Development of in Vitro Resistance to Daptomycin in Enterococcus Faecium and Estimation of Its Fitness Cost

Daptomycin has broad-spectrum antibacterial activity against Gram-positive pathogens, but recent studies have revealed cases where daptomycin has failed to treat multidrug-resistant bacteria, such as vancomycin-resistant Enterococcus faecium. However, the resistance evolution of E. faecium to daptomycin in vitro and tness cost remain unclear. In this study, we sought to analyze the resistance development and mechanism of E. faecium to datomycin, and futher to investigate the relationship between daptomycin resistance and tness cost.


Abstract Background
Daptomycin has broad-spectrum antibacterial activity against Gram-positive pathogens, but recent studies have revealed cases where daptomycin has failed to treat multidrug-resistant bacteria, such as vancomycin-resistant Enterococcus faecium. However, the resistance evolution of E. faecium to daptomycin in vitro and tness cost remain unclear. In this study, we sought to analyze the resistance development and mechanism of E. faecium to datomycin, and futher to investigate the relationship between daptomycin resistance and tness cost.

Methods
To investigate the development of daptomycin resistance in E. faecium, 6 daptomycin-susceptible (DAP-S) clinical isolates, including 3 vancomycin-resistant E. faecium (VRE) and 3 vancomycin-susceptible E. faecium (VSE), were exposured to daptomycin in vitro by serial passage experiment. Then the different resistance mechanisms of daptomycin-resistant (DAP-R) mutants were analyzed by polymerase chain reaction (PCR), cytochrome C binding assay and transmission electron microscopy. Furthermore, we also estimated the changes of tness cost among each highly DAP-R mutants by bacterial growth curve measurement, in vitro competition experiments, infection model of Galleria mellonella larvae and bio lm formation assays.

Results
In vitro, a total of 21 DAP-R mutants with minimal inhibitory concentration (MIC) of 4 to 512 μg/mL were obtained, and these mutants carried more than one mutation of LiaFSR and YycFG system encoding genes. More positive charges were detected among highly DAP-R mutants than parent isolates, and the cell walls of SC1174-D and SC1762-D mutants were remarkly thicker than those of the parent isolates. In comparison with parent isolates, besides, the growth, competition ability and virulence were signi cantly reduced, while the bio lm formation capacity was markedly elevated among each highly DAP-R mutants.

Conclusions
Our ndings suggest that E. faecium isolates are able to rapidly acquire DAP resistance in vitro through different dynamic resistance mechanisms, which often accompany by signi cant tness cost. Intriguingly, DAP and glycopeptide antibiotics may present collateral-sensitivity during E. faecium acquired DAP resistance in vitro.

Background
Enterococcus faecium is an ubiquitous bacteria in nature. It is also an opportunistic pathogen that can cause severe hospital infections, like bacteremia, as well as less severe infections, like urinary tract infections, in the hospital and community settings [1]. It is reported that infections caused by E. faecium is ~20% and is increasing gradually [2]. Moreover, E. faecium, as a member of the 'ESKAPE' pathogens which have an ability to escape the biocidal action of antimicrobial agents, can develop antibiotic resistance and cause deadly clinical outbreaks, making signi cant challenges to the health care system [3]. In the past, vancomycin and teicoplanin were considered to be the most effective glycopeptides for the treatment of enterococcal infections. However, widespread use and unreasonable use of antibiotics have increased the emergence and prevalence of vancomycin-resistant E. faecium (VRE), which poses a great threat to human public health because of high mortality rate [3,4].
Daptomycin (DAP) is a cyclic lipopeptide which targets bacterial cell membranes [5]. In 2003, the US Food and Drug Administration approved its use for treatment of complex skin and soft tissue infections, infective endocarditis and osteomyelitis [6], owing to its wide range of antibacterial activities to most Gram-positive pathogens, including methicillin-resistant Staphylococcus. aureus and vancomycinresistant enterococci [7]. Hence, DAP has been recognized as one of the last line antibiotics against multidrug-resistant Gram-positive pathogens, like VRE [8]. In recent years, an increase number of treatment failure cases with DAP has been reported abroad [9,10].
To date, most studies in the eld of DAP resistance focused on Staphylococcus aureus and Enterococcusfaecalis [11,12], only a few focus on E. faecium. Several resistance mechanisms to DAP in Gram-positive pathogens, like Staphylococcus aureus and Enterococcus faecalis, have been identi ed, including the alteration of the cell surface charge and cell membrane components [5,13]. Mutations in encoding genes of three-component regulatory system LiaFSR, two-component regulatory system YycFG, cardiolipin synthase (Cls) and glycerophosphoryl diester phosphodiesterase (GdpD) have been shown responsible for the DAP resistance [14]. Additionally, increased thickness of the cell-wall and abnormal septations also contribute to DAP resistance in Gram-positive pathogens [15].
At present, it is not clear whether E. faecium can acquire DAP resistance in vitro, if so, by what pathways or mechanism. And what changes in biological characteristics of E. faecium will occur after obtaining daptomycin resistance. In this study, hencein, we investigated the development of DAP resistance by serial passage experiment in vitro, and further analyzed the reisistance mechanism and tness cost. We hope to deepen the understanding of the process and pathways of resistance to DAP in E. faecium, and to provide a new insight to mitigate or prevent DAP-R E. faecium that may appear in China in the future.

