Optimum cultural conditions to achieve the best biolm formation and high level icaA transcription by Staphylococcus aureus

The aim of this study was to investigate the inuences of different broth culture media supplemented with glucose, on the biolm formation and ica expression of Staphylococcus aureus. The phenotypic ability to adhere to a polystyrene surface and to produce slime layer were evaluated using microtiter plate test (MtP) and Congo red tube test, respectively. Using PCR, the presence of ica locus in S. aureus strains was conrmed and subsequently, quantitative real-time RT-PCR was performed to investigate transcription of icaA in various media including Tryptic soy broth (TSB), Brain-heart infusion broth (BHIB), (Nutrient broth) NB and (Muller-Hinton broth) MHB contained 0, 0.25, 0.5, 1 and 2% glucose. Our results showed that although all of the studied strains adhered to the wells of polystyrene microtiter plates, the optimum rate of biolm formation was observed for TSB medium contained 1% glucose, but biolm formation was not signicantly different in NB, MHB and BHIB media. Supplementation of all media with 1% glucose led to the highest production of biolm formation and in all of media transcription of icaA was increased with glucose addition to one present.


Results
Our results showed that although all of the studied strains adhered to the wells of polystyrene microtiter plates, the optimum rate of bio lm formation was observed for TSB medium contained 1% glucose, but bio lm formation was not signi cantly different in NB, MHB and BHIB media. Supplementation of all media with 1% glucose led to the highest production of bio lm formation and in all of media transcription of icaA was increased with glucose addition to one present.

Conclusions
The results of the present study indicated that TSB medium supplemented with 1% glucose was the most appropriate medium for evaluation of bio lm formation by S. aureus isolates.

Background
Bio lms is an adherent community of bacteria that irreversibly attached to a substratum or to each other. Bio lm infections, such as pneumonia in cystic brosis patients, chronic wounds, chronic otitis media and implant-and catheter-associated infections, affect millions of people in the developed world each year and many deaths occur as a consequence [1,2]. Structural analysis of bio lms often reveals that bacterial bio lms are often surrounded by an extracellular polymeric substance (EPS) [3]. The gene expression pattern of bacteria in a bio lm is different from planktonic cells. This difference in gene expression along with other characteristics including physical barrier of exopolysaccharides can cause increased tolerance against antibiotics, disinfectants, and host immune mechanisms in bio lms [4]. S. aureus is one of the most important bacterial species which can form bio lms on biotic and abiotic surfaces [5][6][7]. For instance, S. aureus bio lms may be involved in infections of central venous catheters, urinary catheters, prosthetic heart valves, orthopedic implants, and dialysis catheters. S. aureus EPS is mainly composed of poly-N-acetylglucosamine polymer which is encoded by icaADBC operon [7,8].
Several phenotypic and genotypic methods are available for the detection of staphylococcal bio lms.
Phenotypic methods such as MtP and Congo red agar (CRA) tests measure bio lm density and production of slime layer, respectively, while, genotypic methods like PCR, determine bio lm related genes e.g. those encoded microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [9,10]. MtP test uses 96-well microtiter plates to measure the optical density (OD) values of stained bacterial bio lms. This method gives a quantitative result [9].
In CRA method, bacteria are cultured on Congo red agar and incubated; thereafter, the color of colonies indicates their ability to produce slime layer. Slime-forming strains appear as black colonies. whereas, non-slime-forming strains stain red [10]. In addition, the MSCRAMMs mediate S. aureus attachment to different surfaces [11]. Actually, intercellular signaling between bacteria, which is also known as quorum sensing, has been shown to be involved in the development of bio lm by S. aureus. Quorum-sensing occurs based on the synthesis and secretion of small hormone-like molecules (autoinducers). These molecules can bind to cognate receptors and contribute to bio lm formation. Quorum sensing in these bacteria is encoded by an accessory gene regulator (agr) locus. Activation of agr system can affect S. aureus bio lm formation through down-regulation of MSCRAMMs as well as production of a protease [12]. It has been described that bacterial species, attachment surface and surrounding medium are three important factors involved in the formation of bio lms [13]. The ability of each strain to adhere and form bio lm is very different in various culture media and environmental conditions [13], However, environmental factors such as pH, temperature, osmolarity, O 2 levels, nutrient composition and the presence of other bacteria can affect the ability of a microorganism to produce bio lm [14]. In this case, it has been shown in a study that glucose may repress agr system by lowering pH which results in increased bio lm formation [15]. Thus, the present study was conducted to introduce an appropriate bacterial culture medium with optimum glucose concentration for examination of the ability of bio lm formation by S. aureus strains.

