Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms
© França et al. 2016
Received: 9 March 2016
Accepted: 14 March 2016
Published: 22 March 2016
Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.
Biofilms play an essential role in many human infections, including those related to the use of indwelling medical devices (Donlan 2001). Extensive research on biofilms has led to the characterization of the biofilm lifecycle, which encompasses three main stages: (a) initial adhesion, (b) accumulation and (c) dispersion (Otto 2009). Biofilm dispersion is the less studied stage of the biofilm lifecycle. Nevertheless, it is known that the active release of biofilm cells by dispersion is influenced by several environmental cues including temperature and pH fluctuations (Boles and Horswill 2011), accumulation of waste (Kaplan 2010; McDougald et al. 2012), nutrients and oxygen availability (Hunt et al. 2004; Sauer et al. 2004), as well as by the production of signaling molecules (Boles and Horswill 2008; Davies and Marques 2009). In addition to dispersion, biofilms may also release cells into the surrounding environment due to shear forces, and this passive process is often referred as detachment (Choi and Morgenroth 2003; Picioreanu et al. 2001). Biofilm disassembly, which includes both active and passive mechanisms, has been associated with the emergence of serious acute infections (Boles and Horswill 2011). Nevertheless, due to the technical challenges in devising valid and affordable methods to collect cells released from biofilms, the study of these cells has lagged behind. Both fed-batch and dynamic systems have long been used to characterize initial adhesion (Cerca et al. 2005b; Isberg and Barnes 2002), biofilm accumulation and structuring processes (Moormeier and Bayles 2014; Periasamy et al. 2012). However, no fed-batch system has been used so far to study biofilm disassembly. Despite the advantages of dynamic over fed-batch systems, such as tight control of hydrodynamic systems and continuous diffusion of nutrients and waste, it presents important drawbacks such as difficulty in assembling systems, frequent formation of air bubbles that destroy biofilm architecture (Crusz et al. 2012), limited number of conditions that can be analyzed simultaneously and higher cost. Hence, the development of a model to study the cells released from biofilms based on the widely used fed-batch systems may provide significant insights into the pathophysiology of biofilm-related infections.
Owing to its ubiquitous presence in human skin, Staphylococcus epidermidis frequently colonizes and form biofilms on skin penetrating devices such as vascular catheters being, thus, considered one of the major nosocomial pathogens associated with biomaterial-associated biofilms infections (Otto 2009). Due to the release of cells from biofilms formed on vascular catheters into the surrounding environment, S. epidermidis is responsible for 22 % of bloodstream infections detected in intensive care units patients, in the USA (Otto 2013). It is, therefore, important to study in more detail the phenotype and the interaction of these cells with the host immune system, in order to conceive effective strategies to control the pathologic events associated with biofilm disassembly. Herein, we describe a simple and affordable fed-batch method to collect cells disassembled from S. epidermidis biofilms that can be used for further functional studies.
Materials and methods
Bacteria and growth conditions
Staphylococcus epidermidis strains used for the validation of glucose influence in biofilm dispersion
ATCC reference strain 35984
Christensen et al. (1985)
Reference strain used in biofilm-related studies
Mack et al. (1994)
Clinical isolate from a patient with infective endocarditis
Cerca et al. (2006)
Clinical isolate from a patient with infective endocarditis
Cerca et al. (2006)
Clinical isolate from patient after a kidney transplant
Clinical isolate from a patient after a stomach surgery
Commensal isolates of the human skin
Oliveira and Cerca (2013)
Influence of different glucose concentrations in biofilm disassembly, under agitation or static conditions
TSB without glucose (TSB0 %G) (1 L) was prepared by mixing the following individual components: 17 g pancreatic digest casein (Alfa Aeser, Karlsruhe, Germany), 3 g papaic digest of soybean (Fluka, MO, USA), 5 g NaCl (ChemLab) and 2.5 g dipotassium hydrogen phosphate (ChemLab). In order to test the effect of varying glucose concentrations on biofilm disassembly, TSB0 %G was supplemented with different quantities of glucose to obtain the following final concentrations (v/v): TSB0.25 %G, TSB0.65 %G or TSB1.25 %G. Each medium was carefully added to 24 h-old biofilms and incubated at 37 °C for additional 24 h, without or with agitation at 120 rpm. The cells present in the biofilm bulk fluid were collected by careful aspiration. Biofilms were washed twice and then suspended in 1 mL of 0.9 % NaCl. Both bacterial cultures were sonicated for 5 s at 40 % amplitude using a 13 mm probe tip (Cole-Parmer 750-Watt Ultrasonic Homogenizer 230 VAC, IL, USA). This sonication cycle did not significantly affect cells viability as demonstrated before (Freitas et al. 2014). Thereafter, biofilms and the cells in the biofilm bulk fluid were quantified regarding their total biomass and the number of total or cultivable bacteria, as described below.
