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Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum
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Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum Nicholas S. Britt , Daniel S. Hazlett , Rebecca T. Horvat , Rachael M. Liesman , Molly E. Steed PII: DOI: Reference:
S0924-8579(20)30037-6 https://doi.org/10.1016/j.ijantimicag.2020.105898 ANTAGE 105898
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
9 August 2019 4 January 2020
Please cite this article as: Nicholas S. Britt , Daniel S. Hazlett , Rebecca T. Horvat , Rachael M. Liesman , Molly E. Steed , Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum, International Journal of Antimicrobial Agents (2020), doi: https://doi.org/10.1016/j.ijantimicag.2020.105898
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HIGHLIGHTS
Vancomycin demonstrated poor activity against planktonic MRSA from CF sputum Vancomycin activity was reduced in biofilms cultivated in CF physiologic media Selection of resistant subpopulations can occur even at high vancomycin exposures Vancomycin monotherapy appears unlikely to eradicate MRSA from the CF lung
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Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum
Nicholas S. Britt,a-b# Daniel S. Hazlett,a Rebecca T. Horvat,c Rachael M. Liesman,c Molly E. Steed,a
Department of Pharmacy Practice, University of Kansas School of Pharmacy, Lawrence, Kansas, USAa; Department of Internal Medicine, Division of Infectious Diseases, University of Kansas School of Medicine, Kansas City, Kansas, USA b; Department of Pathology and Laboratory Medicine, University of Kansas School of Medicine, Kansas City, Kansas, USAc
Running head: Pulmonary vancomycin MRSA activity in CF
Keywords: methicillin-resistant Staphylococcus aureus; cystic fibrosis; biofilms; multidrug-resistance; antibiotic tolerance; vancomycin
# Address correspondence to
Nicholas S. Britt, [email protected]. 3901 Rainbow Blvd, Mailstop 4047 University of Kansas Medical Center Kansas City, KS 66160-7231 Tel: (913) 588-5391; Fax: (913) 588-2355
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ABSTRACT Vancomycin is commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection in patients with cystic fibrosis (CF) lung disease; however, there are limited data to support the in vitro activity of this agent against MRSA isolated from CF sputum. The primary objective of this study was to evaluate the activity of vancomycin at pulmonary concentrations (intravenous and inhaled) against 4 clinical MRSA CF sputum isolates in planktonic and biofilm time-kill (TK) experiments. Vancomycin minimum inhibitory concentrations (MICs) were determined for these isolates at standard inoculum (SI; ~106 colony-forming units [CFU]/mL) and high inoculum (HI; ~108 CFU/mL), and in biofilms cultivated using physiologic media recapitulating the microenvironment of the CF lung. Vancomycin concentrations of 10, 25, 100, and 275 µg/mL were evaluated in TK experiments against planktonic MRSA at varying inocula and versus biofilm MRSA. Vancomycin MICs increased from 0.5 µg/mL at SI to 8-16 µg/mL when tested at HI. Vancomycin MICs were further increased to 16-32 µg/mL in biofilm studies. In TK experiments, vancomycin displayed bactericidal activity (≥ 3 log10 killing at 24 h) against 1/4 and 0/4 planktonic MRSA isolates at SI and HI, respectively. Against MRSA biofilms, vancomycin was bactericidal against 0/4 isolates. Based on these findings, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of patients with CF, even at high concentrations similar to those observed with inhaled therapy. Novel vancomycin formulations with enhanced biofilm penetration or combination therapy with other potentially synergistic agents should be explored.
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1. INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive genetic disorder associated with persistent lung infection [1, 2]. Staphylococcus aureus is commonly isolated from individuals with CF and infections due to methicillin-resistant S. aureus (MRSA) have increased in recent years [3-5]. A longitudinal study of patients with CF by Dasenbrook et al. found an association between MRSA infection and more rapid decline in lung function than those without MRSA [6]. More importantly, persistence of MRSA in the respiratory tract is associated with an increased risk of death compared to transient or cleared MRSA infection [7]. Intravenous vancomycin is a recommended treatment for symptomatic culturepositive MRSA in CF patients [8]. However, vancomycin only distributes into the lungs at roughly 50% of serum concentrations [9]. The ability of vancomycin to penetrate the thick mucus and biofilm characteristic of the CF lung in order to reach efficacious concentrations is also unclear. Inhaled vancomycin presents an attractive alternative to intravenous therapy due to the ability to deliver increased concentrations of the antibiotic to the site of infection while minimizing toxicity from systemic exposure. The antibacterial activity of these pulmonary vancomycin concentrations, however, has not been fully assessed. Therefore, the objective of this study was to evaluate the in vitro activity of pulmonary vancomycin exposures (intravenous and inhaled) against MRSA isolates from the sputum of patients with CF lung disease.
