Detection Of Biofilm Producing Staphylococci: Need Of The Hour

*,(M.D)Professor of Microbiology,**search Assistant, Deptt of Microbiology,***(M.D)Professor of Microbiology,****(M.B.B.S)Tutor,Deptt. of Microbiology,Jawaharlal Nehru Medical College,Wardha(M.S)

Abstract

Biofilms are group of microorganisms encased in an exopolymeric coat. They have been associated with a variety of persistent infections that respond poorly to conventional antibiotics. In this study detection of biofilm productions by Staphylococcus spp. was done by using Congo red agar (CRA) methods, tube methods (TM) and tissue culture plate (TCP) methods. Out of 179 Staphylococcus spp., 111 were S.epidermidis and 68 were S.aureus. 44.69% of S.epidermidis and 32.96% S.aureus were slime producers. 97 isolates were detected as slime producer by TCP method, 76 by TN and 11 by CRA method. High resistances to conventional antibiotics were shown by biofilm producers.
This study summarized the prevalence, antibiotic sensitivity pattern and suitable and reproducible method for detection of biofilm producing Staphylococci.

Biofilm detection, Staphylococci, Congo red agar, Antibiotic resistance

Introduction
Biofilms are a group of microorganisms attached to a surface and covered by an exopolysaccharide matrix. Various changes occur during their transition from planktonic to a surface attached community. In response to certain environmental signals, new phenotypic characteristics develop in such bacteria. The first recorded observation concerning biofilm was probably given by Henrici in 1933, who observed that water bacteria are not free floating but grow upon submerged surfaces (1). Certain surface protein, extracellular proteins, capsular polysaccharides, adhesins (PS/A) and autolysin (encoded by atIE gene) are involved in regulation of biofilm production. The ica gene codes for intracellular adhesion (ICA) and may also code for TS/A and is required for biofilm production (1),(2),(3).

Biofilms are often site for quorum sensing influencing their formation. Availability of key nutrients, chemotaxis towards surface, motility of bacteria, surface adhesins and presence of surfactants are certain factors which influence biofilm formation.(3),(4). Using Bacillus subtilis from soil, Dr Stanley Wall has shown that a protein called Deg U helps the individual bacteria to decide whether to form a biofilm or not (5). Biofilm producing Staphylococci frequently colonize catheters and medical devices and may cause foreign body related infections. They easily get attached to polymer surfaces.(4),(5),(6) Crampton et al showed that like S epidermidis, S aureus also has ica locus encoding the function of intracellular adhesion and biofilm formation (7). According to a recent public announcement from National Institute Of Health, more than 60% of all infections are caused by biofilm (8). Biofilm organisms have an inherent resistance to antibiotics, disinfectants and germicides. The use of synthetic material for implantation is widely associated with “Implant associated infection� due to biofilm production. In the long run they may be very damaging because of immune complex disease (2),(9),(10).

Aims & Objectives
Keeping all these things in mind, the present study was undertaken to detect the prevalence of biofilm producer and nonproducer Staphylococci isolated from clinical materials in our laboratory by three different methods, viz. tissue culture plate (TCP) method, tube method (TM) and Congo red agar (CRA) method and to compare the above mentioned three different methods for biofilm production.

Material and Methods

A total of 179 clinical isolates of Staphylococci spp. were isolated from blood, infected devices, skin surface, urine, pus etc. from Indoor patient department (IPD) of a rural hospital with tertiary care in Central India over a period of 1 year. Isolates were identified by Gram staining, catalase and coagulase tests. Reference strains of Staphylococcus epidermidis ATCC 35984 (high slime producer),

ATCC35983 (moderate slime producer) and ATCC 12228 (nonslime producer) were also included in this study (11). Detection of biofilm production of 179 Staphylococci spp. was done by following three methods.