Quantification of bacterial biomass or bacterial cells
Bacterial biomass was determined by optical density (OD), at 640 nm of wavelength, as previously described (Freitas et al. 2014). The number of cultivable cells was determined by the standard colony forming units (CFU) counting. The number of total cells was assessed by flow cytometry (EC800, SONY, San Jose, CA, USA) using SYBR Green (Invitrogen, Glasgow, UK) and propidium iodide (Sigma, St. Gallen, Switzerland) staining as previously optimized (Cerca et al. 2011b).
Biofilm-released cells dynamics under static or agitation conditions
After discarding the spent medium and washing twice the preformed 24 h-old biofilms, 1 mL of TSB0.65 %G was carefully added. Immediately after, in half of the wells, the newly added TSB0.65 %G, together with the cells released from the biofilm due to the shear forces exerted by medium addition, was transferred into empty sterile wells. The cells in the biofilm bulk fluid were incubated, in the presence or absence of the originating biofilm, at 37 °C with shaking at 120 rpm or under static conditions. At different time points, a 20 µL aliquot was collected from both conditions and sonicated for 2 s at 20 % amplitude. The number of total and cultivable cells was determined, respectively, by flow cytometry and CFU counting. Four independent experiments with three technical replicates were performed.
Susceptibility of S. epidermidis populations to vancomycin, tetracycline and rifampicin
One mL of fresh TSB0.65 %G was added to the preformed 24 h-old biofilms and these were allowed to grow for additional 24 h at 37 °C with agitation at 120 rpm. At time points 0, 12 and 24 h, the bulk fluid of 10 individual biofilms was carefully collected by aspiration and pooled together. For comparative purposes, planktonic cultures obtained after 6 h (exponential) or 24 h of growth (stationary), as well as biofilm cells obtained by disrupting the established biofilms as described elsewhere (Cerca et al. 2011a), were also analysed. Cells from all populations were harvested by centrifugation, suspended in 0.9 % NaCl and sonicated for 5 s at 40 % amplitude. Finally, the number of total cells was quantified by flow cytometry and the concentration adjusted to 1 × 107 cells/mL in TSB0.25 %G with or without antibiotics. Antibiotics (all from Sigma) of three different classes, and with different mechanisms of action, were used at their peak serum concentrations: 40 mg/L of vancomycin (a cell wall synthesis inhibitor), 16 mg/L of tetracycline (a protein synthesis inhibitor) and 10 mg/L rifampicin (DNA-dependent RNA polymerase enzyme inhibitor) (Cerca et al. 2005a). These cells were incubated for 2 h, in 10 mL tubes, at 37 °C and agitation at 120 rpm. Thereafter, bacteria were collected by centrifugation, suspended in 0.9 % NaCl and sonicated for 5 s at 40 % amplitude for posterior CFU counting. This experiment was repeated three independent times with two technical replicates.
