Stress conditions in the host induce persister cells and influence biofilm formation by S taphylococcus epidermidis RP 62 A

Introduction: Studies have demonstrated that pathogens react to the harsh conditions in human tissues by inducing mechanisms that promote survival. Methods: Persistence and biofilm-forming ability were evaluated during stress conditions that mimic those in the host. Results: Carbon-source availability had a positive effect on Staphylococcus epidermidis RP62A adhesion during hypoxia, accompanied by a decrease in pH. In contrast, iron limitation led to decreased surface-adherent biomass, accompanied by an increase medium acidification and lactate levels. Interestingly, iron starvation and hypoxia induced persister cells in planktonic culture. Conclusions: These findings highlight the role of host stress in the virulence of S. epidermidis.

The pathogenesis of Staphylococcus epidermidis in medical device-associated infections is mainly attributed to its ability to form adherent biofilms 1 , and more recently to its persistence 2 ; thus, both are important virulence factors.During the invasion of human tissues, microorganisms are exposed to different hostile conditions.
Biofilm development in staphylococci is a two-step process consisting of the adherence and accumulation and maturation phases.Staphylococcal biofilms can form in a polysaccharide intercellular adhesin-dependent manner.
Polysaccharide intercellular adhesin (PIA/PNAG) is a beta-1,6-N-acetylglucosamine polymer that is essential for bacterial cell-to-cell adhesion.The synthesis of PIA/PNAG is mediated by the intracellular adhesion (ica) operon, which encodes three enzymatically active membrane proteins (IcaA, IcaC, and IcaD) and one extracellular protein (IcaB) that are involved in polysaccharide synthesis 3 .Host environmental factors, including osmotic stress, oxygen limitation, temperature, and carbonsource availability [glucose, N-acetylglucosamine (GlcNAc), or glucosamine], may also influence PIA expression 4 .Studies have demonstrated that pathogens react to the harsh conditions encountered in the host by regulating their metabolism in response to the stress to survive.Many studies have focused on the effects of the host environment on the virulence of S. aureus.However, relatively few such studies on S. epidermidis have been reported.The absence of free iron in host tissues and the mechanisms of host iron restriction demand that pathogens develop strategies to combat the nutritional limitation.While some varying results have been reported for the genus Staphylococcus, many studies have demonstrated that iron positively regulates biofilm formation.In staphylococci, biofilm formation is a complex process that is strongly influenced by the external conditions encountered during its interactions with host tissues, including nutrient availability, osmolarity, temperature, and oxygen availability 5 .
One way that bacterial cells overcome stress conditions is by producing persisters.Persister cells are antibiotic-tolerant cells that increase dramatically in number from mid-exponential phase to stationary phase 6 , probably due to decreasing nutrient availability 7 .It is not clear how the stress conditions in the host induce persistence in S. epidermidis, but understanding the mechanisms involved in survival during infection is very important.The main goal of this research was to provide comprehensive insight into the sophisticated machineries of S. epidermidis virulence in response to the stress encountered in the host by studying the host-bacteria interaction in vitro.
The S. epidermidis strains used in the study included a known slime-producing INCQ 00650 strain, ATCC 35984 (RP62A), and a non-slime-producing strain, ATCC 12228, both were provided by CMRVS, FIOCRUZ-INCQS (Rio de Janeiro, RJ, Brazil).The bacterial strains were grown on Mueller-Hinton agar overnight at 36.5°C, and bacterial suspensions in 0.9% NaCl corresponding to a 0.5 McFarland standard (1.5 × 10 8 CFU mL -1 ) were used in the experiments.The bacterial cultures were subjected to different stress conditions, which can be encountered during the host infection, such as hypoxia and glucose and iron starvation.Iron deprivation was analyzed using brain heart infusion (BHI) medium supplemented with an iron chelator (50 µM bathophenanthroline disulfonate, BPS; Sigma-Aldrich).The control cells were incubated in prechelated medium supplemented with 3.5 µM Fe(NH 4 ) 2 (SO 4 ) 2 .Carbon-source availability was evaluated by culturing the cells on BHI, which contains residual amounts of glucose (carbon starvation), or BHI supplemented with 0.5% glucose (carbon supplemented).The cultures were also subjected to either hypoxic conditions by incubation in an environment with low levels of available oxygen (1% O 2 ) and 5% CO 2 or normoxia (20% O 2 ).In vitro biofilm production was assessed by a crystal violet (CV) assay in 96-well microtiter plates, as previously described for staphylococci 8 .Biofilm formation was determined over time and was correlated with the presence of each stressor.All assays were performed in triplicate, and comparisons between groups were performed using Student's t-test or one-way ANOVA.Differences among groups were considered significant when the p-values were less than 0.05.Lactate levels were assessed by VITROS ® dry technologies (Ortho-Clinical Diagnostics, Johnson & Johnson), and the pH was measured using a microelectrode coupled to a pH meter (Thermo Scientific Orion 2-Star Plus pH meter).
We observed biofilm accumulation with the addition of 0.5% glucose (Figure 1A), which was associated with the acidification of the culture medium and increased lactate levels in the supernatant (Figure 1B-C).It has been established that staphylococci growing in a biofilm shift their physiology toward microaerobic or anaerobic metabolism.Thus, the effect of carbon-source availability on S. epidermidis adhesion was assessed under low oxygen availability, and an increase in biofilm accumulation, along with medium acidification, was observed under hypoxia with low glucose (Figure 1).
Sequence similarity searches were performed to identify the main homologous proteins in the S. epidermidis RP62A strain that have been reported to play a role in the regulation of biofilm formation in S. aureus under low glucose, oxygen, or iron availability.The S. aureus protein sequences of interest were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/) in FASTA format.To infer homology, we used BLASTp, and sequences with expectation values (E-values) ≥10 -5 were considered to be similar.
In the host environment, human pathogens are exposed to fluctuating oxygen levels, and adaptive responses to these sudden changes often occur at the metabolic level 10 .Hence, we deemed it important to examine the effect of 0.5% glucose on S. epidermidis adhesion during low oxygen availability.The results showed biofilm accumulation as well as acidification during hypoxia.This acidosis may have been the result of extracellular lactic acid accumulation, which is produced by fermentative metabolism.In addition, our results showed that the presence of a carbon source had a positive effect on S. epidermidis adherence.Rbf and SarX are part of a regulatory cascade that promotes PIA-dependent biofilm formation in response to glucose in S. aureus.Interestingly, homologs for both proteins were identified in our in silico analysis (Table 1), suggesting that this regulatory system might also exist in S. epidermidis RP62A and that there is substantial overlap between the biofilm responses to glucose availability n S. aureus and S. epidermidis.