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2. MATERIALS AND METHODS 2.1. Bacterial Strains. Four consecutive clinical MRSA sputum isolates (CF005, CF006, CF007, and CF009) from 4 separate adult patients with CF were collected from the University of Kansas Hospital clinical microbiology laboratory (Kansas City, Kansas, USA) and frozen at -80oC prior to subsequent analysis. Methicillin resistance was confirmed by polymerase chain reaction for the mecA gene. ATCC® 25923 was used as the control strain for susceptibility and bactericidal activity testing. Mu3 (ATCC® 700698) was used as the reference strain for hVISA determination [10]. 2.2. Antimicrobials. Vancomycin powder was commercially purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fresh vancomycin stock solutions were prepared daily. 2.3. Media. Cation-adjusted Mueller-Hinton broth (CAMHB; Becton-Dickinson, Sparks, MD, USA) was used as media for susceptibility testing and time-kill experiments. Brain heart infusion agar (BHIA; Becton-Dickinson, Sparks, MD, USA) was used for all population susceptibility analyses. Tryptic soy agar (TSA; Becton-Dickinson, Sparks, MD, USA) was used for all other colony counts. In addition to conventional CAMHB, we performed susceptibility testing and biofilm time-kill experiments using artificial sputum media (ASM), a physiologic culture media developed to recapitulate the microenvironment of the CF lung [11, 12]. This media contains amino acids, mucin, and exogenous DNA, and has been validated as a biofilm model for CF sputum [13]. Artificial sputum media was prepared according to a previously published protocol and stored refrigerated at 4oC for up to 30 days [14].
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2.4. Planktonic susceptibility testing. Vancomycin minimum inhibitory concentrations (MICs) were determined by broth microdilution by testing concentrations of 0.125-64 μg/mL at a standard inoculum of approximately 1 × 106 colony-forming units (CFU)/mL (acceptable range, 5.5-6.5 log10 CFU/mL) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [15]. Vancomycin MIC ≤ 2 μg/mL were considered susceptible, per CLSI. All samples were incubated at 35oC for 24 h under aerobic conditions and read by visual inspection. High inoculum MIC testing was performed by broth microdilution at a starting inoculum of approximately 1 × 108 CFU/mL
(acceptable
range,
7.5-8.5
log10
CFU/mL)
by
testing
vancomycin
concentrations of 2–1,024 μg/mL. Following 24 h of incubation at 35 oC under aerobic conditions, bacterial growth was visualized by addition of 20 μL of a 0.5 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue; Sigma-Aldrich, St. Louis, MO, USA) to each well, as previously described [16]. After an additional 20 min of incubation at 35oC, a color change from pale yellow to blue indicated the presence of viable bacterial cells [16]. All testing was performed in duplicate to ensure reproducibility. 2.5. Biofilm susceptibility testing. For biofilm susceptibility testing, bacterial suspension (0.5 McFarland standard) was added to ASM in 2 mL flat-bottom culture plates and biofilms were cultivated for 72 h at 35oC under aerobic conditions and continuous shaking at 75 rpm. Following biofilm conditioning, vancomycin was added at concentrations of 2-1,024 μg/mL and plates were incubated for an additional 24 h at 35oC under aerobic conditions. Biofilms were disrupted by addition of cellulase and a series of manual pipetting and vortex steps to displace any viable bacterial cells from
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mature biofilm structures prior to MIC determination [14]. Bacterial viability was assessed by the addition of MTT and MICs determined as the lowest vancomycin concentration without a visible color change from pale yellow to blue in the corresponding well. All susceptibility testing was performed in at least duplicate to ensure reproducibility. 2.6. Minimum bactericidal concentration (MBC) determination. Planktonic vancomycin MBCs were determined at standard and high inoculum following the broth microdilution method recommended by CLSI guidelines for bactericidal activity testing [17]. Vancomycin tolerance was defined as an MBC/MIC ratio ≥ 32 per CLSI guidelines [17]. For MBC determination, a 100 μL aliquot of all wells corresponding to vancomycin concentrations above the MIC was plated onto TSA, allowed to visibly dry at room temperature, and cross-streaked using a sterile cotton-tipped swab to account for antibiotic carryover effect common to vancomycin MBC assays [17]. Vancomycin MBC was defined as the lowest concentration with ≥ 99.9% killing (as compared to initial measured inoculum) after 24 h incubation at 35oC. The maximum evaluated concentration was 1,024 μg/mL in high inoculum assays. 2.8. Vancomycin time-kill assays. Time-kill assays of planktonic MRSA were performed at standard inoculum and high inoculum. We evaluated intravenous vancomycin pulmonary concentrations approximating the Cmedian (10 μg/mL) and Cmax (25 μg/mL) from a previous study of lung epithelial lining fluid [9]. To evaluate the activity of pulmonary concentrations observed with inhaled vancomycin, we tested the approximate Cmedian (100 μg/mL) and Cmax (275 μg/mL) obtained from published results of a Phase I trial of inhalable vancomycin [18]. For time-kill assays versus planktonic
