Introduction
The genus Citrobacter, whose members are known to cause significant nosocomial infection particularly involving the urinary and respiratory tracts [9], is a distinct group of aerobic, gram-negative, non-spore-forming, rod-shaped bacteria commonly found in water, soil, food, and the intestinal tracts of animals and humans [18]. At the same time, some biofilm-immobilized Citrobacter sp. have been used for the bioremediation of heavy metals via the activity of an acid-type phosphatase enzyme or their ability to accumulate heavy metals [6,13,20]. Nevertheless, biofilm formation is an important virulence trait of Citrobacter sp. pathogens in most instances.
Microbial biofilms are defined as matrix-enclosed bacterial populations, which adhere to each other and to surfaces [2]. Biofilm development is a multistep process involving an initial surface attachment, followed by cluster and microcolony formation, and subsequently resulting in a mature biofilm [33]. It has been proven that these processes are governed by the activities of regulatory networks or pathways, including chaperone-usher fimbrial assembly [35], quorum-sensing regulation [15,36], catabolite repression and stress response [4], that coordinate the gene expression of various motility, adhesion, and exopolysaccharide production processes in response to inter-and intracellular signaling molecules and environmental cues [25]. Although our understanding of the processes of biofilm formation remains incomplete, all the above processes and even biofilm architecture could be affected by nutritional and environmental parameters, including media [8], temperature, pH values [26], metallic ions, and bactericides [5,40].
In particular, metallic factors, such as CaCl2 [3,24,29], MgSO4 [26], MgCl2 [30], FeCl3 [41], ZnCl2 [16], CoCl2, Mn2(SO4)3, FeSO4 [3], ZnO [39], Al2(SO4)3, and CuSO4 [1], can influence biofilm formation in some microorganisms, but it is still not exactly clear how these factors interplay, or which factors dominate, or what their molecular mechanisms are. C. werkmanii BF-6 with a higher capacity for biofilm formation was recently isolated from industrial putrefaction in our laboratory. In the present report, we have expanded our analysis to study C. werkmanii biofilm formation under various conditions of environmental stress and nutrient status. The primary aim of this study was to determine the extent to which a single change in growth conditions affects the formation of a biotechnological monospecies biofilm and the possible molecular regulation mechanism of this process.
Materials and Methods
Bacterial Strain and Culture Conditions
The bacterial strain C. werkmanii BF-6 used in this study was originally isolated from industrial putrefaction and was maintained in our laboratory. It was routinely grown in Luria Bertani (LB) medium containing 1% (w/v) sodium chloride, 1% peptone powder (Oxoid, Hampshire, England), and 0.5% yeast powder (Oxoid), or M9 minimal medium [10] consisting of 1 g/l NH4Cl, 11 g/l Na2HPO4·7H2O, 3 g/l KH2PO4, 5 g/l NaCl, 4 g/l glucose, 120 mg/l MgSO4, and 10 mg/l CaC12, at 30℃ or 37℃ under static conditions throughout the experiments. All chemicals used in this study were reagent grade and purchased from Sigma (St. Louis, MO, USA) unless otherwise indicated.
Microtiter Plate Assay for Biofilm Formation
C. werkmanii BF-6 biofilms were formed on commercially available, presterilized polystyrene flat-bottomed 96-well microtiter plates (Corning Incorporated, Corning, NY, USA), based on previously described methods [32] with minor modifications. Briefly, a column of eight wells was filled with 200 µl of a bacterial suspension reaching an optical density (OD600) of 0.03. Negative control wells contained LB or M9 broth only. The plates were covered to prevent evaporation and incubated in a static incubator for the time indicated in each experiment at 30℃ or 37℃. Then, the contents were discarded and each well was washed three times with 250 µl of sterile water. The plates were vigorously shaken during each wash in order to remove all non-adherent bacteria. After drying for 30 min at room temperature, the plates were stained with 250 µl of 0.1% crystal violet (Shanghai Chemical Reagents Co. Ltd, China) for 40 min. Subsequently, excess stain was rinsed off by washing the plates three times with sterile water. After drying for 30 min again, 260 µl of 95% ethanol (Shanghai Chemical Reagents Co. Ltd) was added to each well to resolubilize the dye bound to adherent cells. The OD of each well was determined at 595 nm using a Multiskan GO Reader (Thermo Scientific, Waltham, MA, USA). Meanwhile, the OD of another plate cultured under the same conditions but untreated as above was measured at 600 nm using the same reader to evaluate bacterial growth.