The sugar nucleotide UDP-GlcNAc, which is synthesized from glucose, is an important precursor for PIA production 11 .Thus, we speculated that the availability of glucose and oxygen might regulate biofilm formation in S. epidermidis via an icadependent pathway, which involves metabolism adaptation as a strategy to improve virulence.We would like highlight this result, since the induction of biofilm formation in response to higher glucose is particularly important in diabetic individuals, who are more susceptible to persistent infections and/or infections associated with catheters during hospitalization, which, given the limited number of effective antibiotics to treat such infections, makes treatment difficult.
The effect of iron limitation on biofilm formation by S. epidermidis was examined by using a static-culture biofilm assay with the iron-chelating compound BPS (50 µM).The addition of BPS led to a decrease in the surface-adherent biomass (Figure 2A), which was accompanied by a decreased pH and increased lactate in the supernatant (Figure 2B-C).Iron starvation has been reported as a requirement for metabolic shifts to anaerobic growth by pathogens in host environments because iron is an important cofactor for the 24 enzymes of the tricarboxylic acid (TCA) cycle.As shown in Figure 2A, S. epidermidis biofilm formation was restricted under ironlimiting conditions, followed by an increase in lactate release 3) or presence of 0.5% glucose (2 and 4).(B) The extracellular lactate concentration in the cell-free supernatant of the culture for the biofilm assay (A) was evaluated by the VITROS350 enzymatic test (Ortho-Clinical Diagnostics).(C) The pH was monitored by a microelectrode coupled to a pH meter.The data showed that both glucose and hypoxia significantly increased biofilm formation, extracellular lactate production, and medium acidification.The differences among groups were considered significant when the p-values were less than 0.05.Legend: 1-BHI with basal glucose in normoxia; 2: BHI with 0.5% glucose in normoxia; 3-BHI with basal glucose in hypoxia; 4-BHI with 0.5% glucose in hypoxia.
(Figure 2B).In S. aureus, the genes regulated by the ferric uptake regulator (Fur) are involved in a synchronized mechanism in response to iron deprivation that inhibits Fur repression and is reflected by increased iron acquisition and glycolysis 12 .Since the enzymes of the TCA cycle are downregulated during iron deprivation, the pyruvate produced by glycolysis is directed toward lactate production by lactate dehydrogenase, which leads to a decrease in the pH, favoring the shutdown of iron binding by transferrin and leading to increased levels of free iron in the host.Other S. aureus virulence factors are positively regulated by Fur under low-iron conditions, including Eap and Emp, which are extracellular proteins that promote adhesion to fibronectin, fibrinogen, vitronectin, and collagen.Although our in silico analysis did not reveal homologous genes in the S. epidermidis RP62A genome (Table 1), we found an ortholog of fur, which suggests that S. epidermidis may possess other Fur-regulated adhesins that promote the virulence of this strain.In contrast, Johnson et al. 13 demonstrated that the expression of the ica-operon was required for biofilm regulation under iron starvation.Nevertheless, the factors involved in iron-mediated adhesion, whether regulated by Fur and/or by the ica operon, have yet to be identified.Here, we performed a biofilm assay with two S. epidermidis strains, one known slime-producing strain, RP62A, and one strain with a deletion of the entire ica operon, ATCC 12228, and we showed that only the RP62A strain was able to adhere to the plate under variable glucose, oxygen, and iron availability.These environmental conditions are relevant to those encountered by S. epidermidis in vivo, The pH was monitored with a microelectrode coupled to a pH meter.The data showed that iron restriction significantly decreased biofilm formation, increased extracellular lactate, and reduced the pH of the medium.The differences among groups were considered significant when the p-values were less than 0.05.Legend: 1-BHI and 2-BHI with BPS.
suggesting that our experimental results correlate with what may be occurring during host-bacteria interactions.
S. epidermidis RP62A persister cells were isolated as described by Shapiro et al 9 .using vancomycin (Sigma).The cells were isolated over time and were correlated with the presence of each stressor.Briefly, a standard culture was used to prepare a 100 mL culture of S. epidermidis RP62A in BHI as described above.At different time points during the growth cycle (0-18 h), one sample was removed for the plating assay, and another aliquot was treated with the optimal concentration of vancomycin (50 µg mL -1 ).To enumerate the persisters, samples collected at each indicated time point were washed twice in 1 mL of cold BHI, centrifuged for 2 min at 10,000 × g, pelleted, and serially diluted in saline.Then, 10 μL aliquots of the dilutions were plated on BHI agar.The number of "persisters" is reported as CFU mL -1 ."Persisters" were defined as the cells that survived at 25 times the MIC of vancomycin (2 µg mL -1 ) for 12 h (stationary phase).Here, we used persister-derived isolates obtained after vancomycin exposure (50 µg mL -1 ) within 12 h to evaluate the influence of hypoxia and iron starvation on persistence.Persisters were not detected after planktonic growth under both low oxygen and iron availability conditions.However, a substantial number of persister cells remained viable after incubation of persister-derived isolates with vancomycin under hypoxia or iron deprivation compared with the number remaining after incubation with vancomycin alone (data not shown).Persisters are important as they have the potential to improve bacterial adaptation to environmental change, and may lead to recalcitrance and relapse of persistent bacterial infections 14 .In this study, we presented evidence that host stress  conditions induce the appearance of persister S. epidermidis RP62A cells, presumably via stringent response.This result corroborates the study by Mordukhova et al. 15 , who reported that the bacterial response to unfavorable environmental conditions, such as methionine starvation, enhances the persistence phenotype in Escherichia coli, while re-establishment of methionine reduced persistence.Here, we described, for the first time, that both oxygen and iron starvation upregulated persister cells, while Fe supplementation and normoxia decreased cell tolerance.Therefore, these results suggest that the stress response induced in host tissues could be a mechanism that leads to high antibiotic tolerance and an increase in resistant strains, contributing to the increased number of chronic infections, which is an emerging problem.Our findings advance our understanding of host-bacteria interactions and present evidence regarding the role of host stress conditions in the regulation of relevant virulence factors of S. epidermidis, namely, biofilm formation and persistence.However, more comprehensive studies are necessary to explain the sophisticated machineries used by S. epidermidis to establish an infection in the host.

