Difference in biofilm development capability of vancomycin and ciprofloxacin resistant Staphylococcus aureus clinical isolates

Background: Staphylococcus aureus has long been recognized as a major pathogen of urinary tract and hospital acquired infections. Over the last decade antibiotic resistance and biofilm formation potential by pathogens is a major challenge. In this study we screened S. aureus clinical isolates by antimicrobial susceptibilty and biofilm assays to determine biofilm capability of vancomycin and ciprofloxacin resistant isolates. Methods: Six clinical isolates of S. aureus were characterized by biochemical tests and further antibiotic susceptibility of vancomycin and ciprofloxacin were tested against S. aureus clinical isolates by disc diffusion method. Biofilm formation capability of these isolates were performed by microtiter plate, coverslip congored agar and tube assays. Results and disscusion: In this study we found that two isolates were resistant to ciprofloxacin and three isolates were resistant to vancomycin, on the basis of CLSI guidelines. Ciprofloxacin resistant isolates showed moderate biofilm formation while vancomycin resistant showed strong biofilm formation. Conclusion: We have concluded that ciprofloxacin and vancomycin resistant clinical isolates were showed differences in biofilm formation. Staphylococcal isolates having biofilm propensity exhibit more resistance to antibiotics, hence are difficult to treat.


Introduction
Staphylococcus aureus is a gram positive cocci normally localize in the skin and mucous membranes in the nasal area of healthy humans and about 30% of the normal healthy population are transiently colonized by the organism [1]. These bacteria are associated with the several syndromes such as skin infections, osteomyelitis, bacteraemia, septicaemia, diarrhea, pneumonia and urinary tract infections [2][3][4][5][6][7][8]. Biofilm formation is the potential and major virulence trait [9] and has been described as a possible attribute to the resistance and major contributor to several infections. Biofilm impairs the action of both host immune system and antimicrobial activities. The formation of biofilms is an excellent example of a phenotypic change in S. aureus to adapt to its surroundings in the presence of environmental challenges and is a recognized method of some microorganism's capability to cause certain infections, and a way by which they increases its levels of antimicrobial resistance [10]. It is now well documented that biofilms are difficult to eradicate and are often resistant to systemic antibiotic therapy and removal of abiotic surfaces becomes necessary [11]. The differentiation of Staphylococci with respect to its biofilm phenotype might help in diagnosis of infections related to hospital devices and prevention of device related infections [12].
The pathogenesis of Staphylococcal infection begins with primary attachment and colonization of the host tissues. Once a biofilm has been formed, it is a major concern for clinicians in the treatment of infectious disease because of multi drug resistance [13]. Therefore, a better understanding of bacterial biofilms is urgently needed, and this may ultimately result in the development of novel therapeutics for the control of infections [14].
In this study we have screened S. aureus clinical isolates by antimicrobial susceptibilty and biofilm assays to determine biofilm capability of vancomycin and ciprofloxacin resistant isolates.

Bacterial isolates
In this study, urine samples were collected the patients of Hamidia hospital, Bhopal, India. Out of 32 isolates, 11 isolates were identified as Gram positive Staphylococci. Further identification of these isolates was done by catalase, coagulase, sugar fermentation tests and cultivation on blood agar, mannitol salt agar, CLED agar. Out of 11 Staphylococci isolates, 6 were Staphylococcus aureus and 5 were coagulase negative Staphylococci. Staphylococcus aureus MTCC 3160 strain was used as a control in the whole study.

Antibiotic susceptibility testing
The antibiotic-resistance profile of S. aureus strains against ciprofloxacin (5 µg) and vancomycin (30 µg) was determined by the disc diffusion assay [15]. Mueller-Hinton agar plates were overlaid with the inoculum (turbidity equivalent to that of a 0.