Bacterial strains and growth conditions
We collected and identi ed 3 VRE strains (SC1174, SC1379, SC1762) and 3 VSE strains (SC1543, SC1706, SC1726), which were daptomycin-susceptible (DAP-S) (DAP MIC=2.0 μg/mL) strains, isolated between 2017 and 2018 from the First A liated Hospital of Wenzhou Medical University, a comprehensive teaching hospital in China. These isolates were used as parent strains to screen DAP-R mutants in vitro. Isolates were stored at -80 ℃, and incubated on the blood agar plates at 37℃ for 18-24 h before using.
Serial passage experiment selection of DAP-R E. faecium To investigate the development of daptomycin resistance in E. faecium, DAP-S strains, including 3 VRE isolates and 3 VSE isolates, were used to select DAP-R variants by serial passage experiments in vitro, according to prior published methods with some modi cations [16]. Speci cally, single pure colony was randomly selected, and then inoculated in 3 mL fresh Luria Broth (LB) with 50 μg/mL calcium (The LB used in this study contains 50 μg/mL calcium, unless otherwise speci ed) and allowed to grow to logarithmic period, 30 μL overnight cultures were transferred to 2.97 mL fresh LB with graded concentrations of DAP: 1/2×MIC, 1×MIC, 2×MIC and 4×MIC. And then the cultures were incubated at 37°C overnight without shaking. Strains were cultured at each concentration for 3 passages before their exposure to the next concentration. This process lasted 36 days.
Genetic stability testing and determination DAP susceptibility Colonies from the highly DAP-R mutants of each parent isolates were picked, inoculated without DAP for 10 passages, and then MIC value against DAP was determined again to check the stability of the phenotype using broth microdilution method. The result was interpreted according to the latest guideline of Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute (CLSI, 2020) (susceptible, ≤4 μg/mL; resistant, ≥8 μg/mL). E. faecium ATCC 29212 was used as a control strain.

Homologous analysis
In this experiment, clone correlation analysis was also performed by multilocus sequence typing (MLST) for parent strains and all DAP-R mutants. The sequences of seven housekeeping genes (gdh, purK, pstS, atpA, adk, ddl and gyd) were ampli ed and sequence types (ST) were assigned based on the MLST databases (https://pubmlst.org/efaecium/). Primers were listed in Table S1 (see Additional le 1).