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MRSA, experiments were performed in 2 mL of CAMHB in flat-bottom cell culture plates. A 100 μL sample at each concentration was removed from incubation (35 oC) at 0, 4, 8, and 24 h and serially diluted 1:10 in cold 0.9% sodium chloride. Appropriate dilutions were drop plated on TSA and colonies were counted by visual inspection after 24 h incubation at 35oC. Mature MRSA biofilms were cultivated for time-kill assays by calibrating bacterial suspensions to a 0.5 McFarland standard in ASM and incubating for 72 h at 35 oC under aerobic conditions and continuous shaking at 75 rpm. Following this conditioning phase, biofilms were treated with vancomycin at concentrations of 100 μg/mL and 275 μg/mL. Vancomycin killing was determined by sampling at 0, 4, 8, and 24 h. For each sample, biofilms were disrupted by adding cellulase and shaking at 150 rpm for 1 h, followed by cycles of manual pipetting and vortex until no biofilm was apparent by visual inspection. Dilutions were drop plated on TSA for final colony count determination after 24 h incubation at 35oC. Time-kill curves were constructed by plotting mean colony counts (log10 CFU/mL) using GraphPad Prism (version 6.04; GraphPad Software, Inc., La Jolla, CA, USA). Bactericidal activity of vancomycin was defined as a ≥ 3 log 10 CFU/mL reduction in viable bacteria after 24 h in comparison with the starting inoculum. Bacteriostatic activity was defined as a < 3 log10 CFU/mL reduction in viable bacteria with an absence of growth in comparison with the starting inoculum. All time-kill assays were performed in triplicate. 2.9. Population analysis profiles. To determine the effect of pulmonary vancomycin exposure on the population susceptibility of MRSA isolates to vancomycin, population analysis profiles (PAPs) were performed before (T 0) and after 24 h exposure
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(T24) to vancomycin 100 μg/mL in high inoculum time-kill assays, as previously described [10]. This concentration was chosen to best simulate the vancomycin concentration achieved for the longest duration of the dosing interval following administration of inhaled vancomycin [18]. A bacterial suspension of approximately 1 × 108 CFU/mL was plated on freshly prepared BHIA plates containing 0, 0.5, 1, 2, 3, 4, and 6 mg/L of vancomycin. After 48 h incubation at 35 oC, colony counts were determined by visual inspection (lower limit of detection 2 log10 CFU/mL). Colony counts were plotted against increasing concentrations of vancomycin and the resultant area under the curve (AUC) was calculated by the trapezoidal method. A shift in the population susceptibility profile was evaluated by calculating the ratio of the before and after PAP AUC (T24:T0 PAP AUC ratio). Any strain with an AUC ratio ≥ 0.9 times that of Mu3 was considered hVISA, as previously defined [10]. 2.10. Scanning electron microscopy (SEM). To confirm mature MRSA biofilm formation in ASM, SEM experiments were performed. Bacterial biofilms were cultivated in 72 h of ASM in a 2 mL flat-bottom cell culture plate, gently transferred to a microcentrifuge tube, and centrifuged at 10,000 rpm for approximately 30 sec to form a bacterial pellet. Following centrifugation, the supernatant was gently removed, and the sample was fixed with 2% glutaraldehyde in 0.1M cacodylate buffer. The primary fixative was then rinsed twice for 10 min with 0.1M cacodylate buffer. Samples were post-fixed in 1% osmium tetroxide in 1% cacodylate buffer for 30 min, thrice rinsed with sterile
deionized
water,
and
dehydrated
stepwise
using
increasing
ethanol
concentrations. Samples were dried using bone dry carbon dioxide in an EMS 850 critical point drier, mounted and sputter coated with gold/palladium using an
9
EMS/Quorum 150 R ES sputter coater. Final preparations were viewed using a Hitachi S-2700 SEM and captured as .TIFF files using the Quartz PCI system. 2.11. Statistical analysis. Quantitative changes in the CFU/mL between tested vancomycin concentrations were compared for each strain at 24 h by two-way analysis of variance with Tukey’s post-hoc test. A P-value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS (version 20.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 6.04; GraphPad Software, Inc., La Jolla, CA, USA) software.
