Analysis of Staphylococcus epidermidis biofilm formation in several types of intravenous fluids based on time

Nosocomial infection is a result of the interactions between pathogens, patients, practices, and places. Medical devices can be the source of infections [1], nowadays, the number of infections is associated with the higher application of medical devices and immunocompromised patients in the hospital [2]. Pathogenic bacteria can penetrate the skin through contamination during the application of a catheter [3] and cause bacteriemia [4, 5]. Generally, microorganisms causing nosocomial infections are biofilm producers [6]. The presence of biofilm poses the risk of decreasing susceptibility to antibiotics [7] and lead resistant infection to conventional drugs [8]. The long-term effects are often related to the length of hospitalization, economic burden, increased patient morbidity and mortality [9]. 
Coagulase-negative staphylococci (CoNS) is the most abundant constituent of the normal flora on the skin and mucous membranes of humans, but it can be opportunistic pathogen bacteria related to nosocomial infection [10]. The most common CoNS species causing the infection is S. epidermidis [11, 12]. Usually, these bacteria are single-celled or planktonic, but in some conditions, they can colonize to form biofilm, especially on moist and nutrient-rich surfaces [13, 14]. Staphylococcus can produce an extracellular polymer substances (EPS) matrix and form biofilm within 18-48 h of incubation in suitable condition [15].
An intravenous catheter is an important therapy for treating surgical patients and critical patients who have lost a lot of fluids such as dehydration due to vomiting, diarrhea, and shock [16], as parenteral nutrition in patients with certain medical conditions [17]. The insertion of an intravenous catheter must pay attention to the aspect of sterility so that microorganisms do not cause local infection in the puncture area and enter the blood vessels [18]. The maximum time for intravenous catheter application is 3 days, but if any signs of phlebitis such as redness, swelling, fever, and pain, the catheter must be removed immediately [19]. Crystalloid intravenous fluid is divided into 3 groups based on tonicity or electrolyte concentration i.e., isotonic such as ringer lactate, 5% dextrose, and normal saline (0.9% NaCl), hypertonic such as 10% dextrose, and hypotonic such as 0.45% NaCl [20]. While colloid intravenous fluid contains animal protein with a higher molecular weight than crystalloid [21].
A study demonstrated an increased prevalence of colonization and bacteremia on intravenous catheters at more than 48 h of installation [22]. However, the study about intravenous fluids and biofilm formation was very rare, the previous study was very old and only used one variable of time [23]. Thus, this study aimed to analyze the biofilm formation of S. epidermidis in several types of intravenous fluids such as ringer lactate (RL), 10% dextrose, 5% dextrose, normal saline (NS), and gelafusal based on incubation time of 24, 48, and 72 h.

Ethical approval
The ethical approval of this research was issued by the Health Research Ethical Clearance Commission (KEPK), Faculty of Medicine, Universitas Airlangga with certificate number 149/EC/KEPK/FKUA/2022 on August 8th, 2022.

Bacterial strain culture and preparation of bacteria
S. epidermidis strain was obtained from the Microbiology Laboratory, Faculty of Medicine, Universitas Airlangga, Indonesia. S. epidermidis strain from the culture stock was grown on nutrient agar plate (NAP, Oxoid, Basingstoke, UK) and incubated in the incubator for about 24 h aerobically at 37⁰C temperature. After verifying the purity of the strain, 3 to 4 identical morphologies were suspended in 5 mL of sterile distilled water [15]. The bacterial suspension was adjusted to match turbidity using 0.5 McFarland standard (1.5 x 10⁸ CFU/mL). Then, the suspension was vortexed for at least 1 min and should be used at least 15 to 60 min after it was made.

Biofilm cultivation
The cultivation of biofilms was grown in a flatbottom microplate or microtiter plate 96-well (Biologix Europe GmbH, Germany). There were 3 microtiter plates prepared for different incubation times of 24, 48, and 72 h. Each well in the microtiter plate was filled with 180 µL of intravenous fluids and inoculated with 20 µL bacterial suspension. The intravenous fluid from the 1st column was RL (Otsuka, Indonesia), the second column was 10% dextrose (PT. Widatra Bhakti, Indonesia), the third column was 5% dextrose (Otsuka, Indonesia), the fourth column was NS (Otsuka, Indonesia), and the fifth column was gelafusal (Serumwerk Bernburg, Germany). The positive control consists of 200 µL trypticase soy broth (TSB, Oxoid, Basingstoke, Hampshire, England) only in the sixth column and the negative control consists of 200 µL RL, 10% dextrose, 5% dextrose, NS, gelafusal only in the seventh to eleventh column respectively. Each treatment in every column was repeated 6 times (rows A to F), so there were 66 filled wells. The microplates were covered with the cover, then incubated in an incubator (Heraeus) at 37⁰C within 24, 48, and 72 h for the first, second, and third microplates, respectively.