Biofilm Development in Different Media
C. werkmanii BF-6 was grown in LB medium at 30℃ or 37℃ for 24 h. Then the culture was diluted to an OD of 0.03 at 600 nm and 200 µl of this cell suspension was transferred into 96-well microtiter plates. The plates were then incubated at 30℃ or 37℃ for 8 days. Biofilm development was evaluated by recording the OD595 at 1-day intervals using the above staining method. Meanwhile, planktonic cells were also transferred into a fresh microtiter plate to determine the bacterial density at OD600. The capacity of the cells to form a biofilm in M9 medium over time was also determined according to the method described in this section.
Effect of pH on Biofilm Formation
The pH of the LB medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 with 1 M HCl or 1 M NaOH before sterilization. Then, a C. werkmanii BF-6 culture was inoculated into sterile LB medium at different pH to give an OD600 of about 0.03. After culturing for 4days at 30℃ in 96-well microtiter plates, planktonic growth and biofilm formation under different pH conditions were measured according to the above-described methods.
Effects of Glucose and Sucrose on Biofilm Formation
In order to assay the effects of osmotic agents on biofilm formation by C. werkmanii BF-6, glucose and sucrose were chosen and added into LB medium so that their final concentration was 25, 50, 100, 200, 400, 800, or 1600 mM. The plates were then transferred to a static incubator and cultured for 4 days at 30℃. Subsequently, biofilm formation as well as planktonic growth of C. werkmanii BF-6 were measured according to the above methods. In addition, cultures without osmotic agents were considered as controls.
Effects of NaCl and KCl on Biofilm Formation
NaCl and KCl were also tested for their effects on biofilm formation. C. werkmanii BF-6 was cultured in LB medium supplemented with NaCl or KCl (50, 100, 150, or 200 mM) and assessed for its ability to form a biofilm using the methods described above. After culturing at 30℃ for 4 days, planktonic growth and biofilm formation were determined at OD600 and OD595, respectively, using a Multiskan GO reader. Cultures without additional salts were considered as controls.
Assessment of Biofilm Structure by Confocal Laser Scanning Microscopy (CLSM)
Biofilms grown on presterilized glass microscope slides were studied using CLSM as described elsewhere [29]. Briefly, 50 µl of C. werkmanii BF-6 cultures with an OD600 of 0.8 was inoculated into 50 ml plastic centrifuge tubes containing 2 ml of LB medium supplemented with 100 mM NaCl or 100 mM KCl. Control tubes contained only LB medium and bacterial suspension. The tubes were incubated at 30℃ for 2 or 4 days under static conditions. On the indicated day, the glass slides were gently taken out and washed with deionized water to remove loosely attached cells. The biofilm on one side was removed and the other side was stained with 5 µM SYTO9 dye (Invitrogen, Carlsbad, CA, USA) and 30 µM propidium iodide (Sigma Chemical Company), in the dark for 15 min at room temperature. Meanwhile, another slide treated with the above procedures was stained with FITC-ConA (Sigma). Then, the slides were washed again three times with deionized water and biofilms were visualized by CLSM (LSM 710 Zeiss, Jena, Germany).