Antimicrobial susceptibility testing
Agar dilution method was carried out to measure the MIC of commonly used antibiotics among parent isolates and highly DAP-R mutants, including ampicillin, cipro oxacin, levo oxacin, nitrofurantoin, penicillin, erythromycin, linezolid, tetracycline, vancomycin and teicoplanin. The results were interpreted according to the criteria established by CLSI. E. faecium ATCC 29212 was used as a control strain. The experiment was repeated in triplicates.
PCR ampli cation and DNA sequencing DNA was extracted from parent strains, all mutants and ATCC 29212 strain using Biospin bacterial genome DNA extraction kit (shanghai boyun biotech co.,ltd), and used as the template in polymerase chain reactions (PCR). Genes were ampli ed with speci c primers [17,18]: (i) LiaFSR three-component regulatory system encoding gene liaF, liaS and liaR; (ii) YycFG two-component regulatory system encoding gene yycF and yycG. Primers were listed in Table S1 (see Additional le 1). The positive PCR products were sequenced by Shanghai MajorbioBio-Pharm Technology Co. (Shanghai, China). The sequencing results were compared with parent strains and the standard strain Enterococcus faecium DO (Accession number: CP003583) using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

Cytochrome C binding assay
The relative positive surface charges of E. faecium strains were determined by previously published methods [12]. Brie y, bacterial suspensions of the parent strains and the highly DAP-R induced strains were allowed to regrow to logarithmic phase after overnight cultures were diluted 1:100 in 20 mL fresh LB. Then cells were washed twice with MOPS (morpholinepropanesulfonic acid) buffer (20 mM, pH=7.0) after centrifugation, and the bacterial suspension was adjusted to an OD 600 of 1.0. Cytochrome C, a positively charged molecule, was prepared in MOPS (5 mg/mL), and then 100 µL was added to 900 µL of bacterial suspension. After 30 min at room temperature, samples were centrifuged at 12,000 g for 5 min. Supernatant free cytochrome C were recovered and the OD 530 was measured spectrophotometrically.
Less free cytochrome C that was detected in the supernatant means more net positively charge on the bacterial surface. The experiment was repeated at least three times.

Transmission electron microscopy
Two parent strains (SC1174 and SC1762) and their last generation of induced strains (SC1174-D and SC1762-D) were randomly selected to measure the cell wall thickness using TEM as described previously with some modi cations [19]. After overnight culture in LB, 1 mL cultures were pelleted and washed 3 times using 0.1 M Millonig's phosphate buffer. The pellet was then resuspended in 1 mL glutaraldehyde in Millonig's phosphate buffer, and further processed in the Electron Microscope Room of Wenzhou medical University. For each strain, Cell wall thickness of at least 25 cells was measured at ×40,000 and 60,000 magni cation, respectively. P values of <0.05 indicated statistical signi cance.

Growth kinetics measurement
Growth kinetics was assessed for all ancestral and the the highly DAP-R mutants. After overnight growth in LB at 37°C for 24 h, cultures were diluted into 20 mL LB with a nal bacterial suspension concentration of 0.5 McFarland standard, and then incubated at 37°C for 24 h with agitation at 200 rpm. Growth curves were generated by plotting OD 600 over time. The experiment was repeated in triplicates, and the averages were used for estimating growth parameters.

In vitro competition experiments
In vitro competition experiments were performed in triplicates to measure tness cost between parent strains and last generation of DAP induced strains [20]. Exponentially growing cells of the DAP-S parent strains and DAP-R mutants were adjusted to 1×10 3 colony-forming units (CFU)/mL, and then equal volumes were combined, thus the initial ratio of the strainpairs was in nitely close to 1:1. Then 1 mL of the mixture was added to 19 mL LB broth and cultured at 37°C with agitation at 200 rpm for 24 h, which corresponds to approximately 20 cell generations. Serial 10-fold dilutions were plated in duplicate on DAP-free LB agar and LB agar containing 4 μg/mL DAP (This concentration inhibited the growth of all parent strains). After overnight incubation at 37°C for 24 h, the CFU of the DAP-R colonies and parent strains were counted, respectively. The competition index (CI) means adaptive difference, which was de ned as the ratio between the CFU of the DAP-R strains and the DAP-S strains. The CI values were calculated for each independent competition assay, and the median values were calculated too.

