Battling the Methicillin-Resistant Staphylococcus aureus Biofilm Challenge with Vancoplus

Of 55 MRSA, 47 isolates were biofilm producers and 8 isolates were non-biofilm producers. Of 47 biofilm producers, 24 (51.0%), 14 (29.8%) and 9 (19.1%) produced strong (OD570 ≥ 0.5), medium (OD570 ≥ 0.2 to <0.5), weak (OD570 0 to <0.2) biofilm, respectively. Of the 47 isolates, eight determinants (genes) (eno, hla, hlb, clfA, fnaA, icaA, agrII and sar) were found predominantly among 70 to 80% isolates whereas cna was observed only in 21.3%, finbB in 10.6% and ebps in 32% isolates. Amongst the strong biofilm producers (51%), the lowest MIC values obtained with Vancoplus (2-4 μg/ml) >linezolid (128 to 256 μg/ml) >daptomycin, clindamycin and teicoplanin (256 to 512 μg/ml). Biofilms eradication rate was also observed in the same order with Vancoplus (87%) >linezolid (51.8%) >clindamycin (31.9%) >daptomycin (27.5%) >teicoplanin (26.5%). Our results showed that the percentage of finbA, hla, eno, clfA and fib genes expressions down-regulation after Vancoplus treatment was 64.0 ± 5.9, 63.8 ± 5.8, 73.0 ± 7.4, 72.8 ± 7.8 and 71.9 ± 7.8%, respectively as compared to the control among strong biofilm producing MRSA whereas teicoplanin produced only 30.3 ± 2.7 to 34.5 ± 3.8% down regulation in fnbA, hla, eno, clfA and fib genes expression. The other comparator drugs, vancomycin, linezolid and daptomycin, demonstrated variable effects on these genes varying from 4.9 ± 3.9 to 30.3 ± 2.7%.


Introduction
Staphylococcus aureus is among the 5 most common pathogens that constitutes normal skin and nasal flora in at least 25 to 30% of healthy humans and is associated with large proportions of hospitalacquired and community acquired infections ranging from superficial wound infections to life-threatening deep infections such as septicemia, endocarditis and toxic shock syndrome [1][2][3]. Phenomenon of S. aureus infection is becoming more worrisome with the emergence of antibiotic resistant strain in particular methicillin-resistant S. aureus (MRSA) [4]. S. aureus has been reported to form biofilms on various surfaces, including medical devices and host tissues [5]. The S. aureus with biofilm forming ability is capable of causing a number of diseases, including infective endocarditis, osteomyelitis, foreign body-related infections, catheters associated Urinary Tract Infections (UTIs), and ventilator associated pneumonia and blood stream infections closely related to intravascular devices [5][6][7][8].
Biofilms, surface associated sessile bacterial communities, are formed when planktonic cells colonize to a surface, aggregate and grow into multicellular colonies, and embed themselves in an exopolysaccharide [9]. In addition to a large number of cell surface associated proteins, secreted proteins, Polysaccharide Intercellular Adhesin (PIA) and intracellular adhesin A, D, B and C (icaA, icaD, icaB and icaC) which are synthesized by products of the intercellular adhesin A, D, B and C (icaA, icaD, icaB and icaC) operon are also required for biofilm formation in staphylococci [5][6][7][8].
The cell surface associated proteins allow S. aureus to interact with host extracellular ligands, such as elastin binding protein (EbpS), laminin binding protein (Eno), collagen binding protein (Cna), fibronectin binding proteins A and B (FinbA, FinbB), fibrinogen binding protein (Fib), clumping factors A and B (ClfA, ClfB) [7,[10][11][12]. The secreted proteins include tissue degrading enzymes and toxins [13]. Recently, α-toxin (Hla) has been shown to play integral role in biofilm formation [14]. Production of these factors in S. aureus which are responsible for adherence, colonization and biofilm formation is controlled by accessory gene regulators including Agr, Sar and others [15]. Based on the agr variations, S. aureus strains can be divided into 4 groups menstrual Toxic Shock Syndrome (TSS) strains belong to agr groups I [16], all the strains causing leucocidin induced necrotizing pneumonia belong to agr group III [17], most intermediate level infections are caused by the organisms that have the capacity of producing biofilms. A study that was carried out in China, also reported 66% prevalence of biofilm-forming MRSA. The increasing incidence of biofilm producing MRSA in clinical infections has received increasing interest due to characterization of genes involved in biofilm formation [21,22]. The antibiotics such as methicillin, oxacillin and nafcillin, macrolides, tetracycline and aminoglycosides are being used to treat the infections caused by biofilm producing MRSA but they are getting resistant [23]. Now, glycopeptide antibiotic vancomycin is the remaining effective therapy. However, 76% treatment failure rate with vancomycin has been reported [24]. The emerging resistance towards vancomycin has limited the use of vancomycin and imposed a major concern to the global health community and reinforced the critical need for new methods of control and treatment of biofilm infections. In view of the growing consequences of biofilm producing MRSA and their resistance to commonly used drug, Venus Remedies Limited, India has developed a combination of ceftriaxone and vancomycin named as Vancoplus (US patent no;7960337, Japan patent no: 4918502). The current study was designed to investigate the biofilm formation ability among MRSA isolates and to assess the activities of commonly used drugs against biofilm producing MRSA. Evaluation of prevalence of genes involved in MRSA biofilm production and their gene expression by semi-quantitative PCR would be more advantageous in determining their relation.