Real-Time RT-PCR Analysis
C. werkmanii BF-6 was cultured at 30℃ in fresh LB medium or LB medium supplemented with 100 mM NaCl or 100 mM KCl in 96-well plates. After 4 days, planktonic bacteria and biofilms formed in each well were collected independently, and the total RNAs of the planktonic or biofilm bacteria were isolated using TRNzol Reagent (Tiangen, Beijing, China) and translated into cDNA using the Quantscript RT Kit (Tiangen) according to the manufacturer’s instructions. Ten-fold dilutions of the cDNA of samples were used as templates for assessing the transcriptional levels of nine genes via qRT-PCR with paired primers and 16S rRNA as an internal standard (Table 1). All qRT-PCRs were performed with SuperReal PreMix SYBR Green (Tiangen) on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) following the user’s guides. The transcription level of each gene in the cDNA was assessed using the 2-∆∆Ct method [19]. The ratio of the gene transcription level of C. werkmanii BF-6 biofilm grown in fresh LB medium or biofilm grown in 100 mM NaCl or KCl to that of the planktonic C. werkmanii BF6 from fresh LB medium was defined as the relative transcription level.
Table 1.aThe primers were designed using Beacon Designer 7.0 (Palo Alto, CA, USA) and synthesized at Beijing Genomics Institute (Guangzhou, China).
Statistical Analysis
Experiments were conducted in a completely randomized design and were repeated at least three times. All data are expressed as the mean ± standard deviation (SD). The data were subjected to one-way ANOVA followed by comparison of multiple treatment levels with the control using Fisher’s LSD test. For the biofilm assay (n = 8),a p value < 0.05 was taken as significant. All statistical analyses were performed with data processing system (DPS) software [34].
Results
Effects of External Factors on BF-6 Planktonic Growth and Biofilm Formation
Since the nutrient content of the growth medium has been found to regulate the development of biofilms by other organisms [8,11], we tested various nutrients as well as environmental conditions for their effects on the capacity of C. werkmanii BF-6 to form biofilms in the wells of microtiter plates. As expected, the growth of planktonic C. werkmanii BF-6 increased with increasing incubation time at 37℃ in M9 medium (Fig. 1A). Although there was a remarkable decrease in biofilm formation on the second day, the maximal biofilm formation in M9 medium was found on the sixth day followed by a marked decrease (Fig. 1A). Similarly, planktonic growth also increased with culture time except for a decline on the second day in LB medium at 37℃ (Fig. 1B). In contrast, the maximal biofilm formation in LB medium appeared on the second day followed by a significant decline by the fourth day. However, 2 days later, on day 6, another peak in biofilm growth was evident followed by a reduction in biofilm formation (Fig. 1B).
Nevertheless, the maximal values were observed on the second day for both planktonic and biofilm growth at 30℃ in M9 medium (Fig. 2A). In LB medium, after an increase from the beginning to the second day, planktonic growth reached a plateau phase until the eighth day (Fig. 2B). However, the greatest amount of biofilms appeared on the fourth day (Fig. 2B). The curves of bacterial growth and biofilm formation might reflect a biofilm developing process from attachment to maturity and finally detachment.
Fig. 1.Quantification of bacterial growth and biofilm formation in C. werkmanii BF-6 in static M9 medium (A) and LB medium (B) during incubation at 37℃. All assays were performed in triplicate
Taken together, C. werkmanii BF-6 biofilm formation was greater at 30℃ than at 37℃. In addition, LB medium is superior to M9 medium for C. werkmanii BF-6 biofilm formation. Thus, this medium and temperature were selected for the next series of analyses.
C. werkmanii BF-6 can grow in a broad pH range. However, excessively acidic and alkaline pHs inhibited planktonic growth except at pH 5.0 (Fig. 3). Interestingly, biofilm formation declined with increasing pH at 30℃ in LB medium (Fig. 3). Thus, an acidic environment is superior for biofilm formation in C. werkmanii BF-6. However, for the convenience of experimental operation and data analysis, the natural pH of LB medium was selected for the following analysis.