Results
All of the isolates were genotypically con rmed as S. aureus. Figure 1 depicts the results of electrophoresis of the species-speci c PCR.
Microtiter Plate Assay (Dup: Abstract ?) The ability of bio lm formation was investigated by measuring OD values of the examined isolates using MtP assay. In all of the tested media, the bio lm formation rate of S. epidermidis 12228 was signi cantly lower than S. aureus 25923 and the other two isolates. However, the results revealed that no signi cant difference was observed in the rate of bio lm formation between S. aureus 25923 and the others. On the other hand, the results showed that the highest and lowest rates of bio lm formation occurred when bacteria were cultured in TSB and NB, respectively. The bio lm formation patterns for two clinical isolates were similar to what was seen for S. aureus 25923 (data are not shown). Addition of glucose from 0 to 1% in all media also showed a signi cant positive effect on the bio lm formation of S. aureus (P < 0.05) and the most bio lm formation was observed in media with 1% glucose. However, increasing of glucose percentage up to 2 percent did not promote bio lm formation. Some reactions of bio lm formation in MtP test are shown in the Figure 2.
Slime Assay (Dup: Abstract ?) In Congo red tube test, S. aureus 25923 and the other two clinical isolates (bio lm producer strains) changed the color of MHB, NB and TSB media from red to reddish grey after the incubation period. However, S. epidermidis 12228 did not change the base red color of all of the examined media. In other words, no signi cant difference was observed among these media in the test. Addition of glucose did not show any visible effect on the tested media ( Figure 3).

Real time-PCR for evaluation of icaA gene expression
The results of the conventional PCR showed that icaA was present in all three tested strains (two clinical isolates and S. aureus ATCC 25923). Sequencing of this PCR product con rmed the accuracy of PCR assay. The sequence obtained for icaA amplicon was 99% identical to the corresponding GenBank sequence (accession number WP_031785277.1). The results of real time-PCR indicated that expression of icaA was increased by addition of glucose to 1% in TSB, MHB, NB and BHIB media. Figure 4 depicts the results of real time PCR for icaA expression.

