Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa

Biofilm production represents an important virulence and pathogenesis factor for Staphylococcus aureus. The formation of biofilms on medical devices is a major concern in hospital environments, as they can become a constant source of infection. Probiotic bacteria, such as Lactobacillus fermentum and L. plantarum, have been found to inhibit biofilm formation; however little is known about the underlying mechanism. In this study, we tested the activity of supernatants produced by L. fermentum TCUESC01 and L. plantarum TCUESC02, isolated during the fermentation of fine cocoa, against S. aureus CCMB262 biofilm production. We measured inhibition of biofilm formation in vitro and analyzed biofilm structure by confocal and electronic microscopy. Additionally, we quantified the expression of S. aureus genes icaA and icaR involved in the synthesis of the biofilm matrix by real-time PCR. Both Lactobacillus supernatants inhibited S. aureus growth. However, only L. fermentum TCUESC01 significantly reduced the thickness of the biofilm, from 14 μm to 2.83 μm (at 18 mg∙mL−1, 90 % of the minimum inhibitory concentration, MIC), 3.12 μm (at 14 mg∙mL−1, 70 % of the MIC), and 5.21 μm (at 10 mg∙mL−1, 50 % of the MIC). Additionally, L. fermentum TCUESC01 supernatant modulated the expression of icaA and icaR. L. fermentum TCUESC01 reduces the formation of S. aureus biofilm under subinhibitory conditions. Inhibition of biofilm production probably depends on modulation of the ica operon.


Background
Staphylococcus aureus is a widely distributed and opportunistic human pathogen. It is the causative agent of both simple skin infections and potentially life-threatening systemic complications such as toxic shock syndrome [1]. S. aureus is a major concern in hospital environments because asymptomatic carriers represent a great risk factor to patients subjected to long hospitalization periods [2].
Some aspects of S. aureus pathogenicity are related to biofilm production, which increases resistance to chemotherapeutic treatments and to the host's defense mechanisms [3]. The biofilm is composed of cells adhering to a surface and held together by a polymer matrix. The main components of the matrix are polysaccharides, proteins, and extracellular DNA (eDNA) [4].
S. aureus secretes polysaccharides of intercellular adhesion (PIAs), which are composed mostly of β-1, 6-Nacetylglucosamine residues. PIA production and excretion is controlled by the icaADBC operon [5]. icaA, the first gene to be transcribed, leads to production of short chains of N-acetylglucosamine oligomers [6]. icaD, coexpressed with icaA, enhances oligomer production by about 20 fold [7,8]. icaC is responsible for increasing oligomer chains and possibly for translocation to the cell surface [8,9]. Finally, icaB is thought to deacetylate poly-N-acetylglucosamines [9]. The icaADBC locus is regulated by a transcriptional repressor located upstream encoded by icaR [10]. This repressor protein can bind to the ica operon promoter region close to the icaA start codon [8]. Additional factors that can negatively influence the ica operon in S. aureus include expression of the weak repressor TcaR, global regulation by SarA, and the insertion sequence element IS256 in the ica locus [8,11]. The ica operon is also regulated by environmental factors, which play an important role in the response to anaerobic growth (SrrAB), in the response to stress (Spx), supplementation with glucose or ethanol, osmolarity, temperature, and low concentrations of antibiotics [8,11,12]. Another way of controlling this operon is through a regulatory protein responsible for biofilm formation, Rbf, which can repress transcription of IcaR and indirectly increase expression of the gene icaADBC [11].
S. aureus biofilms are commonly treated with antibiotics, such as protein synthesis inhibitors that target the cell membrane and cell wall, as well as inhibitors of DNA and RNA synthesis [4], or antimicrobials like Cu 2+ that lead to cell membrane breakage and subsequent cell lysis [13]. The indiscriminate use of antibiotics for the treatment of bacterial infections has been suggested to be responsible for the appearance of multidrug-resistant bacteria such as methicillin-resistant S. aureus strains [14]. In this context, alternatives to antibiotic therapy are more than welcome. Probiotics represent a possible option, as the microorganisms that produce them have been proven effective in the prevention and control of human pathogens [15].
Lactobacillus fermentum TCUESC01 and L. plantarum TCUESC02 strains have been recently isolated from the fermentation of fine cocoa seeds. Our group has demonstrated their anti-inflammatory potential and technological properties. We observed that these strains exhibited probiotic characteristics in vivo in an experimental colitis model, reducing histological damage and the systemic concentration of inflammatory cytokines. Additionally, they inhibited the growth of pathogenic bacteria and displayed high resistance to the stressful conditions of the gastrointestinal tract and industrial environments (unpublished observations). In this study, we evaluated the activity of cell-free supernatant from L. fermentum TCUESC01 and L. plantarum TCUESC02 on S. aureus CCMB262 biofilm formation.

