Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms.

Three-dimensional reconstruction of confocal laser scanning microscopy (CLSM; see supplementary information for experimental details) images showed a large number of survivors residing in biofilms after exposure to vancomycin and oxacillin at MBC biofilm for 24 h ( Fig. 1A ); the percentage of survivor cells is inconsistent with that of persister cells in biofilms proposed by other researchers and ourselves 3 34 35 . The modified broth recovery-based biofilm MBC defined in our previous study was referred to as MBC biofilm in this study 35 ( Table 1 ). This concentration of antibiotics was expected to eradicate normal cells and also “slow-replicating” cells residing in staphylococcal biofilms, leaving only very few viable persister cells 35 . We reasoned that dissociating biofilms and recovering single-cell population would provide a better measure of the proportion of persister cells than using intact biofilms. After mechanical disruption of S. epidermidis RP62A biofilms by scraping, pipetting, sonication, and vortexing, and passing the acquired cell suspensions through a 1.2 μm syringe filter, mostly single cells and diplococci were observed by SEM ( Fig. 1B ). In contrast, bacterial clumps consisting of more than 10 cells were frequently observed when the filtration step was not included ( Fig. 1B ). Viable counts performed on the bacterial suspensions obtained with or without filtration showed that approximately 45–65% of bacteria in the original suspension passed through the filter ( Fig. 1C ). GFP-expressing S. epidermidis RP62A cells were constructed and used in flow cytometry (see supplementary information ) to measure levels of active and dormant subpopulations of biofilm-embedded cells. Biofilm cell suspensions collected with or without filtration showed similar percentage of GFP + (active) cells (70.9% versus 71.9%), suggesting that these ~45–65% cells are representative of the total population of embedded cells in the biofilm. In contrast to the conventional notion that two subpopulations, antibiotic-susceptible bacteria and persister cells comprise a biofilm 8 15 44 , S . epidermidis biofilm cells presented three subpopulations in response to ciprofloxacin challenge ( Fig. 2A ). Most cells (>99.99%) were susceptible to ciprofloxacin at concentrations of 0.25–1 mg/L. The remaining cells, namely persister cells (~0.01% for the laboratory reference strain S. epidermidis RP62A and ~0.002% for a S. epidermidis clinical strain isolate 3), were tolerant of ciprofloxacin at concentrations ranging from 1 to 128 mg/L; most of these persister cells (~96.9% of RP62A persister cells and ~96.3% for isolate 3) could be further eradicated by ciprofloxacin at concentrations of 256–1024 mg/L. The third fraction of cells, dormant cells (~3.1% of RP62A persister cells and ~3.7% of isolate 3 persister cells), survived ciprofloxacin at a concentration as high as 1024 mg/L. Typical two-subpopulation patterns were established when vancomycin was used in place of ciprofloxacin. Over 99.9% of biofilm cells were susceptible, responding to vancomycin at concentrations below 4 mg/L. Persister cells (~0.06% for RP62A and 0.03% for isolate 3) demonstrated tolerance to vancomycin at concentrations ranging from 4 to 2048 mg/L (for RP62A) or 128 mg/L (for isolate 3). However, the two-subpopulation pattern could be extended as a three-subpopulation pattern when the incubation period was continued for vancomycin at MBC biofilm for another 24 h ( Fig. 2B ). Upon 48 h exposure, only very few dormant cells from the persister cell population, approximately 1.8% for RP62A and 4.6% for isolate 3, survived vancomycin challenge. A similar pattern was found when oxacillin was used to select biofilm persister cells (data not shown). Thus we chose 24 h exposure for ciprofloxacin, or 48 h for vancomycin or oxacillin, at a concentration of MBC biofilm ( Table 1 ) to select for biofilm dormant cells. Dormant cells were not detected after exposure of log-planktonic cell cultures to any of the three antibiotics at the levels of MBC biofilm for 24 or 48 h [limit of detection 10 colony forming units (CFU)/mL], however