Congo red agar assay
Production of slime was studied by cultivation of all S. aureus strains on congo red agar media [16]. Briefly, the medium was composed of Brain Heart Infusion Agar (37 gms/L), sucrose (50 gms/L), and congo red stain (0.8 gms/L). Plates were inoculated and incubated aerobically for 24 to 48 hours at 37°C. Positive result was indicated by black colonies with a dry crystalline consistency. Weak slime producers usually remained pink, though occasional darkening at the center of colonies was observed.

Tube assay
A qualitative assessment of biofilm formation was determined by tube assay [17]. Briefly, overnight culture of S. aureus isolates were inoculated in tryptic soya broth with 1% sucrose and incubated for 24h at 37°C. The tubes were removed and washed with phosphate buffer saline and dried. Tubes were stained with 0.1% crystal violet for 10 min. Excess stain was removed and tubes were washed with distilled water. Tubes were than dried and observed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. Tubes were examined and the amount of biofilm formation was scored as 0-absent, 1-weak, 2-moderate or 3-strong. Experiment was performed in triplicate and repeated three times.

Microtiter plate assay
Microtiter plate method is considered as a gold standard method for the quantification of biofilm formation. Briefly, overnight culture of S. aureus strains was grown in brain heart infusion broth (BHI) (Himedia Laboratories, India) for 18 h at 37°C. One ml of overnight culture was transferred to 10 ml of sterile BHI broth with the addition of 1% Sucrose for biofilm development. The suspensions were adjusted with the same BHI medium

Coverslip assay
In coverslip assay [18], biofilms of S.aureus clinical isolates were grown as follows, individual sterile culture dishes were filled with 2.5 ml of BHI broth with 1% sucrose and sterile glass coverslip was added to each petriplate. Each sample was inoculated with defined volume of overnight culture. The dishes were incubated aerobically at 37ºC for 72 h. Planktonic cells were rinsed off with phosphate buffer saline and sessile cells or biofilm were stained with 0.1% crystal violet for 5 min. Finally stained biofilms were observed microscopically.

Results and discussion
Staphylococcus aureus is a gram positive coccal bacterium which is highly adaptive to human environment and are resistant to several antibiotics including vancomycin, which is the last effective antibiotic against S. aureus. There are many resistant species of S. aureus out of which the most infectious strains are vancomycin resistant and ciprofloxacin resistant S. aureus. In this study we found that two isolates were resistant to ciprofloxacin and three isolates were resistant to vancomycin (Tables 1 and 2), on the basis of CLSI guidelines. Over the last few decades the continuous incresing of bacterial resistance to antimicrobials has been a cause of worldwide concern and a great challenge. This situation is provoked by over the counter accessibility, indiscriminate and inappropriate use of antimicrobial agents. Ciprofloxacin and vancomycin are the most effective known drugs against urinary tract infections. In addition biofilm formation is known doi: 10.7243/2052-5958-1-8 major virulence determinant of S. aureus. In our study biofilm forming capability of ciprofloxacin and vancomycin resistant isolates showed differences in biofilm assays (Tables 1 and 2). Ciprofloxacin resistant isolates showed moderate biofilm   formation while vancomycin resistant showed strong biofilm formation (Figures 1 and 2). Staphylococci are bacterial pathogens that usually produce biofilms during different infe-ctious processes, which are generally difficult to treat or control. It has been estimated that about 65 per cent of the nosocomial infections are associated with biofilm formation. These infections are 10 to 1000 times more difficult to eliminate with an otherwise successful treatment. The mechanism for enhanced antimicrobial resistance is believed to involve alteration or change in gene expression leading to a phenotypic difference between the planktonic and sessile condition. The sessile forms are more resistant as they produce exopolysaccharide, have different growth characteristics and take up nutrients and drugs differently from their planktonic counterparts [19].

Conclusion
We have concluded that ciprofloxacin and vancomycin resistant clinical isolates were showed differences in biofilm formation. Vancomycin resistant S. aureus isolates showed strong biofilm formation while ciprofloxacin resistant isolates showed moderate biofilm formation.

Competing interests
The authors declare that they have no competing interests.