Microorganisms and culture conditions
Pure cultures of L. fermentum TCUESC01, L. plantarum TCUESC02, and S. aureus CCMB262 were used. TCUESC01 and TCUESC02 strains were previously isolated by our group during fine cocoa fermentation [16]. S. aureus CCMB262 was obtained from the Microorganisms Culture Collection of Bahia (CCMB), Brazil. The strain, which is resistant to streptomycin and dihydrostreptomycin, was cultured in tryptic soy broth (TSB; DIFCO, Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 1 % (w/v) glucose (Glc) at 37°C with agitation (250 rpm) for 18 h. For inoculum standardization, S. aureus cells were homogenized in saline solution (NaCl 0.85 %) and the suspension was diluted to 0.5 × 10 8 CFU•mL −1 using a spectrophotometer (Evolution 60, Thermo Fisher Scientific, Waltham, MA, USA).

Supernatant preparation and lyophilization process
L. fermentum TCUESC01 and L. plantarum TCUESC02 were cultured in 30 mL of MRS broth. Following centrifugation at 10,000 × g for 15 min at 4°C, culture supernatants were collected and filtered through a 0.22 μm nitrocellulose membrane. Supernatants and samples containing medium culture only (control) were frozen at −80°C for 24 h and lyophilized (Lyophilizer LS3000, Terroni, São Carlos, Brazil). Following lyophilization, the samples were weighed and stored at −20°C. They were then rehydrated with sterile deionized water prior to use.

Minimum inhibitory concentration (MIC) assay
MIC assays were performed by microdilution in 96-well plates (Costar®, Corning, NY, USA), in accordance with recommendations from the Clinical and Laboratory Standards Institute [18]. A serial dilution was performed starting with 40 μg•mL −1 of TCUESC01 and TCUESC02 supernatants on Mueller Hinton (MH) medium containing 5 × 10 5 CFU•mL −1 of S. aureus CCMB262 per well. The same procedure was performed with the following controls: lyophilized medium without Lactobacillus (MRS control); MH without inoculum (medium sterility control -MC); MH containing 5 × 10 5 CFU•mL −1 of S. aureus CCMB262 (positive control); MH containing 5 × 10 5 CFU•mL −1 of S. aureus CCMB262 and 12.5 μg•mL −1 chloramphenicol (negative control). The microplate was incubated for 24 h at 37°C and revealed with 20 μL of Resazurin (0.01 %) for 30 min at 37°C. At the same time, 5 μL of each suspension (samples and control) was cultured on MH agar (MHA; DIFCO) for 24 h at 37°C, after which the inhibitory concentrations were classified as bactericidal or bacteriostatic. The entire experiment was performed three times with three independent repetitions.

Preparation of subinhibitory concentrations of TCUESC01 and TCUESC02 supernatants
To avoid killing all S. aureus, lyophilized supernatants of the two Lactobacillus strains were weighed and diluted to concentrations below the MIC. Thus, dilutions were made using subinhibitory concentrations of 90, 70, and 50 % of MIC.

Biofilm formation on a polystyrene plate
The ability of S. aureus to form biofilms following treatment with Lactobacillus supernatant was analyzed according to the methodology proposed by Oliveira et al. (2014) [19]. Briefly, S. aureus CCMB262 was cultured in 5 mL TSB with 1 % Glc for 18 h at 37°C under agitation (250 rpm) and treated with Lactobacillus supernatants (90 %, 70 %, or 50 % of the MIC) or control medium. The cultures were diluted (1:100) in the same medium, 200 μL was inoculated in a 96-well plate (Costar®), and plates were incubated at 37°C for 24 h. The plates were washed twice with phosphate-buffered saline (PBS), dried for 1 h at 65°C, 1 % crystal violet was added, and the plates were incubated for a further 30 min at 25°C. Each well was washed twice with PBS and 200 μL PBS was added prior to measuring absorbance at 492 nm (A 492nm ) using a microplate reader (VersaMax; Molecular Devices®, Sunnyvale, CA, USA). The experiment was carried out in quadruplicate with at least two independent experiments. Biofilm production was compared to that of Streptococcus pyogenes ATCC75194 (A 492nm = 0.07). The biofilm formation index (BFI) was calculated as follows: where x is the optical density at A 492nm of the biofilm and y is the optical density at A 492nm of Streptococcus pyogenes (0.07). Based on the BFI, S. aureus cultures were classified as non-producers (less than or equal to zero), weak producers (less than 1), moderate producers (between To confirm that the concentration used to inhibit the biofilm was not lethal to S. aureus, a growth curve was constructed. S. aureus (75 μL, 0.5 × 10 8 CFU•mL −1 ) was inoculated in 75 mL TSB with 1 % Glc and cultivated in the presence or absence of 750 mg of TCUESC01 supernatant (50 % of MIC) for 24 h at 37°C under agitation (250 rpm). Aliquots were collected every 4 h to record optical density at 660 nm and count CFU•mL −1 in mannitol salt agar.