a substantial number of dormant cells (approximately 1 out of 10 6  CFU to 1 out of 10 4  CFU, corresponding to cell proportions 0.0001% to 0.01%) remained viable after exposure of single biofilm cells to these antibiotics ( Fig. 3A,B , right panels). Fewer dormant cells were detected after exposure of RP62A or isolate 3 biofilm cells to ciprofloxacin than to vancomycin or oxacillin, with average cell proportions reached 0.00025% and 0.0001% for ciprofloxacin, 0.002% ( P  = 0.01) and 0.0013% ( P  = 0.004) for vancomycin, and 0.013% ( P  = 0.005) and 0.004% ( P  < 0.001) for oxacillin respectively ( Fig. 3A,B ). TBK cells were isolated from both log-planktonic cultures and biofilms. Cell proportions of TBK cells ranged from 46.7% to 0.023% for individual isolates, different growth mode and antibiotics ( Fig. 3A,B , left panels). For both S. epidermidis RP62A and isolate 3, biofilm cell populations generally produced a higher proportion of TBK cells than log-planktonic growth modes ( P  < 0.05) ( Fig. 3A,B , left panels). It has been widely reported that generally defined “persister cells” are only transiently resistant to antibiotics. To determine whether the more stringently selected dormant cells were not genetically resistant to antibiotics, resuscitated dormant cells isolated from a S. epidermidis RP62A biofilm were examined for antibiotic susceptibilities using standard micro-dilution method. Regardless of the method used to isolate dormant cells, the MICs were not different from those of their parent cells ( Table 2 ). Biofilm persister cells selected with either ciprofloxacin (3 h, 8 X MICs) or oxacillin (24 h, MBC biofilm ) and comprising mostly TBK showed normal cell morphology when assessed by scanning electron microscopy (SEM, Fig. 4A,B ). Some cells were in the process of cell division, indicating the presence of metabolically active dividing bacteria ( Fig. 4A,B ). In contrast, no dividing cells were found in the more stringently selected dormant cell population isolated using vancomycin and oxacillin at MBC biofilm for 48 h. These cells appeared to have a smaller size, but with a normal shape ( Fig. 4C,D ). We hypothesized that choosing antibiotic regimens to target dormant cells might lead to eradication of S. epidermidis biofilms. These regimens can be achieved by using effective antibiotics at adequate concentrations for an extended period, as population analysis suggested the proportion of persister cell is related to the selecting antibiotic, its concentration and treatment period ( Fig. 2 ). We examined the efficacy of ciprofloxacin and vancomycin, as single agents or in simultaneous combination for 48 h in completely killing persister cells and intact biofilms. At MBC biofilm , ciprofloxacin alone or combinations of ciprofloxacin and vancomycin, but not vancomycin alone, killed nearly all persister cells or biofilm cells of S. epidermidis isolate 3 (see signs in bold, Table 3 ). Survivors were found for S. epidermidis RP62A, which forms biofilms with more biomass than isolate 3 (data not shown). When 72 h and sequential combinations replaced 48 h and simultaneous combinations, ciprofloxacin or its combination with vancomycin at MBC biofilm as the primary agent killed nearly all persister cells or biofilm-embedded cells of both isolates (in bold, Table 4 ). Sequential combinations with ciprofloxacin at MBC biofilm as the primary agent for the first 24 h, followed by vancomycin at serum-achievable concentration ( Table 1 ) for another 48 h on one occasion eradicated the persister cells and biofilms of isolate 3 ( Table 4 ). In contrast, when antibiotics were used at serum-achievable concentrations, none of the treatments eradicated either persister cells or S. epidermidis biofilms, leaving a substantial number of survivor cells ( Tables 3 and 4 ). To be noted, intact biofilms were occasionally more tolerant to antibiotic treatments than isolated persister cells. For instance, sequential combinations with ciprofloxacin (MBC biofilm ) as the first agent followed by vancomycin (MBC biofilm ) as the second agent killed almost all persister cells but left a substantial number of survivor cells within the biofilms.

