Survey on Phenotypic Resistance in Enterococcus Faecalis: Comparison of Expression of Biolm Related Genes in Persister and Non Persister Cells

Background Currently, phenotypic resistance is a serious therapeutic challenge, and a denitive remedy has not been discovered yet. Biolm formation and persister cells are two well-studied phenotypic resistance, leading to the recalcitrance and relapse of different chronic infections. It appears that the presence of persister cells in biolm is the main factor in the relapse of infections and treatment failure. Thus, we aimed to evaluate the expression of biolm-associated genes in persister and non-persister E. faecalis isolates. microtiter plate assay 91 vancomycin-sensitive E. faecalis isolates (biolm producers) were screened by PCR to determine the presence of biolm-related genes (gelE, esp, and agg). The vancomycin-tolerant isolates were determined by MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biolm-related genes was evaluated in persisters and non-persister isolates of E. faecalis by real-time PCR assay.


Introduction
Enterococcus faecalis emerges as the most common hospital-acquired pathogen (1). A major attribute of enterococcal infections is phenotypic and genotypic resistance (2,3). The phenotypic resistance, recognized as a prominent achievement of bacteria, which dramatically enhances the tolerance against a variety of antibiotic classes (4). Currently, investigating the phenotypic resistance remains as a controversial concern that has received particular attention in microbiology.
Bio lm formation, the rst well-studied phenotypic resistance, is associated with a broad range of infections ranging from exogenous device-related to chronic tissue infections (5)(6)(7). Now, the question arises that what factor(s) do (es) increase the bio lm tolerance. The existence of persister cells de ned as dormant cells or sub-population with low-metabolic activity and also known as other phenotypic resistance in bio lms might represent the reason for the drug tolerance (8). Indeed, persisters are dormant (slow-growing) or growth-arrested phenotypic variants of normal cells in bacterial populations and are transiently antibiotic-tolerant cells. The presence of persister cells in bio lm is deemed as the main factor responsible for the relapse of infections and treatment failure (8- 10). Given that persisters are dormant and inert cells, they can be prey for the immune system factors. Bio lm formation would be a survival strategy for defenseless persister cells (11). Consequently, these observations raised the probability that persister isolates might show more proclivity to bio lm formation. The combination of these phenotypic resistances (bio lm/persister infections) are thought to underlie the dramatically enhances the antibiotic tolerance, treatment failure and relapse of chronic infections. Thus, research in this eld is valuable for public health (12). Herein, we aimed to investigate the antibiotic-tolerant bio lm to identify the persisters and evaluate the expression of bio lm-associated genes in persisters and non-persisters of E. faecalis isolates.

Bio lm Assay
Bio lm formation was performed by the microtiter plate (MTP) and crystal violet assay, as described elsewhere (14).
Brie y, E. faecalis overnight cultures were inoculated in TSB (Merck) containing 0.25% glucose. The culture was adjusted to obtain 0.5 McFarland standard, and bacterial suspension was diluted 1:100 in a fresh TSB. Thereafter, 200 µl of each bacterial suspension was distributed to three wells of the sterile 96-well polystyrene MTP, and the content was incubated at 37°C for 48 h. After three times washes with phosphate-buffered saline (PBS), unattached bacterial cells were removed and then left to dry. Bio lm was stained with 200 µL of crystal violet 2% (w/v) for 15 min, and wells were gently rewashed with water. After the wells were dried, 200 µL of 33% (v/v) glacial acetic acid was used to re-solubilize the dye bound to the adherent cells. The optical density (OD) value was measured at 545 nm using an ELISA reader (Biotech, USA), and the mean OD of the three wells was then calculated. The adherence capabilities of the isolates were classi ed into four categories; three standard deviations (SDs) above the mean OD of the negative control (broth only) was considered as the cut-off optical density (ODc). The isolates were categorized as follows: OD ≤ ODc, ODc < OD ≤ 2 × ODc, 2 × ODc < OD ≤ 4 × ODc and 4 × ODc < OD, implying that the bacteria were non-adherent, weakly adherent, moderately adherent, and strongly adherent, respectively.

Molecular Detection of Bio lm-Related Genes
Genomic DNA was extracted from the fresh overnight cultures of the isolates as explained before (15). PCR was carried out for the detection of bio lm-related genes (esp, agg and gelE) as previously described (16-18). The primers were synthesized by Macrogene (Macrogene, South Korea), and speci c primer pairs for different genes are shown in Table 1.
PCR was performed in a thermocycler (Bio-Rad, USA) with 25 µl of reaction mixture containing 2 µL of each forward and reverse primer, 12.5 µL of Master Mix 2× (Solis BioDyne, Estonia), 7 µL of nuclease-free water, and 1.5 µL of template DNA.
The PCR program is represented in Table 2. PCR products were analyzed by electrophoresis (Bio-Rad, USA) on 1% (w/v) agarose gels (SinaClon, Iran), and the ampli ed products were subjected to DNA sequencing by Bioneer (South Korea).