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3. RESULTS All MRSA sputum isolates were susceptible to vancomycin at standard inoculum when tested using conventional culture media (Table 1). Three of the 4 strains (CF006, CF007, and CF009) met the threshold for vancomycin tolerance (minimum bactericidal concentration [MBC]/minimum inhibitory concentration [MIC] ratio ≥ 32). When tested at high inoculum, vancomycin MICs increased from 0.5 μg/mL to 8-16 μg/mL, consistent with the inoculum effect. Additionally, vancomycin MBC values increased beyond the maximum evaluated concentration of 1,024 μg/mL. Vancomycin MIC and MBC did not differ when tested in artificial sputum media (ASM) at either standard or high inoculum, suggesting that drug was stable in this physiologic media. Vancomycin MICs further increased to 16-32 μg/mL against biofilm MRSA cultivated in ASM. Mature MRSA biofilm formation was confirmed by scanning electron microscopy (SEM; Figure 1). In standard inoculum time-kill experiments, vancomycin was bactericidal at all concentrations against only 1 of the 4 planktonic MRSA isolates (CF005; Figure 2A-D). Against strain CF009, vancomycin was bacteriostatic at all concentrations except 275 μg/mL (Figure 2D). For all other isolates and concentrations, vancomycin was bacteriostatic with no significant dose-response relationship (P > 0.5; Figure 2A-D). In high inoculum time-kill experiments versus planktonic MRSA, bacterial growth was observed at a vancomycin concentration of 10 μg/mL for 3 of 4 strains, consistent with the MIC shifts observed in high inoculum susceptibility testing (Figure 3B-D). As expected based on the results of high inoculum MBC determinations, vancomycin 25, 100, and 275 µg/mL was bacteriostatic against all 4 strains analyzed (Figure 3A-D).
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Vancomycin time-kill curves versus biofilm-embedded MRSA are displayed in Figure 4. Vancomycin concentrations of 10 μg/mL and 25 μg/mL were not tested because they were subinhibitory against the majority of strains. In an ASM biofilm timekill model, vancomycin was bacteriostatic at concentrations of 100 μg/mL and 275 μg/mL against all strains tested and there were no differences in antibacterial activity between these concentrations (P > 0.5). The results of T0 and T24 vancomycin population analysis profiles (PAPs) are depicted in Figure 5. None of the strains analyzed met criteria for heterogenous vancomycin intermediate S. aureus (hVISA) classification by population analysis (AUCtest/AUCMu3 ≥ 0.9) before or after vancomycin exposure. However, a right-shift in the population susceptibility profile to vancomycin was noted in 3 of the 4 strains (Figure 5A-C). 4. DISCUSSION In this report, we describe the pharmacodynamic (PD) effects of vancomycin pulmonary concentrations against MRSA isolated from the sputum of adult CF patients. Generally, vancomycin demonstrated poor activity against these strains, particularly at high bacterial inoculum and in biofilms mimicking those which would be observed in the CF lung. We showed that vancomycin susceptibility is lost against these strains with increasing bacterial density and that vancomycin is minimally active even at high concentrations that would be observed with inhaled therapy. Lack of activity may be further explained by the fact that most of the strains tested were tolerant to vancomycin at standard inoculum and all strains were vancomycin tolerant at high inoculum. Among vancomycin tolerant strains, increased
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exposure to vancomycin may result in collateral inhibition of murein hydrolases, resulting in a loss of autolytic activity [19]. The lack of vancomycin bactericidal activity we observed does not appear to be due to a slow-growing phenotype, as a ~2 log10 CFU/mL growth was consistently observed after only 4 h in antibiotic-free media. To our knowledge, there are no data on the prevalence of vancomycin tolerance among MRSA isolated from the sputum of individuals with CF, however, we previously reported vancomycin tolerance to be present in 26.7% of MRSA bloodstream isolates at our institution [20]. The only patient-specific factor associated with this phenotype was previous exposure to vancomycin, which is frequently used to treat pulmonary MRSA among CF patients [20, 21]. A contributing factor to the high rate of vancomycin tolerance we observed in these clinical strains may also be chronic co-colonization with Pseudomonas
aeruginosa.