Two biofilm-positive S. epidermidis strains: RP62A (ATCC 35984), resistant to oxacillin but sensitive to vancomycin; and isolate 3, sensitive to oxacillin and vancomycin, were used in this study. S. epidermidis isolate 3 is a clinical isolate, originally obtained from a blood culture from a baby being cared for in the Neonatal Intensive Care Unit, Royal Women’s Hospital, Melbourne 55 . First line antibiotics, oxacillin, vancomycin, and ciprofloxacin used in this study were purchased from Sigma (Sigma–Aldrich, Sydney, Australia). In order to isolate single cells from S. epidermidis biofilms, we combined a filtration step with mechanical disruption to separate biofilm cells from biofilm-associated factors that might impact on antibiotic efficacy. Staphylococcus epidermidis biofilms were grown in 6-well microplates with Tryptone Soya Broth (TSB, Oxoid, Hampshire, England) as described by Deighton et al. 56 . Single biofilm cells were isolated as follows: after preparing and washing biofilms, one milliliter of Muller-Hinton broth (MHB, Oxoid, Hampshire, England) was added to each well, and the wells were scraped thoroughly with a FALCON ® cell scraper (Corning, Mexico). The resulting suspensions were mixed well by pipetting several times and transferred into a sterile tube, followed by vigorous vortexing. This step was repeated three times and the final volume of each suspension was adjusted to 25 mL with MHB. The tube was sonicated for 10 min using a sonication bath (42 KHZ, BRANSON 1510) and then vortexed at the highest speed for 2 min (30″ × 4) to break up cell aggregates. The suspensions were firmly pressed through 1.2 μm Acrodisc syringe filters. Muller-Hinton broth was chosen as it is recommended by Clinical Laboratory and Standard Institute (CLSI) for antibiotic susceptibility testing and has been used previously in persister cell studies 23 . Planktonic cultures at mid-log phase were prepared by inoculating one colony of overnight-grown S. epidermidis into 5 mL nutrient broth (NB, Oxoid, Hampshire, England). Cultures were grown at 37 °C for 4–5 h until the turbidity was equivalent to McFarland 0.5 standard (~1 × 10 8  CFU/mL). Bacterial suspensions were then centrifuged at 4400  g for 5 minutes, washed twice with sterile phosphate buffered saline (PBS), re-suspended in MHB, followed by the same vortex and filtration procedures described for biofilm cells. A published method for population analysis profiling was modified for this study as persister cells are unable to pass their “resistant” trait to descendants under a stress-free condition and cannot re-grow into a new population such as a colony on agar plates containing antibiotics 51 57 . To profile the population of S. epidermidis RP62A and isolate 3 biofilm cells upon antibiotic exposure, suspensions of single cells at ~10 7  CFU/mL were prepared with MHB containing vancomycin or ciprofloxacin at increasing concentrations ranging from planktonic MBCs to MBC biofilm (see below). Viable counts were performed after exposure of biofilm cells to vancomycin and ciprofloxacin for 24 h respectively, or exposure to vancomycin at MBC biofilm (2048 mg/L) for 48 h. The rationale of selecting such concentrations of antibiotics was to ensure the effective killing of antibiotic-sensitive cells and TBK cells in the bacterial populations. MBCs of antibiotics for planktonic cells at mid-log phase were determined by the broth micro-dilution method recommended by CLSI 58 . MBC biofilm of oxacillin, vancomycin, and ciprofloxacin for biofilms formed by S. epidermidis RP62a and isolate 3 were determined in our previous study ( Table 1 ) 35 . To avoid antibiotic carryover which is related to the usage of very high concentration of antibiotics that might hinder the recovery of viable cells on agar plates, bacterial suspensions were pelleted by centrifuging at 20,000  g for 5 minutes, washed three times with PBS before being plated on Typtone Soya Agar (TSA) plates. Colonies were counted after 72 h of incubation at 37 °C. The log 10 of CFU/mL was plotted against the antibiotic concentration. The difference in the number of survivors after exposure of cells to two antibiotic concentrations represents the fraction of cells that remains sensitive to the higher concentration but tolerant to the lower concentration of antibiotics. We also used a previously reported method to quantify the generally defined persister cells from planktonic cultures at mid-log phase and single biofilm cell populations 6 41 . To isolate the generally defined “persister cell” population, which might comprise both TBK cells and dormant cells, suspensions of single cells at densities of ~10 8  CFU/mL from planktonic cultures at mid-log phase and biofilms were exposed to antibiotics at 8 × MIC for 3 h 6 41 . This method has been shown to eradicate the majority of bacteria with normal susceptibilities 8 . Eight times of MIC of the selected antibiotics are slightly higher than their MBCs, but remain achievable in human serum ( Table 1 ). Before and after the antibiotic challenge, 100 μL volumes of suspension were sampled, serially diluted with PBS and 10 μL was spotted on a TSA plate for cell counting. In some cases 100 μL volumes of the suspension were directly spread on agar plates to increase the sensitivity for detecting persister cells. Cultures were