FIGURE 1 :
FIGURE 1: Quantification of S. epidermidis biofilm formation, lactate production, and medium pH changes in response to hypoxia and the addition of 0.5% glucose.(A) A biofilm assay was performed in the absence (1 and 3) or presence of 0.5% glucose (2and 4).(B) The extracellular lactate concentration in the cell-free supernatant of the culture for the biofilm assay (A) was evaluated by the VITROS350 enzymatic test (Ortho-Clinical Diagnostics).(C) The pH was monitored by a microelectrode coupled to a pH meter.The data showed that both glucose and hypoxia significantly increased biofilm formation, extracellular lactate production, and medium acidification.The differences among groups were considered significant when the p-values were less than 0.05.Legend: 1-BHI with basal glucose in normoxia; 2: BHI with 0.5% glucose in normoxia; 3-BHI with basal glucose in hypoxia; 4-BHI with 0.5% glucose in hypoxia.

FIGURE 2 :
FIGURE 2: Quantification of S. epidermidis biofilm formation, lactate production, and medium pH in response to iron starvation.(A) A biofilm assay was conducted in BHI medium supplemented with an iron chelator (BPS), and control cells were incubated in previously chelated medium supplemented with Fe (NH 4 ) 2 (SO 4 ) 2 .(B) The extracellular lactate concentration in the cellfree culture supernatant during the biofilm assay was evaluated by an enzymatic test (VITROS350; Ortho-Clinical Diagnostics).(C)The pH was monitored with a microelectrode coupled to a pH meter.The data showed that iron restriction significantly decreased biofilm formation, increased extracellular lactate, and reduced the pH of the medium.The differences among groups were considered significant when the p-values were less than 0.05.Legend: 1-BHI and 2-BHI with BPS.

TABLE 1 :
Identification of S. epidermidis RP62A proteins with similarity to S. aureus proteins involved in biofilm formation by BLASTp.