Discussion
The chemical components of growth media and their supplements such as NaCl, ethanol, glucose and sub-inhibitory concentrations of antibiotics, strongly affect bio lm development [16]. In particular, the presence of glucose in the growth medium has been reported to enhance bio lm formation [17]. Various methods have been introduced to measure the bio lm-forming capability of S. aureus isolates, among which, MtP is one of the most common methods in bacteriology laboratory. In this technique, TSB medium containing various glucose concentrations is often used to study the adherence ability of S. aureus to 96-well polystyrene tissue culture plates [18].
In the present study, the ability of bio lm formation by S. aureus strains was investigated in four bacterial culture media (NB, TSB, MHB and BHIB) containing various glucose supplements (0, 0.25, 0.5, 1 and 2%) using MtP assay. The results showed that among all of the tested broth media, TSB was the most suitable medium to examine the ability of bio lm formation. However, some studies reported that bacterial bio lm formation in BHIB is sometimes occurred better than TSB [17,19,20]. Nevertheless, it has also been shown that some staphylococcal strains produce greater bio lm in TSB compared to BHIB [21]. Moreover, researchers commonly use TSB with various glucose supplements for evaluation of bio lm formation by S. aureus strains [20]. It has been reported that addition of glucose to culture media lead to the increase of the ability of staphylococci bio lm formation [19,21]. In agreement with the previous ndings, the present study showed signi cant increase in S. aureus bio lm production when the bacterium exposed to the increasing concentrations of glucose. The best concentration of glucose for bio lm formation of S. aureus on polystyrene surfaces in all of the tested culture media was 1%.
In addition, Congo red tube test and real time-PCR were used to detect extracellular exopolysaccharide and demonstrate icaA expression, respectively. RT-PCR results showed that icaA was expressed in all of the media with different glucose concentrations but the most expression occurred when one percent glucose was added to the medium (P<0.05). Within the icaADBC operon, the icaA gene encodes the enzyme N-acetylglucosaminyl transferase, capable to catalyse the synthesis of poly-N-acetylglucosamine polymer which is called PIA and is important for cell to cell adhesion and accumulation [22]. The icaADBC and products of the ica locus [icaR (regulatory) and icaADBC (biosynthetic) genes] have been demonstrated to be necessary for bio lm formation [23]. Consequently, in the present study, the expression of icaA locus was evaluated in different culture media supplemented with 0, 0.25, 0.5, 1 and 2% glucose. The results revealed that among all culture media (TSB, MHB, NB and BHIB) the most icaA expression level was detected in 1% glucose addition. In addition, the color of Congo red tube culture was not changed was not changed by glucose addition, indicating that this medium is not suitable for measurement of precise amount of PIA or measurement of icaADBC operon expression.
The results of the present study revealed that in fact, glucose increases the ability of bio lm formation by promoting icaA transcription and subsequently, PIA production. Hence, additional ica-independent factors may be involved in increasing of phenotypic bio lm formation by staphylococci. For instance, it was reported that Agr system is down-regulated by addition of glucose [15], and S. aureus agr mutants also show enhanced bio lm formation [24]. Therefore, glucose-induced promotion of bio lm formation may be related to a suppression mechanism of agr. Using phenotypic analysis of wild type and mutant strains of S. aureus, Lim et al also described that rbf gene is another gene that mediates the bio lm formation of S. aureus at the multicellular aggregation stage in an ica independent manner [25], suggesting that the genetic basis of bio lm formation in staphylococci is multifactorial, and much remains to be explored.

Conclusions
The results of the present study suggested that among various examined media, TSB supplemented with 1% glucose was the most appropriate medium for evaluation of bio lm formation of S. aureus strains. However, further studies are necessary to elucidate the precise mechanisms of increasing S. aureus bio lm formation following glucose induction to culture media.

Methods
Bacterial strains, culture media and growth conditions S. aureus ATCC 25923 was used as a reference bio lm producer strain in all of the experiments. Besides, two S. aureus strains isolated from tracheal tube samples of two patients (45 and 63 years old) admitted to the ICU of a hospital in Hamedan (west of Iran) were also included in the present study. These bacterial isolates were identi ed by culture characteristics and biochemical tests and their identity was also con rmed by a species-speci c PCR assay which investigated femA gene of S. aureus [26]. Meanwhile, S. epidermidis ATCC 12228 was used as a non-bio lm producer control in the assays. All culture media were obtained from Merck (Darmstadt, Germany) and the bacteria were cultured under aerobic conditions at 37°C for 24 h.