Minimal Bactericidal Concentration for Bio lm (MBCB) of E. faecalis Isolates
The MBCB of E. faecalis isolates was determined as per a method described formerly (19) with modi cations in the isolation of persister cells. In brief, 200 µL of each diluted culture (as previously noted) was distributed to the individual wells of 96-well MTP and incubated at 37°C for 48 h. The following day, the suspension was discarded and washed with sterile PBS. Afterward, 200 µl of the desired antibiotic dilution was added (diluted in normal saline, ranging from 0 to 2,500 µg/ml in 250-µg/ml increments).
After the incubation of the plates at 35°C for 24 h, the antibiotic was discarded, and then the bio lms were slowly sonicated. The content of each well was diluted with fresh media and cultured on blood agar (BA; Condalab, Spain) plates.

Persister Assay
Page 5/13 The enzymatic lysis method was carried out as previously described (20). Brie y, for persister isolation, 1 mL of overnight culture and 200 µl of the lysis solution (SinaClon, Iran) were added to a 15 mL falcon.

Expression of Bio lm-Related Genes
Quantitative real-time PCR (qPCR) assay was performed to determine the expression level of bio lm-related genes (esp, agg, and gelE) in persister and non-persister cells in triplicate. RNA was extracted with the RNA extraction kit (SinaClon, Iran). The quality and integrity of the total RNA assessed with a NanoDrop spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) were electrophoresed on 1% agarose gel. In following, cDNA was synthesized by oligo dT primers according to the manufacturer's instructions (cDNA synthesis kit; Takara, Tokyo, Japan). The speci c primers are presented in Table 1. To carry out qPCR, the 16srRNA gene was used as the internal reference gene. The reaction mixture, in a total volume of 25 µL, contained 2 µL of each forward and reverse primer, 12.5 µL of 2× real-time PCR Master Mix (SYBR Green; Fermentas, Lithuania), 7 µL of nuclease-free water, and 1.5 µL of cDNA. Moreover, negative controls including all the elements of the reaction mixture except the template cDNA were performed in every analysis, and no ampli ed cDNA product was ever detected. Real-time PCR system (CFX96 real-time PCR detection system, Bio-Rad, California, USA) was carried out in triplicate according to the following conditions: an initial holding at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 45 seconds, annealing at 58°C for 60 seconds, and extension at 68°C for 30 seconds.
The relative expression fold changes of mRNAs were calculated using the 2 −ΔΔCt method.

Bio lm Formation and detection of related genes
The Based on the results, esp, agg, and gelE genes were detected in 72 (79.12%), 74 (81.32%) and 91 (100%) isolates, respectively (Table 3). Fifty-six (61.53%) isolates had these genes simultaneously, and all the isolates included at least one bio lm-related gene. Coexistence genes, esp, agg, and gelE, were observed in 22 strong bio lm producer E. faecalis isolates. The relationship between the bio lm formation and related genes in E. faecalis isolates is presented in Table 4.

Minimum Bactericidal Concentration for Bio lms (MBCB)
About determination of the MBCB of E. faecalis isolates for identi cation of tolerant bio lm and persister cells, the cells resided in all of the bio lms indicated extremely high tolerance to vancomycin with MBCB > 2500 µg/ml.

Persister Assay
Although all bio lms had a high tolerance to vancomycin, but the enzymatic lysis assay displayed that 3 isolates (strong bio lm) were persister and other 88 isolates were non-persister cells. About persister killing assay, the results suggested that 5× MIC (5 µg/ml) of MMC could completely eradicate the planktonic persister cells at 3 h-interval incubation and led to the reduction of viable cells in the bio lm. However, it could not fully eliminate the bio lm at 24 h-interval incubation. We tested the 10 µg/ml but could not due to dispersal of bio lm. The MBC of MMC for E. faecalis persister cells in bio lm state was > 10 µg/ml.

Bio lm-Associated Gene Expression in Persisters and Non-Persisters
In following, we determined the expression of the esp, agg, and gelE genes in three persisters (Ef 2, Ef 18 and Ef 50) and seven non-persisters isolates (Ef 3, Ef 12, Ef 30, Ef 37, Ef 43, Ef 46 and Ef 58). Overexpression means that the isolates have fourfold increase in the expression level of genes compared with the reference strain E. faecalis ATCC 29212. The esp, agg, and gelE genes were overexpressed in three persisters isolates, but their expression did not change in seven non persisters isolates. In persister isolates, the agg gene had lower expression than the esp and gelE genes (Fig. 1).