incubated for 72 h to maximize recovery of persister cells. To isolate dormant cells, suspensions of single cells at mid-log phase or single biofilm cells were statically exposed to antibiotics at MBC biofilm for the designated exposure time (37 °C, 24 h for ciprofloxacin and 48 h for vancomycin or oxacillin, based on our population analysis results). The same treatment periods were used for both log-planktonic cultures and biofilm cells to enable valid comparisons to be made. The numbers of survivors were determined by viable count as described above. The proportion of persister cells and dormant cells in different bacterial populations was calculated as follows: (bacterial density after antibiotic treatment)/(bacterial density before antibiotic treatment). The proportion of TBK cells was calculated as the proportion of persister cells minus the proportion of dormant cells. In order to determine whether there was a resistance determinant passed from parent cells to the strictly selected dormant cells, antibiotic susceptibility tests were performed for descendants of dormant cells. Three to five colonies from the dormant cell quantification procedure (grown on TSA plate after antibiotic treatment) were inoculated directly into MHB and grown for 5 h. The resulting suspensions were used for the MIC tests, following the CLSI guideline. Scanning electron microscopy was employed to examine the presence/absence of bacterial cell aggregations before and after filtration when isolating single cells from biofilms, and also to examine the morphology of biofilm persister cells and dormant cells. After isolating persister cells with ciprofloxacin (8 X MIC for 3 h), oxacillin (8 X MIC for 24 h), and dormant cells with oxacillin (MBC biofilm for 48 h) and vancomycin (MBC biofilm for 48 h) respectively, 100 μL of the suspensions were placed on filter membranes (0.2 μm or 0.45 μm) and air-dried overnight. The samples were sputter coated with 200 angstroms of gold using a Magnetron sputter coater (Dynavac, SC100M) and the trapped cells were viewed and imaged with scanning electron microscopes (Philips XL30 or Hitachi S570). Intact S. epidermidis biofilms were formed in 96-well microplate 56 and single biofilm cells were prepared as described above. Both biofilms and single biofilm cell populations were challenged with vancomycin and ciprofloxacin for 48 h, alone or in simultaneous combinations at MBC biofilm or serum achievable concentration (see Table 3 ). In parallel, we challenged intact biofilms or isolated single biofilm cells with single agent for 72 h or two antibiotics sequentially for a total exposure period of 72 h. Two protocols were examined: vancomycin for 48 h and then ciprofloxacin for 24 h, or vice versa ( Table 4 ). Viable counts were carried out for survivor persister cells as described earlier. To increase the detection sensitivity, a one mL volume of the treated suspension was pelleted, washed three times with PBS, and resuspended into 100 uL of PBS, followed by spreading and recovering on TSA plates. To perform viable count for survivors from intact biofilms, biofilms cells were scraped and removed from the bottom of a 96-well microplate and transferred in an Eppendorf tube. After dissociating the cell clumps by vortex and washing with PBS three times, the cells were plated on TSA plate and grown at 37 °C for three days.

How to cite this article : Yang, S. et al. Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms. Sci. Rep. 5 , 18578; doi: 10.1038/srep18578 (2015).

Highlights of the current study include 1) development of a new method to recover single S. epidermidis biofilm-embedded cells free of biofilm-related antibiotic-compromising factors, 2) demonstrating the presence of three subpopulations of biofilm cells with regard to antibiotic susceptibility, and 3) suggesting strategies using antibiotic regimens to eradicate S. epidermidis biofilms. To our knowledge, this is the first study that successfully isolated large numbers of single cells from bacterial biofilms to study their persister cell proportions. A combination of scraping, pipetting, sonication and vortexing was used to dissociate biofilm cells from the extracellular matrix, followed by a filtration step to select the single cells. The method was qualitatively and quantitatively validated by SEM, fluorescence-activated cell sorting (FACS), and viable counts before and after filtration. Antibiotic-compromising effects of some other biofilm-associated factors, such as extracellular hydrolytic enzymes and QS factors 27 28 29 30 31 , though not specifically examined, were believed to be minimized by resuspending the biofilm cell into fresh medium after removing from the substratum and before sonication, voxtexing and filtration. Using the successfully isolated single biofilm cell population, this study provides quantitative and qualitative insights into the presence of antibiotic-tolerant cells within S. epidermidis biofilms. Population analysis suggested that biofilms contain TBK cell and dormant cell subpopulations in addition to normal cells. We showed that dormant cells constitute a very small subpopulation of cells that show little susceptibility to antibiotics, irrespective of antibiotic concentration. These cells were isolated under very stringent conditions that eradicated TBK cells. The