Microtiter Plate Assay
All S. aureus isolates were examined for the ability of bio lm formation using a previously described microtiter plate assay [18]. Brie y, 50 µl of an overnight culture of each isolate was added into 96-well microplates (SPL, Korea) containing 50 µl of each of the Nutrient broth (NB), Tryptic Soy broth (TSB), Mueller-Hinton broth (MHB), and Brain-heart infusion broth (BHIB) media supplemented with 0.25%, 0.5%, 1% and 2% glucose (Merck, Germany). NB, TSB, MHB and BHIB media without glucose were also used as control group. The microplates were aerobically incubated at 37°C for 24 hours. The supernatants were then discarded and microplates were washed twice with phosphate-buffered saline (PBS). The plates were left to dry for an overnight and stained with 200 µl of 0.1% Safranin dye for 15 min followed by three times washing with 300 µl of PBS and air-dried for an overnight. Afterwards, 100 µl of ethanol-acetic acid (95:5 V/V) was added to each well to dissolve bounded Safranin in adherent bacterial cells. Finally, optical density values were measured at 490 nm by a plate reader. The experiment was performed in triplicates. The bio lm-producer S. aureus ATCC 25923 and the bio lm non-producer S. epidermidis ATCC 12228 were used as control strains [27].

Slime Assay
Congo red tube test was performed to evaluate the ability of slime production in different media supplemented with various glucose concentrations. To do this, a single colony from an overnight culture was inoculated into 10 ml of each of NB, TSB, MHB and BHIB media contained 0.04% Congo red dye and supplemented with 0.25, 0.5, 1 and 2% glucose. NB, TSB, MHB and BHIB media without glucose were used as control group. The culture tubes were incubated on a shaker (100 rpm) at 37°C for 24 hours. The cultured media were then categorized into three groups based on their colors: black, weak black and red. As exopolysaccharides directly react with Congo red and produce black color, the level of black color is considered as the amount of exopolysaccharides or polysaccharide intercellular adhesins (PIA) [28].Therefore, the black and red media were considered to be related to normal slime producing and nonslime producing isolates, respectively.

Rna Extraction And Cdna Synthesis
To evaluate transcription of icaA gene, the presence of its encoding DNA was rst investigated in S. aureus isolates and the reference strain by a PCR assay using previously introduced primers [29]. The sequence of forward primer was 5′-TATTCAATTTACAGTCGCAC-3′ and of reverse primer was 5′-GATTCTCTCCCTCTCTGCCA-3′. These primers amplify a 407 bp DNA fragment of icaAD gene based on a published sequence (AF086783). A PCR product was sequenced to con rm PCR speci city. For removing DNA contamination, the extracted RNA was treated with RNase-free DNase I (Thermo Scienti c, USA).
The quality and quantity of the extracted RNA were determined by agarose gel electrophoresis and con rmed by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer ND-1000 (Thermo Fisher Scienti c, Wilmington, DE, USA). Extracted RNAs were stored at -70°C for next experiments. Then, the puri ed RNA was converted to cDNA according to the manufacturer's instructions (cDNA Synthesis Kit, Takara, Japan), and stored at -20°C to use as the template for real time RT-PCR.
Relative Quantitative Real-time Rt-pcr SYBR Green real-time PCR Master Mix (Amplicon, Denmark) was used for real-time RT-PCR, according to the manufacturer's instructions. The primers (Takapouzist, Iran) used are listed in Table 1. The reactions were conducted in a Corbett Life Science Rotor-Gene 6000 Cycler (Qiagen, Germany) and 16S rRNA housekeeping gene was considered as an internal control to normalize the expression levels of icaA. The ampli cation proceeded as follows: denaturation at 95°C for 10 min and then 40 cycles including denaturation at 95°C for 30 sec, annealing at 53°C (for icaA) and 59°C (for 16S rRNA) for 30 sec, and 72°C for 30 sec. A negative control was included in each run. All the samples were analyzed in triplicate and nally, relative gene expression was calculated using the 2 −ΔΔCT method [30].

Consent for publication
Not Applicable.

Availability of data and materials
The raw data supporting the conclusions of this article will be made available by the corresponding author, without undue reservation.

Figure 2
Measurement of the ability of bio lm formation using microtiter plate assay (MtP). Lanes A to C indicate the reaction of weak, moderate and strong bio lm producers, respectively.