Antibacterial agents
The following antibiotics were used in this study: a novel antibiotic adjuvant entity ceftriaxone sodium and vancomycin hydrocloride with VRP1020 (Vancoplus), teicoplanin, linezolid, daptomycin and clindamycin. All the drugs were reconstituted in water for injection except Vancoplus which was reconstituted in solvent provided with the pack as per manufacturer's instructions. Working solutions were prepared using Mueller Hinton broth (MHB, Himedia, Mumbai, India), and serial two fold dilutions were made using Cation-Adjusted Mueller-Hinton broth (CAMH, Himedia, Bombay, India) in wells of 96-well plate.

Bacterial isolates collection and their identification
A total of 70 clinical isolates of S. aureus were collected from various hospitals of North India including Vijayanagara Institute of Medical Sciences, Bareilly, Utter Pradesh, India; Sanjay Gandhi Postgraduate Institute, Lucknow, Utter Pradesh, India and Government Medical College and Hospital, Chandigarh, India. Initial inoculum of these isolates was made on blood agar plates and incubated at 37°C for 24 h. Identification of the S. aureus was confirmed by Gram staining, catalase, oxidase, coagulase and growth characteristics on mannitol-salt agar. MRSA isolates were detected by the presence of the mecA gene as described earlier [25].

Antimicrobial susceptibility testing
Minimum Inhibitory Concentration (MIC) of each drug was determined by the broth dilution method according to the Clinical and Laboratory Standards Institute [26]. The MICs of the drugs represent the lowest concentration at which bacteria fail to grow.

In vitro biofilm formation assay
Biofilm forming ability of MRSA was investigated according to the method described earlier [27]. Briefly, 250 µl of each MRSA isolate diluted 1:1000 in medium were inoculated in 96 wells polystyrene microtitre plates and incubated for 94 h at 37ºC without shaking. After development of biofilm, non-adherent bacteria cells were removed and washed twice with 200 μl of sterile phosphate buffer saline (PBS; pH 7.0), dried in an inverted position at room temperature under laminar air flow and stained with 300 μl of crystal violet (2%) for 45 min. It is then washed 3 times with distilled water to remove excess stain. For destaining, 300 μl of ethanol: acetic acid (95:5 v/v) was added to each well. Hundred microlitre of this solution was transferred to another 96 wells plate and absorbance was measured at 570 nm using Elisa reader (Merck, USA). As a control, uninoculated medium was used. The mean OD570 value from control wells subtracted from the mean OD570 value of tested wells. The degree of biofilm production was classified in three categories: strong (OD570 ≥ 0.5), medium (OD570 ≥ 0.2 to <0.5) and weak (OD570 0 to <0.2) [28]. Each test was done in triplicate.