rationality of using these stringent conditions is supported by our SEM findings. Both replicating and non-replicating persister cells were found when isolation was carried out following the method published by others 6 8 41 ; however only non-dividing dormant cells were observed when specific concentrations and exposure times based on a pre-determined population analysis profile were introduced ( Figs 2 and 4 ). TBK cells represent a much larger subpopulation than dormant cells. These cells have reduced antibiotic susceptibilities but remain eradicable with selected antibiotics. Viable but non-cultivable cells constitute the fourth cell population that might reside in biofilms 14 , however, this subpopulation is out of the scope of this study. Our population profiling of biofilm persister cells based on antibiotic tolerance matches the persister classification by Babalan et al. and others 7 10 42 45 . Babalan et al. categorized persister cells as either type I or type II 10 . Type I persister cells are a fully dormant sub-population that can only be isolated from stationary-phase cultures; type II persister cells are a fraction of cells in a slow-growth state that can be obtained from any stages of growth, including log-planktonic cultures 10 45 . Dormant cells described in the present study are similar to type I persister cells. These cells were mainly isolated from the biofilm, a growth mode that has similar levels of resistance to antibiotics as stationary-phase cultures; SEM images of dormant cells showed no evidence of cell division. TBK cells might characteristically match the continuously generated type II persister cells. In our study, TBK cells could be isolated from both biofilms and log-planktonic cultures and SEM clearly showed dividing cells. Our finding that a single cell population isolated from S. epidermidis biofilms contained more persister cells than log-planktonic cultures is consistent with other published studies 3 4 39 , however this study goes into greater detail than previous studies. The observation presented in this study that dormant cells are only found in the biofilm growth mode suggests their important role in the tolerance of biofilms to high concentrations of antibiotics. Zhang et al. has suggested a key principal for successful treatment of chronic infections: to reduce the persister cells quickly to a sufficiently low number with effective drugs so that reversion from persister cells to a normal bacterial population will not occur before the treatment is continued 46 47 . By introducing this principal into our study, we proposed a new strategy to treat S. epidermidis biofilm: killing TBK cells with effective antibiotics within a short period, followed immediately by a dormant-cell-targeted antibiotic regimen. Our population profiling suggested that high doses of ciprofloxacin but not vancomycin or oxacillin eradicate S. epidermidis TBK cells within 24 h. This is in accordance with the high efficacy of ciprofloxacin against staphylococcal biofilms 48 49 . We also observed that the critical concentration, MBC biofilm must be reached to completely kill the TBK and to expose dormant cells. To further eradicate dormant cells, we propose extending the treatment period to a continuous 48 h or 72 h ( Tables 3 and 4 ). In our in vitro study, eradication of dormant cells and intact biofilms were both achieved with ciprofloxacin at MBC biofilm, or on some occasions, combinations with ciprofloxacin at MBC biofilm as the major agent. Extended incubation might “wake up” the persister cells from dormancy and recover their susceptibility to antibiotics before they can establish a new population 50 51 . We however cannot exclude the possibility that the killing of dormant cells is simply a consequence of a longer exposure to antibiotics. We initially proposed that combining vancomycin and ciprofloxacin would be more efficient in killing persister cells than ciprofloxacin alone, based on the staggered tolerance spectrum of persister cells to vancomycin and ciprofloxacin, and also on conclusions from other studies 8 . Surprisingly, combinations of vancomycin and ciprofloxacin at MBC biofilm generally demonstrated a lower efficacy in eradicating persister cells or intact biofilms than ciprofloxacin alone. This might be explained by the finding by Dorr et al. that stress from antibiotics can stimulate the formation of persister cells 52 . To be noted, there is also a slight difference in antibiotic tolerance between single biofilm cell populations and intact biofilms ( Tables 3 and 4 ), suggesting the possible involvement of other biofilm-related factors, such as the presence of EPS, QS factors, and extracellular hydrolytic enzymes. In summary, S. epidermidis biofilm persister cells comprise TBK cells and dormant cells; both subpopulations play important roles in biofilm drug tolerance. Selection of a suitable antibiotic, at adequate concentration, and an extended exposure time, should be considered in the treatment of S. epidermidis biofilm-related infections. Although the concentrations of antibiotics and treatment period used in this in vitro study might not be suitable for intravenous administration, they could be applied for other applications such as antibiotic lock solutions, inhaled aerosolized antibiotics or wound dressings 53 54 .