Previous
research
demonstrates
that
S.
aureus
downregulates penicillin-binding protein 4 in response to quorum sensing molecules released from P. aeruginosa, resulting in vancomycin tolerance [22]. Specifically, P. aeruginosa has been shown to “protect” S. aureus planktonic and biofilm populations from vancomycin through exogenous release of 2-n-heptyl-4-hydroxyquinolone N-oxide (HQNO) and siderophores without small-colony variant reversion [23]. This is of high clinical significance given the near universal prevalence of P. aeruginosa and MRSA cocolonization in patients with CF. The present research suggests that the vancomycin tolerant phenotype may persist even in monoculture, resulting in poor vancomycin activity. To our knowledge, this was the first study to evaluate the antibacterial activity of clinically achievable vancomycin concentrations versus MRSA biofilms mimicking those
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observed in patients with CF. Previous studies of vancomycin activity and penetration of biofilm-embedded MRSA have used models in which cells are cultivated on abiotic surfaces, such as polystyrene pegs or coupons, which are not representative of biofilm formation in patients with CF [23]. Nonetheless, these models have consistently shown vancomycin to have poor activity against biofilm-producing S. aureus. [24-27]. This is consistent with the findings of the current study, in which ASM biofilm-embedded MRSA isolates demonstrated reduced in vitro susceptibility to vancomycin. All strains tested were highly sensitive to changing bacterial density, as increasing the inoculum from 106 to 108 CFU/mL resulted in a profound loss of vancomycin susceptibility (16- to 32-fold MIC increase). This is consistent with previous in vitro analyses of the inoculum effect on vancomycin activity versus MRSA [16]. Even more pronounced was the decrease in bactericidal activity with vancomycin MBCs increasing to >1,024 μg/mL for all 4 strains. Clinically, CF lung disease is characterized by a large overall bacterial density (~1010 CFU/mL), of which the composition varies significantly between patients and over an individual’s lifetime [28, 29]. Bacterial density of MRSA in any single CF patient is difficult to estimate in the clinical setting, as bacteria are often embedded in complex polymicrobial mucus biofilms. For this reason, we tested vancomycin activity against varying MRSA inocula (standard and high) and modes of growth (planktonic and biofilm). We found vancomycin activity against MRSA isolated from CF sputum was consistently poor across multiple inocula and was further decreased against biofilm-entrenched MRSA characteristic of CF lung disease. We further evaluated the influence of pulmonary concentrations of inhaled vancomycin on the corresponding population susceptibility profiles. The observed right-
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shift in the T24:T0 PAP AUC ratio suggests that selection of MRSA subpopulations with reduced vancomycin susceptibility can occur even at high vancomycin exposures. The concentration-dependent nature of population susceptibility shifts has been previously demonstrated by Rose and colleagues, with the largest shifts observed at lower vancomycin AUC/MIC ratios [30]. Therefore, although we observed a significant rightshift in the population susceptibility profile after only 24 h of vancomycin 100 μg/mL exposure, this shift may be even more pronounced at lower vancomycin concentrations relative to the MIC, such as those that would be observed with intravenous vancomycin, or with prolonged vancomycin exposures. The clinical implications of our findings are further supported by a recent doubleblind, randomized, placebo-controlled trial that investigated the addition of inhaled vancomycin therapy to an intensive MRSA eradication protocol that included oral trimethoprim-sulfamethoxazole or doxycycline combined with oral rifampin and MRSA decolonization with nasal mupirocin and chlorhexidine gluconate cleanses [32]. The addition of inhaled vancomycin to this multimodal strategy did not result in significant differences in MRSA eradication rates nor improved lung function [32]. Furthermore, 28% of inhaled vancomycin-treated patients experienced bronchospasm despite pretreatment with albuterol, requiring discontinuation with the trial [32]. Novel vancomycin formulations with enhanced biofilm penetration and tolerability, such as those currently in development (ClinicalTrials.gov identifier: NCT03181932), or prolonged suppressive combination therapy with other potentially synergistic agents will likely be required to optimize outcomes in these patients.