Minimum biofilm eradication concentrations (MBECs) determination
MBECs of drugs were determined as described earlier [29]. Briefly, after development of biofilms in wells, the wells were washed with phosphate buffer saline (PBS; pH 7.0) to remove non-adherent bacterial cells and added 200 μl of each antibiotic of 2-fold dilutions (from 2 to 4096 μg/ml) and incubated for 24 h at 37°C. The MBECs values represent the lowest dilution at which bacteria embedded in biofilm failed to regrow. All samples were run in duplicate and one lane served as a positive control and another lane with media served as negative control.

Effect of drugs on biofilm
To study the effect of drugs, after development of biofilms, the biofilms were treated with half concentrations of MBECs of drugs for 12 hrs. After treatment, assessment of drug efficacy was performed by two independent methods: counting of bacterial cells before and after treatment with different antibiotics and crystal violet staining.
For counting of bacteria embedded in biofilm, free floating bacterial cells from the wells after 12 hrs of drug treatment were completely removed and 200 μl of PBS were added to each well to remove remaining biofilm from the wells by ultrasonic disruption for 5 min. The viable counts were determined on MHA plates. The same procedure was used for control plates to calculate the numbers of cells/well. For staining of biofilm, after drug treatment and incubation, the antibiotics were removed from wells carefully without disturbing the cells in biofims and stained as mentioned above.

DNA isolation
DNA from selected clinical isolates was isolated by using the method explained below. Five ml of each MRSA overnight grown culture in soybean casein digest medium (SCDM; Hi-Media, Mumbai, India) was centrifuged at 5000 rpm for 5 min at 25°C, washed twice in phosphate buffer saline (pH 7.2) and transferred to a 2 ml micro-centrifuge tube. Then 0.2 ml of the solution containing 25 mm Tris buffer (pH 8), 10 mM EDTA (pH 8) and 50 mM glucose was added. To this, 0.4 ml of solution containing 1% sodium dodecyl sulfate (SDS) and 0.2 N NaOH was added. The tubes were then gently inverted for 2-3 times and allowed to stand for 5 min at 28 ± 2°C. Finally, 0.3 ml of chilled solution of 3 M potassium acetate and 5 M glacial acetic acid were added and allowed to stand on ice for 10 minutes. After centrifugation (14000 rpm, 2 min) pellet was dissolved in 0.5 ml of 0.05 M Tris-EDTA buffer (pH 8.0), incubated at 65°C for 2 min and 0.5 ml of phenol-chloroform-isoamyl alcohol (25:24:1) was added and shaken thoroughly for 10 min and then centrifuged (14000 rpm, 3 min). The DNA was precipitated by adding 1 ml of ice-cold 70% ethanol to the supernatant. The quality of DNA was checked on 1.0% agarose gel electrophoresis. DNA purity and concentration were assayed in a spectrophotometer (260/280).

Polymerase Chain Reaction (PCR) assay
PCR assays were carried out to detect the various genes in the biofilm producing MRSA isolates. The primers used in this study are summarized in Table 1. All the respective primers were obtained from Sigma Aldrich Chemicals Pvt. Ltd., Bangalore, India. For PCR amplifications, about 200 pg of DNA was added to 20 µl mixture containing 0.5 mM of dNTPs, 1.25 µM of each primer and 0.5 µl/unit of Taq polymerase (Bangalore Genei) in 1x PCR buffer. Amplification was performed in an Eppendorf thermal cycler (Germany). The amplified products were separated in 1.0% agarose gel containing 2.5 µl of 10 mg/ ml ethidium bromide. The gel was run at 70 volt for 1 h. The gel images were taken under ultraviolet light using gel documentation system (Bio-Rad, USA). A 100 bp ladder (Bangalore Genie) was used to measure the molecular weights of amplified products.