Reversibility of antimicrobial susceptibility phenotype of biofilm-released cells
The concentration of the cells in the biofilm bulk fluid obtained immediately after medium replacement (Brc.0) or after 24 h of incubation in the presence of the originating biofilms (Brc.24), was adjusted to 1 × 107 cells/mL in TSB0.25 %G after quantification by flow cytometry. Both cultures were transferred into 25 mL sterile Erlenmeyer and incubated, up to 4 h, at 37 °C with agitation at 120 rpm. At time points 0, 2 and 4 h, 20 µL aliquots were collected, counted by flow cytometry and their concentration adjusted to 1 × 107 cells/mL in TSB0.25 %G or TSB0.25 %G containing 40 mg/L of vancomycin. The susceptibility to vancomycin was then assessed under the same conditions described above. Three independent experiments were performed with two technical replicates.
Quantification of gene expression levels in S. epidermidis populations
Quantitative PCR (qPCR) analysis was performed, as described elsewhere (Franca et al. 2012), in order to determine the levels of transcription of genes involved in quorum sensing (agrB), biofilm accumulation (icaA) and dispersion (atlE, psmβ). In brief, after pooling together the bulk fluid of 10 biofilms, the 10 originating biofilms were washed twice and pooled together as well, in order to reduce the characteristic heterogeneity of this population (Sousa et al. 2014). Thereafter, 2 mL of planktonic (24 h stationary phase), biofilm bulk fluid or biofilm cells were collected by centrifugation and total RNA immediately isolated using FastRNA® Pro blue Kit (MP Biomedicals, Santa Ana, CA, USA). Henceforth, 0.5 µg of total RNA was reverse transcribed into complementary DNA in the presence of the enzyme RevertAid™ Premium reverse transcriptase (Thermo Scientific, Lisbon, Portugal). Random primers (NZYTech, Lisbon, Portugal) were used as priming strategy. The primers used for qPCR experiments were designed using Primer3 software (Rozen and Skaletsky 2000) having S. epidermidis RP62A complete genome (PubMed accession number NC_002976.3) or ATCC 12228 (PubMed accession number NC_004461.1) as a template. The size of the amplicon and the sequence of the primers used for the quantification of the transcripts encoding icaA, psmβ, agrB and 16 ribosomal RNA have been previously described (Franca et al. 2012). The sequence of the primers used for the detection of atlE transcripts, which yield a 180 base pair amplicon, are: Fw5′-GTAGATGTTGTGCCCCAAGG-3′ and Rv: 5′-TGGAAGAGGAACAGTTTGGAC-3. The qPCR run was performed using CFX96™ thermal cycler (Bio-Rad, Hercules, CA, USA) with the following cycling parameters: 10 min at 95 °C followed by 40 repeats of 5 s at 95 °C, 10 s at 58 °C and 20 s at 72 °C. The qPCR reaction was prepared by mixing 5 µL of iQ™ SYBR Green Supermix (Bio-Rad), 0.5 µL of each forward and reverse primers at 10 µM, 2 µL of ultrapure water and finally, 2 µL of 1:400 diluted cDNA. Melting curves were analyzed for the detection of unspecific products or primer dimer formation. The efficiency of the qPCR reactions (with all set of primers) was calculated using several dilutions of cDNA from biofilm samples. Fold change expression was determined by applying a variation of the Livak and Schmittgen method, the Efficiency∆Ct method, using 16 rRNA as reference gene and biofilm cells as control sample. Data analysis was based on at least four independent experiments.
Graphs and statistical analysis were performed with GraphPad Prism version 6 (CA, USA). Data are depicted as means and standard deviation. Unpaired T tests with Welch’s correction or One-way ANOVA with Tukey’s, Dunnett’s or Bonferroni’s multiple comparison tests were used when appropriate (the statistical test used is indicated in each figure legend). For gene expression studies, since a normal distribution of the values determined was not found, data are depicted as medians with interquartile range and statistical differences among populations were determined using Kruskal–Wallis with Dunn’s multiple comparison test. Differences among groups were considered significant when P was less than 0.05.