1. Tissue culture plate (TCP) method (8),(11)
2. Tube method (TM) (11),(12)
3. Congo red agar (CRA) method (11),(13)

1. Tissue Culture Plate Method
10 ml of Trypticase soy broth with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar. The broth was incubated at 370C for 24 hours. The culture was further diluted 1:100 with fresh medium. 96 wells flat bottom tissue culture plates were filled with 0.2 ml of diluted cultures individually. Only sterile broth was served as blank. Similarly control organisms were also diluted and incubated. All three controls and blanks were put in the tissue culture plates. The culture plates were incubated at 370C for 24 hours. After incubation, gentle tapping of the plates was done. The wells were washed with 0.2 ml of phosphate buffer saline (pH 7.2) four times to remove free floating bacteria. Biofilms which remained adherent to the walls and the bottoms of the wells were fixed with 2% sodium acetate and stained with 0.1% crystal violet. Excess stain was washed with deionized water and plates were dried properly. Optical densities (OD) of stained adherent biofilm were obtained with a micro ELISA autoreader at wave length 570 nm. Experiment was performed in triplicate and repeated thrice. Average of OD values of sterile medium were calculated and subtracted from all test values (8),(11).

2. Tube Method
10 ml Trypticase soy broth with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar individually. Broths were incubated at 370C for 24 hours. The cultures were decanted and tubes were washed with phosphate buffer saline (pH 7.3). The tubes were dried and stained with 0.1% crystal violet. Excess stain was washed with deionized water. Tubes were dried in inverted position.

In positive biofilm formation, a visible stained film was seen lining the wall and bottom of the tube. Experiments were done in triplicate for 3 times and read as absent, weak, moderate and strong.(11),(12)

3. Congo Red Method
The medium composed of Brain heart infusion broth (37 gm/l), sucrose (5 gm/l), agar number 1 (10 gm/l) and Congo red dye (0.8 gm/l). Congo red stain was prepared as concentrated aqueous solution and autoclaved at 1210C for 15 minutes. Then it was added to autoclaved Brain heart infusion agar with sucrose at 550C. Plates were inoculated with test organism and incubated at 370C for 24 to 48 hours aerobically. Black colonies with a dry crystalline consistency indicated biofilm production (11),(13).

Antibiotic sensitivity test was done on Muller-Hinton agar (MHA) using following antibiotic discs- penicillin (10units), ampicillin(10µg), ofloxacin(5µg), ciprofloxacin (5µg), cefotaxime(30µg), erythromycin(15µg), co-trimoxazole(25µg), amikacin(30µg), gentamicin(10µg), Netillmicin(30µg), linezolid(30µg), vancomycin(30µg), Antibiotics discs were procured from HiMedia Laboratories Pvt. Ltd, India. ATCC Staphylococcus aureus 25922 was used as control. Antibiotic sensitivity test was done as per Kirby-bauer disc diffusion method. (14)

Results

(Table/Fig 1) A total of 179 Staphylococci were isolated from various clinical materials. Out of 179 Staphylococcus spp. 111 S.epidermidis and 68 S.aureus. Among 111 S.epidermidis isolated from different clinical samples, 44.69% were slime producers and 17.32% non-slime producers, whereas among 68 S. aureus, 32.96% were slime producers and 5.03% were non-slime producers. Among S.epidermidis maximum biofilm producers were from catheter (51 out of 111). Among 51 catheters from which S.epidermidis were isolated, 40 were intravenous catheter and 11 were foley’s catheters. From two of such catheters adherent slimy growth were seen. Maximum numbers of biofilm producing S.aureus were from orthopedic implants (19 out of 68). We found high resistance pattern among biofilm producers in comparison with non-biofilm producers. Two strains of S. aureus were intermediate Vancomycin sensitive. Both the strains were biofilm producers (Table/Fig 2).

(Table/Fig 3) OD value of stained adherent biofilm was obtained with a microELISA autoreader at wave length 570nm. OD value less than 0.120 was considered as non-biofilm producers, 0.120 -0.240 as moderate biofilm producers and more than 0.240 as strong biofilm producers.

(Table/Fig 4) Among 179 clinical isolates of Staphylococci, 15.64% were high biofilm producers by TCP methods,12.30% by TM, and 4.47% by CRA method , whereas 38.55% are moderate biofilm producers by TCP method, 30.16% by TM and 1.68% by CRA method. In TM, 2 were found to be false positive and 23 false negative. In CRA method 3 were false positive and 89 false negative(Table/Fig 5),(Table/Fig 6),(Table/Fig 7).