RNA isolation
Total RNA from untreated and treated MRSA strains with various drugs at their half of MIC was extracted using the method described elsewhere [30]. Briefly, two milliliter of overnight grown MRSA strains was centrifuged at 5000 rpm for 5 min at 4°C and pellet was washed with 1 X TE buffer (pH 8.0), suspended in 1 ml of TE buffer containing 0.2% Triton X-100. The suspension was incubated at 100°C for 10 min and thereafter immediately placed on an ice bath. After incubation, an equal volume of chloroform : methanol (2:1) mixture was added, mixed thoroughly and centrifuged at 12000 rpm at 4°C for 10 min. This step was repeated twice. Finally, RNA was precipitated by addition of 2 volumes of pre-chilled 100% ethanol into the supernatant and mixture was incubated at -20°C for 4 hour and then centrifuged at 12000 rpm for 10 min at 4°C. The pellet was air dried for 5 min and re dissolved in 50 µl of Diethyl pyrocarbonate (DEPC) water. The purity of RNA was confirmed by measuring the absorbance at 260/280 ratio which came approximately 1.9 as indicator of pure RNA. RNA was stored at -70°C until use.

cDNA synthesis
Total RNA (2 µg) was then converted to first strand cDNA as follows: two microgram of RNA was combined with 1 µl of oligodT primer and 9.2 µl of water and then mixture was incubated at 65°C for 5 mins. After incubation, following reagents were added sequentially: 4.0 µl of 5X RT buffer, 1.0 µl of 0.1 M DTT, 0.5 µl of 10 mM dNTP and 0.3 µl of 20 U/µl Moloney Murine Leukemia Virus Reverse Transcriptase (MMLVRT) mixed well and the mixture was subsequently incubated at 37°C for 60 min. The reaction was stopped by heating at 70°C for 10 min. The resultant solution was cDNA which can be used for further study.

RT-PCR analysis
RT-PCR was performed using the cDNA. The primers for the selected genes were the same as mentioned in Table 1. β-actin primer was used as internal control and following sequences were used: β-actin-F-5'-GAAGCATTTGCGGTGGACCAT-3' and β-actin-R-5'-TCCTGTGGCATCCACCAAACT-3' . For PCR amplifications, about 3 μl of cDNA was added to 20 μl mixture containing 0.5 mM of dNTPs, 1.25 μM of each primer and 3.0 U of Taq polymerase (Bangalore Genei) in 1x PCR buffer. Amplification was performed in an Eppendorf thermocycler (Germany) with the same cycling parameters: 2 min at 94°C and 25 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s and one cycle for 10 min at 72°C. PCR products were then electrophoresed on a 1.0% agarose gel containing ethidium bromide. After electrophoresis, density of PCR products was measured using image J software.

Effect of drugs on genes finbA, fib, clfA, eno and hla expression
To evaluate the effects of drugs on expressions of fnbA, fib, clfA, eno and hla genes, MRSA strains were treated with all the selected drugs, Vancoplus, clindamycin, linezolid, teicoplanin and daptomycin, at their half of MIC for 24 h. Following treatment,first strand cDNA from RNA was synthesized as mentioned above. First strand cDNA was synthesized as mentioned above. The same strain without drug treatment served as a control. All experiments were carried out in triplicate and representative data are presented.

Relationship between biofilm formation and virulence determinants
The icaA gene was excluded from the evaluation as all the isolates possessed this gene. As shown in Table 2, the mean OD570 value was higher in eno, hla, clfA, finbA and fib positive isolates compared to eno, hla, clfA, finbA and fib isolates. The percentage of hla, hlb and fnaA was 100% among strong biofilm producers whereas other determinants vary from 87 to 95%. The percentage of hla was the highest in medium biofilm producing MRSA (78.6%) while other ranged from 33 to 77%. None of the isolates was positive with hlb. The percentage of hlb and fnaA in weak biofilm producing MRSA was 100% and 33.3% while none of the isolates was positive with hla. These results suggest that MRSA isolates possessing hla, hlb, fnaA and clfA genes had greater capacities for biofilm formation than those of lacking these 3 genes (Figure 2). Table 3, Vancoplus emerged as the most effective antibacterial agent with MIC values 2 to 4, 0.5 to 1 and 0.125 to 0.25 µg/ ml in strong, medium and weak biofilm producing isolates, respectively. Second most active agent was linezolid followed by teicoplanin, daptomycin and clindamycin.