Crystal violet staining
After incubation, the contents of the wells were decanted into a discarded container. Microplate was washed 3 times with 300 µL phosphate buffer saline (PBS, Laboratorium Bio Analitika, Surabaya, Indonesia) in each well using a micropipette with a gentle tap to remove planktonic bacterial cells that were not adherent to the microplate. Before staining, the microplate was drained in an inverted position. Then, 200 µL of 1% crystal violet dye (Laboratorium Bio Analitika, Surabaya, Indonesia) was added to each microplate well until it was submerged to stain the biofilm biomass in the microplate wells and left at room temperature for 20 min. The dye was removed, and the wells were washed again 3 times using PBS with the same method and then dried air in an inverted position. The last step before the reading of optical density was adding 200 µL of 96% ethanol (Laboratorium Bio Analitika, Surabaya, Indonesia) into each well and incubating for about 20 min. 

Measurement of biofilm formation 
The total biomass of biofilm after staining was measured by using a microplate reader (BIO-RAD iMarkTM) with 595 nm to get the OD value. The results obtained were interpreted by first looking for the cut-off value to distinguish biofilm-producing from non-biofilm-producing strains. There are 4 categories of grouping abilities to form biofilm [24], including OD < OD_cut is non-biofilm-former (NBF), OD_cut < OD < 2 OD_cut is weak-biofilm-former (WBF), 2 OD_cut < OD < 4 OD_cut is moderate-biofilm-former (MBF), and OD > 4 OD_cut is high-biofilm-former (HBF). 

Statistical analysis
OD value was analyzed using Statistical Package for the Social Sciences (SPSS) 25.0 for Windows. Normality test with One Sample Kolmogorov-Smirnov Test and homogeneity test with Levene Test were used to find out the data distribution. The level of significance was determined using Kruskal-Wallis, then continue with the Mann-Whitney test and P < 0.05 was considered significant.

Based on the research, the average biofilm biomass (OD value) of S. epidermidis grown in intravenous fluids of RL, 10% dextrose, 5% dextrose, NS, and gelafusal within 24, 48, and 72 h of incubation time are presented in Table 1. The OD values of biofilm formation of S. epidermidis in TSB media as positive controls at every incubation time always increased, with the lowest value of 1.009+0.146 at 24 h and the highest value of 2.633+0.444 at 72 h. The OD values of biofilm of positive controls were significantly compared to inoculated intravenous fluids and negative controls (Figure 1-3).

Biofilm formation within 24 and 48 h of incubation times
All the inoculated intravenous fluids incubated for 24 and 48 h had similar OD values compared to negative controls (Table 1). Based on statistical test with Kruskal-Wallis, the OD values of biofilm formation within 24 and 48 h of incubation time were 0.002 and 0.008, respectively (P < 0.05), then continued with the Mann-Whitney test to determine the significance between treatments. The results of Mann-Whitney test on the formation of bacteria biofilm during incubation time of 24 and 48 h were not significant (Figures 1 and 2). The same letter in the graph indicated insignificant OD values between inoculated intravenous fluids and negative controls. The interpretations of biofilm formation showed that S. epidermidis grown in RL, 10% dextrose, 5% dextrose, NS, and gelafusal within 24 and 48 h of incubation time were NBF.

Table 1. Biofilm OD values obtained for S. epidermidis based on incubation time.

 

Biofilm formation within 72 h of incubation time
The average OD values of S. epidermidis biofilm formation within 72 h of incubation time grown in intravenous fluids had a slightly higher values than the negative controls (Table 1). The result of the Kruskal-Wallis test was 0.000 (P < 0.05), there were 3 treatments (10% dextrose, 5% dextrose, and NS) which had different significance compared to negative controls based on the Mann-Whitney test. The different letters in the graph indicated significant OD values between inoculated intravenous fluids of 10% dextrose, 5% dextrose, and NS compared to negative controls (Figure 3). However, the interpretation results of bacteria biofilm formation grown in RL, 10% dextrose, 5% dextrose, NS, and gelafusal were considered as NBF. This could be due to differences in standards between statistic and interpretation formulas.