Crystal violet bio lm assays
Bio lm-bound crystal violet was quanti ed for parent strains and the last generation of DAP-R mutants. For the bio lm formation assays, isolates were incubated at 37°C with shaking at 200 rpm overnight, and culture suspensions were diluted 1:100 into fresh LB, then inoculated (200 µL per well) into 96-well polystyrene microtiter plate for 48 h at 37°C. After incubation, planktonic cells were removed, and 200 µL 0.1% crystal violet was added to each well. The plate was incubated for 20 min at 37°C before it was washed twice with sterile water. Finally, 95% ethanol was used to dissolve the stained bio lm and the absorbance at OD 600 was measured for quanti cation. Additionally, untreated well supplemented with sterile LB was served as control. All experiments were conducted in triplicates.

Galleria mellonella killing assays
Infection model of Galleria mellonella larvae was used to compare the virulence differences between parent strain and the last generation of DAP-R mutants as described previously with slight modi cations [21]. Twelve caterpillars weighing between 200 and 250 mg were randomly selected for each isolate. Bacterial suspension or normal saline (NS) were injected into the last left proleg by 25 μL Hamilton precision syringe. Larvae were injected with 10 μL of bacterial suspension containing 10 8 CFU/mL as an experimental group, and injected with nothing or 10 μL NS as a control group. Worms were incubated at 37°C in the dark, and their survival or mortality rates were observed and recorded every 24 h over a period of 6 days to plot killing curve. Those worms that did not move when touched and their body colors were dark brown were considered dead. The result of infection model was rapidity and extent of mortality of Galleria mellonella, as assessed by Kaplan-Meier analysis and Logrank test. All experiments were performed in 3 independent replicates.

Statistical analysis
Statistical analysis of growth rate was performed with the GraphPad Prism 8.0 software using one-way analysis of variance (ANOVA). The independent Student's t-test (two-tailed) was performed using SPSS software version 26.0 for statistical analysis, P values of <0.05 were considered as signi cance.

DAP-R E. faecium mutants
To identify the development and pathways of DAP resistance among E. faecium strains, 3 VRE isolates and 3 VSE isolates were exposed to graded levels of the DAP, with a starting concentration of 1 μg/mL. As shown in Table 1, a total of 21 mutants from the 6 original strains were obtained. The resistance of mutants to DAP was not changed after passaging without DAP for 10 days in LB, and the MIC of these strains against DAP enhanced by 16-256 folds (Table 1). Besides, mutants with low level of daptomycin resistance were isolated for the rst time on the fourth day of daptomycin induction, and we also obviously observed that the resistance of mutants to daptomycin increased gradually with the prolongation of DAP exposure day (Figure 1), demonstrating that resistant mutants can be rapidly selected by serial passaging across sub-lethal gradient.
Moreover, the results of homologous analysis showed that the ST types of SC1174, SC1379, SC1762, SC1543, SC1706 and SC1726 were ST761, ST78, ST976, ST32, ST1025 and ST32, respectively ( Table 1). The ST type of the parent strains was consistent with that of the respective induced strains, which illustrated the parent strains and their induced strains had high homology.