Phenotypic evaluation of biofilms by scanning electron microscopy (SEM)
The phenotype of S. aureus CCMB262 biofilm was evaluated by SEM according to Pitino et al. (2012) with some modifications [21]. S. aureus was cultured in TSB with 1 % Glc in the presence or absence of TCUESC01 supernatant in 12-well culture plates (Costar®) containing 18 mm coverslips. After incubation for 24 h at 37°C, the coverslips were washed twice with 0.85 % NaCl and dehydrated with 50, 60, 70, 80, and 90 % acetone for 10 min each, and twice with 100 % acetone for 10 min. The coverslips were subsequently dehydrated in a critical point dryer CPD 030 (BAL-TEC®, Balzers, Germany) using liquid carbon dioxide as transition fluid. The samples were placed in aluminum pieces, metallized with a SCD-050 (BAL-TEC®, Alzenau, Germany), and viewed under a scanning electron microscope (Quanta 250, FEI, Hillsboro, OR, USA).
Total RNA extraction S. aureus CCMB262 was cultured in TSB with 1 % Glc for 18 h at 37°C under agitation (250 rpm), in the presence or absence of L. fermentum TCUESC01 supernatant (50 % of the MIC). The samples were then diluted in the same medium (1:100) and incubated at 37°C for another 18 h, after which aliquots were collected and used immediately for RNA extraction.
Each sample (1 mL) was centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was discarded and RNA from the pellet was extracted with the RNAqu-eous® Phenol-free total RNA isolation kit (Ambion®, Waltham, MA, USA), according to the manufacturer's  Gene expression analysis was carried out on a 7500 Fast real-time PCR system (Applied Biosytems, Waltham, MA, USA), using 10 ng cDNA and the KAPA SYBR® qPCR Master Mix ABI Prism™ kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer's recommendations. The PCR reaction was carried out in quintuplicate with an initial denaturation at 95°C for 5 min followed by 40 cycles of amplification at 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. Data were normalized to16S rRNA (endogenous control) and the relative quantification (RQ) was calculated with 7500 Software v 2.3 (Applied Biosytems).

Statistical analysis
Average, standard deviation, t-test, analysis of variance, Tukey's multiple comparison test, graphics, and other statistical analyses were performed using the Graphpad® prism 5.0 software (GraphPad Software, San Diego, CA, USA).