MBEC
The MBEC of Vancoplus for strong, medium and weak biofilm producers MRSA was approximately 2X, 3X and 2X of MIC, indicating that 2 to 3 times more Vancoplus is required to kill the bacteria in biofilms than that was required to inhibit planktonic bacterial cells. The MBEC of linezolid varied from 1024 to 2048, 256 to 512 and 64 to 128 µg/ml for strong, medium and weak biofilm producing MRSA, respectively. The MBECs of daptomycin, teicoplanin and clindamycin were >2048, 1024 to 2048 and 128 to 512 µg/ml for strong, medium and weak biofilm producing MRSA, respectively which is 3 to 4 times higher than their respective MIC values (Table 4).

Biofilm breaking
Drugs were treated with half MBEC on preformed biofilms of selected strong biofilm producing strains, the data (Table 5)

Effects of drugs on finbA, fib, clfA, eno and hla gene expression
For gene expression study, we selected those genes which were present predominantly among strong and medium biofilm producers strains. Our results showed that the percentage of finbA, hla, eno, clfA and fib genes expression down-regulation after Vancoplus treatment was 64.0 ± 5.9, 63.8 ± 5.8, 73.0 ± 7.4, 72.8 ± 7.8 and 71.9 ± 7.8%, respectively as compared to the control among strong biofilm producers MRSA whereas teicoplanin produced only 34.5 ± 3.8, 25.09 ± 2.3, 25.6 ± 2.5, 28.9 ± 2.5 and 30.3 ± 2.7% down regulation in fnbA, hla, eno, clfA and fib genes expression. The other comparator drugs, clindamycin, linezolid and daptomycin, demonstrated variable effects on these genes varying from 4.9 ± 3.9 to 30.3 ± 2.7% (Figure 3). Overall, Vancoplus was found to be significantly down regulating the genes required for biofilm formation hence controlling the biofilm formation.