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The current study was strengthened by its use of MRSA strains isolated from the sputum of “real-world” patients with CF. Additionally, clinical pulmonary concentrations from CF patients treated with both intravenous and inhalable vancomycin were evaluated to enhance external validity and clinical relevance. Our study also had several noteworthy limitations. First, the use of a 24 h time-kill methodology did not allow for replication of the true vancomycin concentration-time profile observed clinically. We attempted to partially mitigate this by using multiple concentrations available in published pharmacokinetic (PK) data of intravenous and inhaled vancomycin [18]. However, due to the methodology used, replication of our findings in animal or in vitro PK/PD models over a longer time period of vancomycin exposure and against additional isolates is warranted. Estimated pulmonary concentrations from intravenous vancomycin were derived from epithelial lining fluid ELF from healthy volunteers, as this data is not yet available in the CF population. Vancomycin PD targets in patients with CF are no different than those without CF; however, patients with CF may require more aggressive dosing due to increased clearance. It is well known that there is a large inter-patient variability in vancomycin exposures observed in the clinic, further complicating experimental design. Ultimately, we chose to evaluate vancomycin concentrations which we believe are representative of clinically achievable exposures in CF patients. An additional point to consider is that correlation of antibiotic susceptibility and in vitro activity with clinical response is problematic in the management of CF pulmonary infections. The CF lung is characterized by thick mucus and most bacterial communities persist firmly entrenched in biofilms, which may limit the clinical applicability of standard planktonic susceptibility testing [31]. We attempted to
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overcome this by employing an ASM biofilm TK model mimicking the biochemical conditions of the CF lung. However, the clinical applicability of biofilm antibiotic susceptibility testing in CF is still uncertain and additional data on clinical outcomes associated with intravenous and inhaled vancomycin in this vulnerable patient population is needed [31]. Only clinical strains from CF sputum were evaluated in this study and it is unclear whether the effects observed are unique to CF isolates or the growth modalities tested. Furthermore, we analyzed only a single isolated MRSA phenotype from each patient included in the study. Clinically, multiple MRSA phenotypes are likely to exist in a complex polymicrobial environment that may affect vancomycin activity. 5. CONCLUSIONS In summary, vancomycin demonstrated minimal in vitro activity against MRSA strains isolated from CF sputum, particularly at high inoculum and in biofilm. While inhaled vancomycin represents an achievement in the armamentarium to treat respiratory MRSA infections, the results of this study highlight the uniqueness and resilience of organisms isolated from CF patients. Inhaled vancomycin affords the advantage of an improved nephrotoxicity profile and higher concentrations localized at the site of infection compared to intravenous therapy. Based on the results of this study, however, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of CF patients.
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ACKNOWLEDGMENTS This study was presented in part at the 55th Interscience Conference on Antimicrobial Agents and Chemotherapy in San Diego, CA, USA. This study was classified as nonhuman subjects research and ethical approval was not required per institutional policy We would like to acknowledge Pat St. John and Larysa Stroganova at the KUMC EMRL for technical assistance, as well as Mallory Schroeder and Daniel Younggren (University of Kansas School of Pharmacy) for data collection.
DECLARATIONS Funding: NSB was supported in part by Clinical and Translational Science Award TL1 TR000120-03 from the National Institutes of Health (NIH). Electron microscopy was performed at the University of Kansas Medical Center Electron Microscopy Research Laboratory (KUMC EMRL), which is supported in part by NIH grant P20GM104936. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Competing Interests: All authors report no relevant conflicts of interests Ethical Approval: Not required
Contributions NSB and MES conceived and designed the study, oversaw all experiments, and analyzed and interpreted resulting data. DSH performed experiments and acquired data. RTH and RML assisted with data interpretation. All authors contributed to drafting the manuscript and have approved the final version of the article.