Glucose modulates biofilm disassembly in S. epidermidis, under agitation or static conditions
The majority of cells present in the biofilm bulk fluid result from both biofilm dispersion and detachment processes
Washing and growth medium addition procedures result in biofilm detachment
Biofilm-released cells are more tolerant to vancomycin and tetracycline than planktonic cells
Biofilm-released cells present a distinct gene expression profile than planktonic and biofilm cells
The release of cells from biofilms formed on the top of medical devices has been associated with the emergence of serious acute infections. However, despite the clinical relevance of these cells, little is known regarding their phenotype or interaction with the host immune system. Hence, more efforts towards the development of affordable models allowing the collection and posterior study of Brc cells are necessary. One of the most studied factors capable of initiating biofilm dispersion is the availability of essential nutrients (Hunt et al. 2004; Sauer et al. 2004). Glucose, an important carbon source for bacterial growth, is known to play a major role in S. epidermidis biofilm formation (Mack et al. 1992). Hence, we evaluated the influence of glucose in S. epidermidis biofilm dispersion. Interestingly, when no supplementary glucose was added to the culture medium, practically no differences were found between 24 and 48 h biofilms biomass, while with the highest glucose concentration tested, biofilm accumulation more than doubled, and disassembly was minimized. This was not surprising as Jäger and colleagues had previously shown that glucose-limiting conditions would result in a decrease in biofilm biomass over time (Jager et al. 2005), although in that study they did not address the cells in the biofilm bulk fluid. In addition, recently, our group has demonstrated that during the shift from biofilm to planktonic mode of growth important alterations in the expression of genes involved in glucose metabolism (icaA and rpiA) occurred having a significant impact on S. epidermidis physiological state (Cerca et al. 2014). Taken together, these data indicate that, depending on the concentration used, glucose can direct S. epidermidis biomass toward biofilm accumulation or disassembly. Importantly, this phenomenon was observed in six additional clinical and five commensal isolates, strengthening the transversal role of glucose in S. epidermidis biofilm accumulation/disassembly modulation. For further characterization of this model, we have selected TSB supplemented with 0.65 % of glucose since this is the most used concentration for biofilm studies.
Although we have shown that low glucose content and the presence of shear forces result in the disassembly of established biofilms, one possible criticism to this model is related to the difficulty in determining if the cells present in the biofilm bulk fluid were mainly originated from biofilm disassembly or proliferation of planktonic bacteria carried-over from the initial 24 h of growth. Since this may depend on the replacement of spent medium and the efficacy of the inherent washing steps, we became interested in assessing the impact of medium replacement on biofilm detachment. By washing the same biofilms sequentially, we demonstrated that not only the number of cells recovered in the washing suspension significantly decreased from the first to the third wash, but stayed essentially constant after that. As this step is highly influenced by the operator, this experiment was repeated by independent investigators and similar results were obtained. Importantly, the two washing steps performed before the addition of fresh medium removed practically all non-adherent cells (~99.01 to 99.99 %). Thus, it is reasonable to assume that the remaining cells (~1 × 105 cells/mL) would not be the main source of the growth detected in the biofilm bulk fluid, since the concentration of cells in the bulk fluid was determined to be at least 5 × 107 cells/mL (Fig. 3). Nevertheless, to properly address this question, the number of cells in the suspensions, in the presence or absence of the originating biofilms, was monitored up to 3 h. Our results showed that the cells obtained using this model were indeed originated from the biofilm attached to the bottom of the plate, as the number of cells in biofilm-dependent cultures increased more than 20-fold in the 3 h-period tested, while it only increased approximately 2-fold when growing in the absence of biofilms. Additionally, by performing the same experiment under static conditions (in the absence of shear forces), it was still possible to observe the release of cells from the biofilm as the number of cells in the biofilm-dependent cultures still increased 6-fold. This demonstrated that, despite the passive released of cells, an active mechanism was also taking place, although at much lesser extent. Taken together, these two experiments suggested that the proposed in vitro fed-batch disassembly model enables the collection of cells released from S. epidermidis biofilms by both passive and active mechanisms.