Fig. 2.Quantification of bacterial growth and biofilm formation in C. werkmanii BF-6 in static M9 medium (A) and LB medium (B) during incubation at 30℃. All assays were performed in triplicate.
Fig. 3.Influence of pH on bacterial growth and biofilm formation in C. werkmanii BF-6 grown in LB medium. All assays were performed in triplicate, and mean values and standard deviations are shown. Values having different letters are significantly different from each other according to Fisher’s LSD test (p < 0.05).
Effects of Glucose and Sucrose on Planktonic Growth and Biofilm Formation
As mentioned above, biofilm formation in C. werkmanii BF-6 was found to be affected by culturing time, media, temperature, and pH. Subsequently, the ability of C. werkmanii BF-6 to form biofilm in the presence of glucose and sucrose was also determined. Both planktonic growth and biofilm formation were reduced with increasing concentration of glucose and sucrose (Fig. 4). From a concentration of 25 to 400 mM, glucose (Fig. 4A) was more efficient than sucrose (Fig. 4B) at inhibiting the growth of bacterial cells and biofilms. At higher concentrations of both glucose and sucrose (800 and 1600 mM), however, most of the planktonic and biofilm growth was repressed (Fig. 4).
Fig. 4.Bacterial growth and biofilm formation in C. werkmanii BF-6 in LB medium supplemented with different concentrations of glucose (A) or sucrose (B). All assays were performed in triplicate, and mean values and standard deviations are shown. Values having ifferent letters are significantly different from each other according to Fisher’s LSD test (p < 0.05).
Effects of NaCl and KCl on Planktonic Growth and Biofilm Formation
Beside glucose and sucrose, two other osmotic agents, NaCl and KCl, were also chosen and evaluated for their effect on planktonic growth and biofilm formation in C. werkmanii BF-6. The results illustrated that biofilm formation was generally inhibited at higher concentrations of NaCl or KCl (Fig. 5). Across a range of NaCl concentrations (50 to 200 mM), planktonic growth was practically unaffected (Fig. 5A). In contrast, biofilm formation was significantly repressed at higher concentrations of NaCl (Fig. 5A), especially at 200 mM. Unlike NaCl, KCl inhibited the growth of C. werkmanii BF-6 at higher concentrations (150 and 200 mM; Fig. 5B). Additionally, the ability to form biofilm decreased with increasing KCl concentration (Fig. 5B), but the effect was less than that of NaCl, except at 50 mM. Taken together, these observations suggest that both NaCl and KCl negatively affect biofilm formation through an osmotic or metal ion effect. In addition, the efficacy of NaCl and KCl to inhibit biofilm formation was generally higher than that of glucose and sucrose. Thus, NaCl and KCl were selected for further study.
Fig. 5Bacterial growth and biofilm formation in C. werkmanii BF-6 in LB medium supplemented with different concentrations of NaCl (A) or KCl (B). All assays were performed in triplicate, and mean values and standard deviations are shown. Values having different letters are significantly different from each other according to Fisher’s LSD test (p < 0.05).
Fig. 6.CLSM images of C. werkmanii BF-6 biofilms grown in the presence of NaCl and KCl and stained with SYTO9 and PI. (A) and (D), control; (B) and (E), 100 mM NaCl; (C) and (F), 100 mM KCl. Scale bar = 50 μm.