Results and discussion
Antimicrobial susceptibility tests revealed that the supernatant from L. plantarum TCUESC02 had a stronger inhibitory effect on S. aureus CCMB262 growth (MIC, 2.5 mg•mL −1 ) than that of L. fermentum TCUESC01 (MIC, 20 mg•mL −1 ) (Fig. 1). The potential of Lactobacillus species to inhibit pathogens of clinical importance such as S. aureus had been evaluated before. Accordingly, Al  showed that L. fermentum CMUL054 and L. plantarum CMUL140 were active against S. aureus ATCC33862 [23]. Moreover, Hor and Liong (2014) analyzed 87 lactic acid bacteria strains and three strains of bifidobacteria, and found that all strains inhibited the growth of S. aureus by 0.5 to 34.2 %; L. fermentum and L. plantarum, which were isolated from milk, inhibited growth by around 20 % [24]. Although inhibition of S. aureus by lactic bacteria has been reported in many studies [23][24][25][26], few of them have calculated the MIC from extracellular bacterial extracts. This has made it difficult to assess the extracts' inhibitory action. Lactobacilli produce various secondary metabolites that exhibit antimicrobial activity, such as organic acids, ethyl alcohol, bacteriocins, hydrogen peroxide, and surfactants [15,[26][27][28]. The strains used in this study were isolated during cocoa fermentation, a process characterized by high temperature conditions, low oxygen concentrations, and low glucose availability. Lactobacilli are probably selected for their ability to withstand these conditions, which makes them an interesting source of compounds with biological activities of human interest.
L. plantarum TCUESC02 was less effective against S. aureus CCMB262 biofilm formation (MIC, 2.5 mg•mL −1 ). We found a significant difference in the BFI only when using 90 % of the MIC (p < 0.01), meaning that S. aureus CCMB262 was still a strong biofilm producer. We did not observe statistically significant differences between treatments with 90 % (2.25 mg•mL −1 ) or 70 % of the MIC (1.75 mg•mL −1 ) for this strain. In a previous report, the supernatant of L. bulgaricus FTDC8611 inhibited significantly S. aureus biofilm, an effect attributed to organic acids [24]. Also, Ait Ouali et al.(2014) demonstrated that L. pentosus LB3F2 had antimicrobial and antibiofilm activity against S. aureus SA3 [29].
A comparison between bacterial growth curves in the presence or absence of TCUESC01 supernatant at 50 % of the MIC (10 mg•mL −1 ) revealed this concentration was not lethal to S. aureus CCMB262. This indicates that the decrease in biofilm formation was not caused by death of the pathogen (Fig. 3). Instead, TCUESC01 appeared to secrete a modulatory substance capable of interfering with the pathogen's capacity to form biofilms.
To confirm the results on biofilm inhibition, we visualized its structural organization by confocal microscopy  (Fig. 5). eDNA is one of the main components of biofilms [4]. It functions as a cohesive factor between bacteria, stabilizing the overall biofilm structure [30]. Moreover, it increases resistance to antimicrobials, such as aminoglycosides [31] and vancomycin [32]. Thus, reduction of eDNA production represents a potential tool for microbial control.
To visualize the intimate structure of the S. aureus biofilm we used SEM. Following treatment with L. fermentum TCUESC01 supernatant (50 % of the MIC), we noticed a decrease in the biofilm matrix (Fig. 7), suggesting possible interference with the production of PIAs.
PIA synthesis depends on the icaADBC locus [7]. Thus, we sought to verify whether the phenotypic modulation of S. aureus biofilm by L. fermentum TCUESC01 was related to the genetic regulation of icaA and icaR genes. We observed that treatment with L. fermentum supernatant (50 % of the MIC) significantly increased the expression of S. aureus regulator gene icaR. Accordingly, RQ increased from 1 (untreated) to 68.45 (treated) (Fig. 8a), while icaA expression dropped from a RQ of 1 (untreated) to 0.39 (treated) (Fig. 8b). These results suggest that the reduction of S. aureus CCMB262 biofilm upon treatment with TCUESC01 is associated with inhibition of PIA production.
Biofilms are formed by planktonic cells that bind irreversibly to a solid substrate. Wrapped in a matrix, cells multiply, differentiate, and modify gene expression patterns, in addition to creating channels for nutrient circulation [33]. After ripening, fragments of this bacterial community scatter, carrying all the "mother" community features such as antimicrobial resistance and virulence, and adhere elsewhere to repeat the cycle [34]. When organized into biofilms, bacteria tend to become more pathogenic. Suci et al. (1994) demonstrated the delayed penetration of ciprofloxacin into biofilms of Pseudomonas aeruginosa ERC-1 [35]. Seemingly, Ceri et al. (1999) found that the MIC of some antibiotics was around 100 to 1000 times greater for microorganisms in biofilms [36]. These findings underscore the importance of developing new strategies to combat biofilms, such as the use of probiotics. Here, L. fermentum TCUESC01 and L. plantarum TCUESC02 showed bactericidal properties against S. aureus CCMB262. Furthermore, TCUESC01 was able to reduce the formation of S. aureus biofilms under subinhibitory conditions. In addition, increased icaR and reduced icaA expression in S. aureus cultures treated with TCUESC01 supernatant indicated that the reduction in biofilm production occurred through modulation of the ica operon.

Conclusions
The results presented in this study suggest an interesting novel application for lactobacilli isolated from fine cocoa. The antimicrobial effect and biofilm inhibition exhibited by the probiotic strain TCUESC01 could be applied to the treatment and prevention of infections with pathogenic bacterial strains.