Antimicrobial susceptibility testing pro les
The agar dilution method was used to determine the MIC of both parent strains and derived strains, according to the latest guideline of CLSI. As shown in Table 2, after acquired daptomycin resistance, the resistance of DAP-R mutants to antibiotics did not change except SC1762-D mutant. In contrast to the parent strain SC1762, SC1762-D mutant was highly sensitive to vancomycin and teicoplanin, which presented collateral-sensitivity that bacteria acquiring resistance to one antibiotic can give rise to sensitivity to other drugs during adaptive evolution [22]. The MIC of vancomycin dropped from 512 to 2 μg/mL, and teicoplanin dropped from 128 to below 2 μg/mL, respectively. LiaFSR and YycFG systems encoding genes mutations were related to DAP resistance To investigate various DAP resistance evolutionary routes, we analyzed the gene mutations of liaFSR and yycFG by PCR and sequencing. Different gene mutations were observed in different mutants, compared with parent strains and standard strain Enterococcus faecium DO (Figure 2). Speci cally, mutations of liaS, liaR and yycF genes were observed among SC1174 induced strains, it should be noted that mutations in liaS and yycF genes only occurred in the last generation induced strain SC1174-D with DAP MIC of 64 μg/mL. Similarly, the accumulation of liaS gene mutations was also found in the last generation induced strain SC1379-D. For SC1762 induced strains, mutations were detected in all genes except liaR, and accumulation of genes mutations was detected in liaF, yycF and yycG. For SC1543 induced strains, liaF, liaS and yycG genes mutations were noticed. SC1706 induced strains mainly carried liaR and yycG genes mutations. And the mutations of liaS and yycF genes were also found in SC1726 induced strains.
Increased cell surface charge and cell-wall thickness mediated DAP resistance DAP resistance mechanisms were also analyzed by cytochrome C binding assay and thickness of the cell-wall assays. cytochrome C binding assays revealed that DAP-R mutants had signi cantly increased positive charge on surface relative to parent strains except SC1379-D (P <0.05) (Figure 3). Furthermore, we found that SC1174-D mutant strain (55.09 ± 6.55 nm) exhibited thicker cell-wall than its parent strain SC1174 (19.97 ± 5.26 versus, P <0.0001) ( Figure 4A and Figure 4C). Similarly, SC1762-D mutant strain (46.456 ± 9.08 nm) presented signi cantly thicker cell-wall than its parent strain SC1762 (18.21 ± 3.36 nm, P <0.0001) ( Figure 4B and Figure 4C). Noticeably, the septum of cell-wall was obviously thickened both in SC1174-D and SC1762-D mutant strains compared to their corresponding parent strains.
E. faecium showed tness costs after acquiring DAP resistance To assess whether the evolution of DAP resistance strains was accompanied by associated tness costs, we analyzed bacterial growth curves, bio lm formation ability and competition experiments for the last generation mutants. Growth rate of DAP mutants slowed down and the bacterial density was lower than their original strains (All P <0.001) ( Figure 5). The results of competition experiments showed that DAP mutants had a marked decrease in tness. The greatest decrease was observed for the SC1726 strain, with a median CI of 0.021, followed by the SC1379, SC1762, SC1706, SC1174 and SC1543 isolates, with median CI of 0.077, 0.096, 0.098, 0.111, 0.141 and respectively ( Figure 6A). Moreover, the bio lm forming ability of tested DAP-induced strains was signi cantly higher than corresponding parent strain, especially SC1706-D (P <0.05) ( Figure 6B).

Infection model of Galleria mellonella larvae
To evaluate the virulence changes of induced strains, an infection model of Galleria mellonella larvae was constructed. Compared with the corresponding DAP-S strains, at 6 days post-infection, the mortality of larvae was lower for DAP-R mutants (P <0.001), and no mortality was observed in the control injected with NS ( Figure 7).

