18

9.      Mathur T, Singhal  S, Khan S, Upadhyay DJ, Fatma T, Rattan  A. Detection of biofilm formation among the clinical isolates of Staphylococcus : an evaluation of three different screening methods. J Med Microbiol  24;2006:25-29.

10.  Triveda L, Gomathi S. Detection of biofilm formation among the clinical isolates of Enterococci: An evaluation of three different screening methods. Int.J.Curr.Microbiol.App.Sci  5(3); 2016: 643-650.

 

 

 

Accepted on 16.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(11): 1877-1879