Discussion
Over the past several decades, the incidence of resistant grampositive organisms has risen throughout the world. Among these, MRSA are predominant pathogens known to form biofilm on various surface [28] and believed to have an enormous impact on healthcare and are estimated to be associated with 65% of nosocomial infections [31].
In this study, 85.4% MRSA isolates were confirmed to be biofilm producers of which approximately 51% (24/47) were strong biofilm producers, 19.1% and 14.5% of MRSA were weak biofilm and nonbiofilm producers, respectively which is in agreement with previous studies [32,33]. S. aureus is capable of adhering to a large variety of surfaces which is frequently mediated by protein adhesins of the family MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules). The collagen binding proteins, fibronectin binding proteins, clumping factors and fibrinogen binding proteins belong to this family [7,10]. In the current investigation, we observed that >70% isolates possessed clfA, eno, fib, hla and finbA which were responsible for strong biofilm production whereas the prevalence of other cell surface proteins were less than 40%. Of the studied determinants, clfA, eno, fib, hla and finbA were predominants in those isolates that had significant greater capacities for biofilm formation than clfA, eno, fib, hla and finbA negative isolates.
We also observed that icaA gene which is known to be involved in Polysaccharide Intracellular Adhesin (PIA) synthesis of staphylococci plays important role in cell to cell interaction during biofilm formation [5] and is present in all biofilm producers.
As universally reported, planktonic cells were found to be more susceptible than cells embedded in biofilm. This is because, once biofilm is formed, the bacteria undergo phenotypic changes that include increased production of extracellular polysaccharide, decreased metabolic rates and decreased multiplication. The decreased multiplication of bacteria is less susceptible to antibiotics by virtue of their reduced growth rates [34,35].
Vancomycin is a time dependent antibiotic and its clinical efficacy depends on various factors including inoculumn size, tissue distribution and protein binding effects [36,37]. Our results revealed that among the tested drugs, Vancoplus (combination of vancomycin plus ceftriaxone along with VRP1020) was found to be more active against planktonic bacteria (MIC 0.125 to 4 µg/ml) as well as bacteria embedded in biofilm (MBECs 0.5 to 16 µg/ml).
The enhanced activity of Vancoplus to planktonic bacteria may be due to synergistic action of ceftriaxone, vancomycin and VRP1020 (a non-antibiotic adjuvant which prevents degradation of antibiotics). Ceftriaxone inhibits bacterial cell wall synthesis by means of binding to the penicillin-binding proteins [38]. The MBECs values for Vancoplus is less compared to previous study where vancomycin MBEC was reported >512 µg/ml against S. aureus [39]. This is probably may be due to VRP1020 which enhanced the penetration of antibiotics into the biofilms by breaking the exopolysaccharide structure through removal of Ca 2+ ions required for biofilm structure [40]. Further, Vancoplus reduced 5.3 logs in bacterial count in bacterial cells embedded in biofilm with 87% biofilm eradication. As in previous studies [41,42], we also observed that teicoplanin and clindamycin are not much effective in bacterial cells embedded in biofilm. Contrary to previous study, our data demonstrated reduced activity of daptomycin and linezolid at high inoculumn of baterial populations [37].    It is interesting that Vancoplus demonstrated activity on these bacteria with MIC 0.125 to 4 µg/ml and MBECs of 0.5 to 16 µg/ml. Since 29% of the Vancomycin is protein bound [43], the calculated maximum concentration of unbound drug in serum is 34 µg/ml [44]. Thus clinical doses of Vancoplus (free drug) will exceed the MBEC and should have activity against bacteria embedded in biofilm. The clinical achievable concentration of other comparator drugs are less compared to their MBECs hence these drugs may not be effective against the bacteria embedded in biofilm [44,45]. The clinical data study supports the use of Vancoplus in the treatment of lower respiratory tract infections, endocarditis, meningitis and bone infections (unpublished data). The animal model data suggest efficacy in the treatment of intra abdominal infection and meningitis [46,47].
Several previous studies have demonstrated the influences of subinhibitory concentrations of antimicrobial agents on the expression of various virulence factors which are produced by S. aureus and required for biofilm formation [48][49][50]. In our study, expression of all the selected genes was down regulated when MRSA positive for these genes were treated with different drugs. Hla is important for S. aureus biofilm formation and deficiency in Hla caused defects in biofilm formation [51]. Howden et al. [52] reported that on treatment of VISA clinical isolates with vancomycin drug significantly downregulated the expression of cell surface adhesion molecules and a number of genes involved in pathogenesis and toxin production (spa, finbA, finbB, efb). Furthermore, Schröder et al. [53] reported that on treatment of S. aureus isolate with novobiocin antibiotic showed decreased expression of finbA when analyzed with microarray and northern blot hybridization. In accordance with our study, Koszczol et al. [54] demonstrated that enolase (eno) was down-regulated following drug treatment. Clumping factor A (ClfA) was shown to be critical in mediating direct S. aureus platelet adhesion and in indirect binding via exogenously added fibrinogen [55]. During the early growth phase, ClfA was identified as the dominant staphylococcal adhesion receptor in both the presence and absence of exogenously added fibrinogen [55]. Numerous studies have demonstrated that treatment of S. aureus with temporin L, ovispirin-1 and dermaseptin K4-S4 peptides caused downregulation of several virulence factors and their regulators (saeRS and agr) including clfA and clfB. Beenken et al. [3] reported that fib gene showed down-regulation when treated with vancomycin in JH9 strain of S. aureus, these results are also in accordance with our study where we reported that fib gene get downregulated when culture of S. aureus treated with different antibiotics. These findings strongly suggest that changes in the regulation of transcription of these particular set of virulence determinants may represent an attractive therapeutic targets.

Conclusion
In conclusion, this work demonstrates combining ceftriaxone with vancomycin in presence of VRP1020 significantly reduces the MIC and MBEC values against strong biofilm producing MRSA isolates. Exposure to a sub inhibitory concentration of the Vancoplus significantly down regulated levels of expression of several genes encoding biofilm associated proteins, it was observed that the regulation and expression of certain virulence factors involved in pathogenesis in S. aureus are markedly downregulated in the presence of the Vancoplus, which is encouraging. Although Linezolid appeared to be the second best option after Vancoplus, but failed to eradicate biofilm effectively. This work also conclude the maximum biofilm eradication efficiency of Vancoplus and thus Vancoplus could be one of the best choice to eradicate the biofilm caused by these organisms and an effective therapeutic option for the treatment of biofilm producing MRSA strains. This study was conducted only on strong biofilm producer strains.