Finally, two sets of experiments were performed in order to provide further evidence of the origin of the cells in the biofilm bulk fluid. One of the major aspects differentiating biofilms from planktonic cultures is their characteristic tolerance to antibiotics and particular gene expression profile. Previously it was thought that soon after being released from a biofilm, bacteria would regain the characteristic planktonic cell’s sensitivity to antimicrobials (Chua et al. 2014; Lauderdale et al. 2010). However, opposing observations have been reported for different bacterial species (Liu et al. 2013; Rollet et al. 2009). Our results showed that S. epidermidis Brc were more tolerant than planktonic cells to the three classes of antibiotics tested. An identical tolerance profile was observed between the cells recently detached from the biofilm (Brc.0) and biofilms cells suggesting that these cells were originated from the established biofilm. Moreover, Brc retained their antimicrobial tolerance phenotype, despite being in the planktonic mode of growth. Curiously, after 12 h (Brc.12), a significant reduction in the susceptibility to tetracycline was observed. This effect was kept constant up to 24 h (Brc.24) after the initial disassembly. Interestingly, we also showed that if Brc are in the presence of the originating biofilm, they could maintain the high tolerance to antibiotics but, if centrifuged, suspended and transferred to an independent flask, they would eventually lose that tolerance, as observed for other bacterial species (Chua et al. 2014). This phenomenon is likely to be related to quorum-sensing regulation, which has been shown to be involved in tolerance to antibiotics (Singh and Ray 2014).
In addition to the antibiotic susceptibility, the quantification of gene expression identified additional particularities associated with Brc. Dai et al. showed previously that a specific group of S. epidermidis clinical isolates had enhanced biofilm dispersion and this is related to increased atlE activity (Dai et al. 2012). They proposed that biofilm dispersion was occurring, at least partially, due to autolysis of biofilm cells and could be mediated by the agr system. While we did not detect significant changes in agrB transcription levels, atlE transcription was significantly upregulated in Brc. Furthermore, in an earlier study, Yao et al. also characterized the transcriptome of stationary planktonic cells, biofilms and the cells in the biofilm bulk fluid (Yao et al. 2005). Similar to our study, they found that psmB was downregulated in biofilms. However, in their study, a significant difference between stationary planktonic and the biofilm bulk cells was detected, which was not observed in our study. Discrepancies in agr regulation have been reported in different strains (Stevens et al. 2009), but this is not surprising since several studies have linked agr to both biofilm accumulation and dispersion, as reviewed by Novick and Geisinger (2008).
Importantly, we have to acknowledge that one disadvantage of this fed-batch system for fundamental studies is that it’s not possible to physically separate the cells released by passive from the ones release by active mechanisms. However, this model is suitable for functional studies, as in vivo disassembly is likely to involve both dispersal and detachment mechanisms and, therefore, the population obtained in this model will more closely relate the population present in an in vivo situation of biofilm infection.
NC, MV and GP designed the experiments. NC, AF and VC performed the experiments. AF and NC drafted the manuscript. All authors critically read and contributed to the final version of the manuscript. All authors read and approved the final manuscript.
This work was supported by European Union funds (FEDER/COMPETE) and by national funds (Fundação para a Ciência e a Tecnologia-FCT) under the project with reference FCOMP-01-0124-FEDER-041246 (EXPL/BIA-MIC/0101/2013). The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit and the project FCOMP-01-0124-FEDER-027462 (RECI/BBB-EBI/0179/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AF and VC were supported by the FCT fellowships SFRH/BPD/99961/2014 and SFRH/BD/78235/2011, respectively. NC is an Investigador FCT.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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