Effects of NaCl and KCl on Biofilm Architecture
Since biofilm formation can be affected by NaCl and KCl, we speculated that biofilm architecture could also be affected by these two agents. Therefore, CLSM studies were carried out to examine the effects of NaCl and KCl (100 mM) on C. werkmanii BF-6 biofilm topography and architecture using glass slides. Live cells, stained with SYTO9, fluoresced green, whereas dead cells, stained with PI, fluoresced red. When cultured for 2 days, a typical biofilm could be observed on the surface of the glass slides in LB medium (Fig. 6). Although some biofilm formed in LB medium supplemented with 100 mM NaCl or KCl, biofilm architecture was sparse and more dead cells were found (Figs. 6B and 6C). Two days later (on day 4), the biofilm grew to maturity, especially in LB medium (Fig. 6D). Meanwhile, the biofilm in LB supplemented with NaCl was more extensive than the one in LB supplemented with KCl (Figs. 6E and 6F). FITC-ConA reacts with exopolysaccharide (EPS) matrices of the biofilm and fluoresces green under CLSM. Compared with the thick PGA matrix of the control, the EPS of C. werkmanii BF-6 treated with NaCl or KCl was thinner and less extensive than that of the control at both days 2 or 4 (Fig. 7).
Expression Levels of Genes Related to Biofilm Formation
Biofilm formation was significantly affected by both 100 mM NaCl and KCl (Fig. 5). Therefore, we selected biofilms cultured in 100 mM NaCl or KCl for RT-PCR experiments. A comparison of relative biofilm formation gene expression levels in C. werkmanii BF-6 with and without NaCl or KCl was illustrated in Fig. 8. Under normal conditions (in LB medium), the expression of all selected genes, except bssR and bssS, which remained stable, were upregulated between 1.13- and 6.35-fold in C. werkmanii BF-6 biofilms when compared with the expression levels in planktonic cells. In NaCl, bssS, csgE, mrkA, and mrkB (especially bssS, the expression of which was enhanced 23.90-fold) were upregulated; bssR, csgD, csgF, and mrkE (especially bssS, the expression of which declined 30.43%) were downregulated; and only bsmA showed no change. Meanwhile, bsmA, bssR, csgD, csgE, and mrkE (especially bsmA, the transcription of which was enhanced for 3.16-fold) were upregulated in KCl; only csgF was downregulated (by 24.87%); and bssS, mrkA, and mrkB remained stable. The results of RT-PCR suggest that different mechanisms may be launched in response to different osmotic agents.
Fig. 7.CLSM images of C. werkmanii BF-6 EPS grown in the presence of NaCl and KCl and stained with FITC-ConA (A) and (D), control; (B) and (E), 100 mM NaCl; (C) and (F), 100 mM KCl. Scale bar = 50 μm.
Fig. 8.Expression levels of C. werkmanii BF-6 biofilm formation genes under normal conditions and in the presence of 100 mM NaCl or KCl, as determined by RT-PCR. All assays were performed in triplicate, and mean values and standard deviations are shown. Values having different letters are significantly different from each other for a given gene according to Fisher’s LSD test (p < 0.05).
Discussion
It is well known that bacterial biofilms have a significant impact in medical, industrial, and environmental settings. Moreover, the successful establishment of biofilms in these settings can be affected by a number of environmental parameters [26].
Biofilm formation represents a survival strategy in a nutritionally limited environment that increases the transition of microorganisms from a planktonic to a sessile mode of life [7,26]. The reason for this phenomenon may be explained by the fact that surface colonization can provide many advantages, such as increased capture of nutrients that may be absorbed to surfaces [37]. It has been reported that some ingredients, such as proteins in a rich medium, may adhere to and then alter the surface, making attachment by bacteria difficult [27]. In this study, LB (a relatively higher-nutrient medium) was generally superior to M9 (a nutrient-poor medium containing 4 g/l glucose) for biofilm formation (Figs.1. and 2.), which suggests that the ability of C. werkmanii BF-6 to form biofilms was greater in higher-nutrient media. However, this study did not explore the mechanism by which poor nutritional parameters inhibit biofilm formation.
Besides nutritional conditions, temperature also affects biofilm formation [8,26]. Extreme temperatures negatively affected both bacterial growth and biofilm formation [26]. Although the planktonic growth of C. werkmanii BF-6 was not significantly different at 37℃ and 30℃, biofilm formation at 30℃ was obviously greater than that at 37℃ (Figs.1 and 2). These results suggest that a lower temperature is superior for biofilm formation by C. werkmanii BF-6.