Discussion
Application of antibiotics has played a crucial role in controlling and prevention of bacterial infections in humans, but with a wide range of continuous and inappropriate use of antibiotics, bacterial resistance to antimicrobial agents has become increasingly common, which poses a serious threat to global public health [23]. Nevertheless, under the pressure of antimicrobial selectivity, the evolution of bacterial resistance to antibiotics largely depends on the changes of genomics background and adaptability. Thus, better understanding of the development and evolutionary trajectories of DAP resistance may potentially inform effective therapeutic strategies in the future.
Experimental evolution, like serial passage experiment in vitro, is an e cient tool to investigate the effects of environment and inherent genetic factors on speci c phenotype changes during evolutionary adaptation, and to provide exact insights into the course of adaptation to antimicrobials, the frequency of mutations, tness costs associated with resistance, and the predictability of evolutionary trajectories and adaptive pathways [24].
In our study, 3 VRE and 3 VSE isolates were selected as parent isolates to evolve resistance to DAP in vitro. We found the DAP MIC of parent isolates was signi cantly and rapidly enhanced, and DAP-R strains were rst detected only 4 days after exposure to daptomycin, indicating that E. faecium was easy to develop DAP acquired resistance in vitro. Therefore, it is necessary to take preventive and control measures to deal with DAP-R E. faecium that may appear in China in the future.
Previous study has been reported that laboratory evolution of bacteria to different antimicrobials can decrease or enhance the sensitivity to multiple other antimicrobials agents, called cross-resistance and collateral sensitivity, respectively [25]. Cross-resistance was not detected between DAP and other antimicrobials agents. But, intriguingly, it was surprising that DAP evolved resistance exhibited collateralsensitivity to vancomycin and teicoplanin in SC1762-D. These data remind us that once DAP is invalid after a period of treatment in clinic, the sensitivity of glycopeptides should be paid close attention to, or the combination of DAP and glycopeptides may be an effective measure to deal with VRE strains.
Previous studies have shown that mutations in LiaFSR, a three-component regulatory system controlling cell-envelope stress response, and in YycFG, a two-component regulatory system regrating cell envelope homeostasis, were directly linked with the emergence of daptomycin (DAP) resistance in enterococci [14,26]. Here, mutations in LiaFSR and YycFG systems encoding genes were found in DAP-induced strains, which were consistent with Munita JM et al. previous work which showed that mutations in these systems could be a pivotal initial event in the development of DAP resistance. [18]. But unfortunately, the same gene mutation sites were not observed in all the induced strains, which may explain the diversity and dynamics of daptomycin resistance evolution pathway in E. faecium.
Intriguingly, the occurrence time of genes mutations was not consistent in different strains, indicating that gene mutations were related to daptomycin exposure time. For example, liaS gene mutations were rstly detected in the rst generation of SC1762 and SC1726 induced strains on the fourth day of daptomycin induction, while it was not detected until the last generation of SC1174 and SC1379 induced strains after the 36 days of daptomycin induction. Moreover, it was obvious that the frequency of genes mutations was different among LiaFSR and YycFG systems, the mutation frequency of liaS gene was the highest, followed by yycG and liaF genes, and nally yycF and liaR genes, which suggested that different genes played different roles in the evolution of daptomycin resistance. Contrary to earlier report, liaS and yycG genes were likely to play leading roles in mediating DAP resistance in these strains, which may be due to different pathways of daptomycin resistance in different strains [27]. In fact, our ndings were not entirely consistent with previous study. There was no evidence showing a rule between gene mutations and increased the MIC of DAP among E. faecium, but accumulation of liaS, liaF and yycF genes mutations may be positively correlated with the MIC of DAP, which demonstrated evolved DAP resistance is dynamic [18].
Other mechanisms should be taken into consideration, such as charge rejection mechanism and cell membrane stress adaptation [11,19]. It was described that DAP resistance is associated with distinct alterations of cell membrane phospholipid content in enterococci [28]. Indeed, we also found cell surface net positive charge and cell-wall thickness were dramatically increased in these mutants compared to parent strains, and the spacing of the cell-wall was markedly thickened.
Notably, laboratory evolution often lead to adapted strains having different physiological characteristics compared with their parent strains, which are usually related to a tness cost that is typically observed as a reduced bacterial growth rate [29,30]. Our results showed a statistically signi cant decreased growth rate and enhanced bio lm formation for DAP-R mutants, which were similar with previous ndings that the ability of bio lm formation was associated with increased resistance [31]. And the lethality and pathogenicity in Galleria mellonella larvae were also weakened for mutants. Besides, the tness cost is most commonly measured by pair-wise competition experiments [32]. Our data suggested that the median CI values for adaptive mutants were associated with a statistically markedly decreased tness in vitro. In summary, we demonstrate that bacteria acquired daptomycin resistance through different evolutionary pathways in vitro, which would appear with varying degrees of tness costs.
Conclusions E. faecium tended to develop DAP resistance in vitro, which was likely to be related to the following three ways: (i) gene mutations in LiaFSR and YycFG systems; (ii) "repulsion" from the cell surface; (iii) thickened cell wall. Besides. Besides, this is the rst study to report that DAP resistance of E. faecium conferred predictable sensitivities to glycopeptides, providing a new way to optimize treatments for multidrug-resistant E. faecium infection. It was also worth noting that with the increase of DAP resistance, the ability of resistant bacteria to propagate was weakened, suggesting that evolving DAP resistance was associated with tness costs.