Discussion
Bio lms are actually the architecture of an intelligent response made by certain bacteria as has long been considered as the main cause of their survival. In contrast to some hypotheses that antibiotics can readily traverse the bio lm layers and resulting in dispersion of them, it has been established in earlier studies, some bio lm-forming bacteria are able to temporarily tolerate extremely high concentration of antibiotics (22,23). Indeed, bio lm barriers are a type of scape strategy to circumvent the immune system factors (8, 12,24). Our results revealed that the expression of bio lm-related genes was higher in persister than non-persister isolates of E. faecalis. Moreover, most of E. faecalis isolates (95.8%) were vancomycin sensitive, which supports the nding of a meta-analysis study (25). The presence of gelE in all of our strains re ected the signi cant function of gelatinase in bio lm formation (26), which has previously been reported in the literature (27,28). Additionally, our ndings, in line with that of Tendulkar et al. (28), unmasked a direct link between the presence of esp gene with the bio lm intensity. Evidence has pointed out that persisters are the main factor underlying drug tolerance (8, 29,30). Interestingly, in the current study, 3 isolates were persisters but, the cells embedded in all of the bio lms could dramatically tolerate vancomycin (MBCB > 2500 µg/ml), which is consistent with the nding of Butini et al. (31) who indicated that the high concentration of vancomycin (i.e. >1024 µg/ml) is required for bio lm eradication. Of note, several investigations has evinced that Gram-positive cocci might have tolerance to vancomycin and other cell wall-effective antibiotics; besides, the molecular and phenotypic mechanisms of this bacterium are still unclear (32-34).
Abranches et al. (35) have reported that tolerance is related to the intracellular level of (p) ppGpp, and mutation in (p) ppGpp-regulator (relQ) leads to reduction of vancomycin tolerance. In another report, Abranches and coworkers (36) directed inherently tolerance to cell wall-effective antibiotic due to metabolic downshift by suppressing of genes that implicated in translation, transport, energy metabolism and binding. Furthermore, Brauner et al. (37) have highlighted discrepancy among resistance, persistence, and tolerance, which the last two has been classi ed as time-dependent and dose-dependent persistence as well as tolerance by slow growth and by lag, respectively. It would also be of interest to know that they have suggested that resistance and tolerance are the characteristics of whole bacterial populations, while persistence is a strategy to survive a subpopulation of a clonal bacterial population. Thus, given that only 3 persister were identi ed, increasing tolerance of bio lm-producing E. faecalis isolates against vancomycin would re ect the obscure mechanism of antibiotic tolerance of non-persister cells. The enzymatic lysis assay is the most appropriate method for persister isolation because it is not affected by environmental conditions and has a potential ability to discriminate between two types of persister cells. Using this method, we identi ed only 3 isolates as persister. Conventional antibiotic assay is dependent on environmental conditions, and more importantly, antibiotics can induce stress response and also leads to increasing persister cells (20).
Persister killing by the anti-cancer drug was subsequent focus of our study. MMC was effectively affected planktonic cells but could not thoroughly eradicated bio lm. Our result was in agreement with that of Kwan et al. (21) who denoted that MMC was unable to completely eliminate bio lm dispersal. Chowdhury et al. (38) have also determined that the anticancer drug such as cisplatin can fully eradicate bio lms which could be an effective treatment. Currently, numerous methods have been designed for the dispersal of bio lm dispersion but it should be noted that the dispersion of persistercontaining bio lm might leads to persister dissemination and recurrent infection that anti-persister treatment needs to be considered.
In this study, we evaluated the expression of bio lm-related genes (esp, agg, gelE) in persister and non-persisters of bio lmproducing E. faecalis isolates. We determined that the expression bio lm-related genes were higher in persister than nonpersisters E. faecalis isolates. There was a difference between the expression of these genes in the persister and nonpersister isolates, which can highlight this function in the persister cells. However, our result would con rm this hypothesis that bio lm is a strategy for survival of persisters, but further studies is necessitated to completely prove this hypothesis, because we only studied 3 persister isolates. If this hypothesis is completely proven, a hypothesis arises whether might be a common mechanism for the simultaneous regulation of these two types of phenotypic resistance. Several studies have been supported that toxin-antitoxin system play a key role in bio lm formation and persistence that could be an evidence to this hypothesis.

Conclusion
This study highlighted that the associated bio lm-related genes in the persister cells are expressed at a higher rate in comparative with non-persister cells.

Declarations
Author contribution