Discussion

Bacterial biofilm has long been considered as a virulence factor contributing to infection associated with various medical devices and causing nosocomial infection (12),(13)

The exact process by which biofilm producing organisms cause disease is poorly understood. However, suggested mechanisms are:
i. Detachment of cells from medical device biofilm causing bloodstream or urinary tract infection.
ii. Endotoxin formation
iii. Resistance to host immune system
iv. Generation of resistance through plasmid exchange (2)

We isolated 179 Staphylococcal spp. from clinical samples, namely, blood, pus, urine, dialysis fluid, catheter, nasal swab etc. All isolates were isolated by standard procedure (15) and tested by three in vitro screening tests for biofilm production namely TCP, TM and CRA methods. Out of 179 Staphylococcal spp. 111 (62.01%) were S. epidermidis and 68 (37.99%) were S. aureus. We found that although the formation of biofilm on indwelling medical devices is generally associated with coagulase negative Staphylococci, S. aureus strains are also capable of production of biofilm (5.03%) which was observed by other workers also. (16),(17)

In this study antibiotic sensitivity pattern of various biofilm producers and non-producer Staphylococci spp. Isolated from clinical materials were obtained. The significant and clinically relevant observation was that the high resistance shown by biofilm producers to conventional antibiotics than non-biofilm producers. This observation was supported by other studies also (2),(10). All strains were sensitive to linezolid and vancomycin except two strains isolated from catheters which were intermediate vancomycin sensitive Staphylococcus aureus (VISA). Both were biofilm producers. Glycopeptides may not be optimal antimicrobial agents for the treatment of foreign body associated infection. This may be due to entrapment of vancomycin by the extracellular mucopolysaccharides because of their high molecular weight (10).

We adopted modified TCP method with extended incubation period for 24 hours instead of 18 hours. Trypticase soy broth with 1% glucose medium was used. This method was claimed superior to other methods by various researchers using Trypticase soy broth without glucose and Brain heart infusion broth with sucrose (11).

In TCP method biofilm formation was observed in 97 (54.19%) isolates and non-biofilm producers were 82 (45.81%). This study is similar to the observation made by Mathur et al (11). In tube test method, 76 (42.46%) isolates were found as biofilm producers whereas 103 (57.54%) were non-biofilm producers. In CRA, 11 (6.15%) strains produced biofilm and 168 (93.85%) were non-biofilm producers. Rate of positivity in CRA method in our study is higher than that of Mathur et al.

For data calculation, OD values obtained for individual strains of staphylococci spp.(11) mean OD values < 0.120 was considered non-biofilm producer, 0.120 – 0.240 was moderate and > 0.240 was considered as strong biofilm producers. Modified TCP method was considered as gold standard for this study as various researchers proved this method superior to standard TCP method using Trypticase soy broth without glucose. (8),(11)

Considering modified TCP as gold standard, data from TM and CRA methods were compared. Parameters like sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were calculated. True biofilm producers were positive by modified TCP, TA and CRA. False positive were biofilm producers by TM and CRA method but not by modified TCP method. False negatives were non-biofilm producers by TM and CRA methods but the same strains were biofilm producers by modified TCP method. True negatives were non-biofilm producers by all the methods. In our study 3 strains gave false positive result and 89 false negative by CRA method. By TM only 2 strains were false positive and 23 false negative considering modified TCP method as gold standard.

Comparative analytical study of TM and CRA methods, with respect to modified TCP method which was considered as gold standard in this study, was as follows: Sensitivity of CRA method was 8.25%; specificity 96.34%; PPV 72.72%; and NPV 47.02%. Sensitivity of TM method was 76.27%; specificity 97.56%; PPV 97.36%; and NPV 77.66%.

Our study shows TCP is the better screening test for biofilm production than CRA and TM. The test is easy to perform and assess both qualitatively and quantitatively. In our study, positivity rate of CRA method was higher than observed by other workers, e.g. Mathur et al. Who has reported 5.26% biofilm producers by CRA method.

There are some highly accurate methods like PCR analysis to detect ica genes as virulence marker of staphylococcal infection. Biofilm non-producers are negative for icaA and icaD and lack the entire ica ADBC operon.[13,17] But in a developing country like ours, a low cost method for detection of biofilm is needed which require inexpensive equipment and less technical expertise.

Conclusion

Biofilm can be composed of a single or multiple organisms on various biotic and abiotic surfaces. There is association between biofilm production with persistent infection and antibiotic failure.(19) Hence, in infection caused by biofilm producing staphylococci, the differentiation with respect to its biofilm phenotype might help to modify the antibiotic therapy and to prevent infection related to biomedical devices. A suitable and reproducible method is necessary for screening of biofilm producers in any healthcare setup and this TCP method can be recommended.