C. werkmanii BF-6 biofilm formation was more sensitive than bacterial growth to pH changes (Fig. 3). In particular, the bacterial growth at pH 9.0 was comparable to that at pH 4.0, but biofilm formation was remarkably inhibited at pH 9.0 (Fig. 3). Similar results were also found by other researchers [26]. However, pH was unexpectedly found to have no effect on biofilm formation in other microorganisms [21]. Taken together, these comparisons suggest that pH effects may differ from one bacterial species to another in terms of establishing a biofilm, thereby enabling each bacterial species to efficiently colonize its preferred environment [26].
Some reports have proven that the cells within a biofilm are under greater osmotic stress than planktonic cells, and also that high osmolarity potentially inhibits biofilm establishment [26]. In this study, we also found that biofilm formation was significantly inhibited by higher concentrations of glucose and sucrose (Fig. 4). Environmental glucose inhibits multilayer biofilm formation in a variety of pathogenic and laboratory strains of Escherichia coli, and a number of clinical isolates of Enterobacteriaceae [14]. In E. coli, the repressive effect of glucose is exerted through catabolite repression via the cAMP-CRP system [12]. Sucrose is a substrate for the synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides [17]. In addition, biofilmassociated gene expression can also be affected by sucrose [28].
Although planktonic cells were not or only slightly influenced by NaCl or KCl, the biofilms were remarkably inhibited by these two osmotic agents when their concentrations were higher (Fig. 5) and this phenomenon generally occurred in a dose-dependent manner. Moreover, these results were confirmed by CLSM photographs (Fig. 6). Besides inhibiting biofilm formation, NaCl and KCl also influenced the expression of EPS (Fig. 7).
Biofilm formation appears to be regulated by signaling processes at the following steps: surface attachment, microcolony formation, and maturation of biofilm. In many bacterial systems, signal transduction proteins have been identified as being involved in regulating biofilm formation [31]. It has been proposed that a quorum-sensing-regulated gene, bsmA, which is involved in biofilm development and stress response, is engaged in fine-tuning the formation of cell aggregates at a specific point in biofilm development [15,36]. bssR and bssS appear to be global regulators of several genes involved in catabolite repression and stress response and regulation of the uptake and export of signaling pathways, including quorum sensing and the putative stationary-phase signal [4]. Meanwhile, the initial steps in biofilm development require the transcription of genes involved in reversible attachment and motility, and subsequent ones are involved in the irreversible attachment of bacteria [38]. The irreversible step might require the synthesis of adhesive organelles, such as curli fibers (csg genes) [16]. In addition, the expression of functional type 3 fimbriae (mrkABCD) is strongly associated with biofilm growth [22,23]. The expression levels of the selected nine genes, which are involved in the signal transduction of quorum sensing (bsmA), catabolite repression and stress response (bssR and bssS), attachment of bacteria (csgD, csgE, and csgF), and biofilm growth (mrkA, mrkB, and mrkE) were different in the presence of 100 mM NaCl or KCl (Fig. 8). Thus, we proposed that all of the above signal processes may be affected by these two osmotic agents. However, in summing up the results of RT-PCR, we found that only csgE and csgF were upregulated and downregulated respectively, at the same time in both 100 mM NaCl or KCl (Fig. 8), implying a conclusion that osmolarity can regulate biofilm formation and this effect may depend on the type of osmolyte in the environment.
In conclusion, a simple system for the analysis of biofilm formation by C. werkmanii BF-6 was utilized to evaluate different nutritional and environmental conditions. We found that biofilm formation by C. werkmanii BF-6 could be affected by the media, temperature, pH, and osmotic agents. The expression levels of some biofilm formation genes were also shown to be regulated by NaCl and KCl. A better understanding of the molecular aspects of adhesion and the biofilm phenomenon will certainly help in developing biofouling control strategies for practical treatment.
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