The data obtained from this study indicates that the length of incubation time results in different biofilm formations in intravenous fluids media. Statistically, in-vitro studies on biofilm formation tests with incubation times of 24 and 48 h did not show significant data, meanwhile at 72 h showed significant data. The biofilm biomass which is adherent on the base or wall of the microplate wells will bind to the crystal violet dye and this staining is performed to determine quantification through the absorbance of stained biofilms by measuring the OD value [25]. 
S. epidermidis used in this study were suspended in sterile distilled water, then inoculated into each intravenous fluid. Characteristics of sterile distilled water are pure, does not contain metals or anions, and has a pH of 7 (neutral) [26] which aims to provide a real condition where intravenous fluids given to patients are sterile. TSB is used as positive control media as it contains casein peptone which is rich in nitrogen, mineral, and vitamin, also soy peptone (papain) which is used as a dual-energy source for biofilm growth and formation [27].
In this research, biofilms were not formed in all intravenous fluids during 24 and 48 h of incubation time. In a previous study, the fluids passing through the catheter can affect microorganism growth but S. epidermidis did not grow well in intravenous fluids such as normal saline and dextrose solutions within 24 h of incubation time [23]. Incubation time is one of the factors influencing the growth of biofilms, time of biofilm formation is an important factor to see how quickly microorganisms can form biofilm in the right media [18]. 
Biofilm is defined as the association of microorganisms and EPS matrix which are sticky and slimy substances for binding bacteria to each other and attaching them to a material surface [13]. Some genes play role in S. epidermidis biofilm formation of the initial attachment stage including the PIA (polysaccharide intercellular adhesin) which functions to induce biofilm formation, the embp gene which functions in intercellular adhesion [28] and the Aap gene, protein related to accumulation gene which functions in primary attachment [29]. The genes involved in the maturation and dispersion stages are the dspB gene which functions as a PIA degrading enzyme [30] and the atlE gene which functions as an autolysin gene [28]. The metabolism of planktonic and biofilm cells of bacteria are different, tricarboxylic acid (TCA) or Krebs cycle is inactive in bacteria cells in the biofilm phase, either due to Krebs cycle inhibitors or gene mutations, which will increase the expression of the icaADBC operon in S. epidermidis [31]. A study proved that S. epidermidis isolates with icaA and/or icaD positively formed biofilm [32].
In this study, S. epidermidis cultivated in 10% dextrose, 5% dextrose, and normal saline started to form biofilms at 72 h of incubation time, although the values were slightly higher compared to negative controls. No previous report described the biofilm formation of S. epidermidis in intravenous fluids at 72 h of incubation time. However, a previous study showed that after 72 h of incubation, the morphology of S. epidermidis had changed, from planktonic phase to cell clusters which are defined as biofilm characteristics [33]. The clinical isolates from center venous catheters had identified the biofilm formation and 90% of the isolates were strong biofilm formers of S. epidermidis [34], so there is still a possibility that biofilm formed on catheter needles in patients’ body for three or more days can grow in intravenous fluids as media to continue forming biofilm and cause bacteremia. 
Biofilm formation of S. epidermidis in 10% dextrose at 72 h incubation had the highest value, followed by normal saline, and 5% dextrose. The glucose molecule is higher in 10% dextrose than 5% dextrose, so there were more nutrients for bacteria to grow and form biofilm. Glucose is a carbon source used for the formation of biofilms [35]. S. epidermidis biofilms can be formed on media rich in carbon sources such as glucose, even maltose, lactose, and sucrose [36]. Research on biofilm formation with 0.5% glucose and 1.0% glucose addition to TSB media produces higher biofilms than biofilms grown on TSB media only [37]. Even the addition of 0.2% and 0.4% glucose can increase the bacteria biofilm biomass significantly [38]. Glucose metabolism produces short fatty acid chains which will decrease the pH of the medium [39] and suppress the agr (accessory gene regulator) quorum sensing system [40]. The acidic condition suppresses the production of extracellular protease [41], induces the association of biofilm matrix proteins on cell surfaces [42], and forms biofilm. Besides, the addition of 1.0% NaCl and 0.2% NaCl to TSB media also can increase the biofilm formation [37]. NaCl can activate the expression of the icaADBC operon [43]. The icaADBC operon is involved in the production of PIA, so biofilm-forming bacteria are able to induce biofilm formation [31].

In this study, we inoculated S. epidermidis in several types of intravenous fluids at three different times. The results showed that biofilm formation did not form at 24 and 48 h of incubation time in all intravenous fluids, whereas biofilms began to form at 72 h in some intravenous fluids. The OD values of all inoculated intravenous fluids were not statistically significant at 24 and 48 h of incubation time compared to negative controls. Within 72 h of incubation time, the OD values of inoculated 10% dextrose, 5% dextrose, and NS were statistically significant, while the inoculated RL and gelafusal were not statistically significant compared to negative controls. The length of incubation time supported by the rich-nutrient media may influence biofilm formation. The longer using of medical devices may cause the higher possibility of biofilm formation. Biofilm formation can be prevented by applying aseptically procedures, carrying out routine checks and maintenance. We have many shortcomings and limitations in this study, so further research can be carried out in the future.

We would like to say thank you to the Institute of Tropical Disease (ITD), Universitas Airlangga, Surabaya, Indonesia for agreeing to assist in measuring research data.

SFJ, ADWW, and RJS designed outlines and drafted the manuscript.  SFJ performed the experiments, analyzed the data, and wrote the initial draft of the manuscript. ADWW and RJS reviewed the scientific contents described in the manuscript.

There is no conflict of interest among the authors.