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with gentamicin, in an in vitro pharmacodynamic model. Antimicrobial agents and chemotherapy. 2004;48:4665-72. [17] Clinical Laboratory and Standards Institute. Methods for Determining Bactericidal Activity of Antimicrobial Agents - Supplement M26A. Wayne, PA1999. [18] Jouhikainen T, Lord J, Hofmann T, Waterer G. Aerovanc - Phase I Study of Vancomycin Inhalation Powder. 26th Annual Cystic Fibrosis Conference. Orlando, FL: Cystic Fibrosis Foundation; 2012. [19] Sieradzki K, Tomasz A. Inhibition of the autolytic system by vancomycin causes mimicry of vancomycin-intermediate Staphylococcus aureus-type resistance, cell concentration dependence of the MIC, and antibiotic tolerance in vancomycinsusceptible S. aureus. Antimicrobial agents and chemotherapy. 2006;50:527-33. [20] Britt NS, Patel N, Shireman TI, El Atrouni WI, Horvat RT, Steed ME. Relationship between vancomycin tolerance and clinical outcomes in Staphylococcus aureus bacteraemia. J Antimicrob Chemother. 2017;72:535-42. [21] Britt NS, Patel N, Horvat RT, Steed ME. Vancomycin 24-Hour Area under the Curve/Minimum Bactericidal Concentration Ratio as a Novel Predictor of Mortality in Methicillin-Resistant Staphylococcus aureus Bacteremia. Antimicrob Agents Chemother. 2016;60:3070-5. [22] Filkins LM, Graber JA, Olson DG, Dolben EL, Lynd LR, Bhuju S, et al. Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model. J Bacteriol. 2015;197:2252-64.
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[23] Orazi G, O'Toole GA. Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection. MBio. 2017;8. [24] Parra-Ruiz J, Vidaillac C, Rose WE, Rybak MJ. Activities of high-dose daptomycin, vancomycin, and moxifloxacin alone or in combination with clarithromycin or rifampin in a novel in vitro model of Staphylococcus aureus biofilm. Antimicrob Agents Chemother. 2010;54:4329-34. [25] Hall Snyder AD, Vidaillac C, Rose W, McRoberts JP, Rybak MJ. Evaluation of High-Dose Daptomycin Versus Vancomycin Alone or Combined with Clarithromycin or Rifampin Against Staphylococcus aureus and S. epidermidis in a Novel In Vitro PK/PD Model of Bacterial Biofilm. Infect Dis Ther. 2014. [26] Rose WE, Poppens PT. Impact of biofilm on the in vitro activity of vancomycin alone and in combination with tigecycline and rifampicin against Staphylococcus aureus. J Antimicrob Chemother. 2009;63:485-8. [27] LaPlante KL, Mermel LA. In vitro activities of telavancin and vancomycin against biofilm-producing Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis strains. Antimicrob Agents Chemother. 2009;53:3166-9. [28] Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TW, Carroll MP, et al. Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation? J Cyst Fibros. 2011;10:357-65. [29] Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, et al. Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci U S A. 2012;109:5809-14.
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[30] Rose WE, Knier RM, Hutson PR. Pharmacodynamic effect of clinical vancomycin exposures on cell wall thickness in heterogeneous vancomycin-intermediate Staphylococcus aureus. The Journal of antimicrobial chemotherapy. 2010;65:2149-54. [31] Waters V, Ratjen F. Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis. Cochrane Database Syst Rev. 2012;11:CD009528. [32] Dezube R, Jennings MT, Rykiel M, Diener-West M, Boyle MP, Chmiel JF, et al. Eradication of persistent methicillin-resistant Staphylococcus aureus infection in cystic fibrosis. J Cyst Fibros. 2019;18:357-63.
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TABLE 1. Microbiological characteristics of 4 clinical methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates a Standard media Physiologic media (CAMHB) (ASM) VAN Biofilm VAN SI MIC VAN HI MIC VAN SI MIC VAN HI MIC Isolate MIC (MBC) b (MBC) c (MBC) b (MBC) c (MBC) d 0.5 8 0.5 8 16 CF005 (1) (> 1,024) (1) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF006 (16) (> 1,024) (16) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF007 (32) (> 1,024) (32) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF009 (16) (> 1,024) (16) (> 1,024) (> 1,024) CAMHB, cation-adjusted Mueller-Hinton broth; ASM, artificial sputum media; VAN, vancomycin; SI, standard inoculum; HI, high inoculum; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration a All units in µg/mL b ~106 colony-forming units (CFU)/mL c ~108 CFU/mL d Determined following 72 h biofilm conditioning in ASM
A
B
FIG 1. Representative scanning electron microscopy (SEM) images of biofilm methicillin-resistant Staphylococcus aureus strain CF007 cultivated in artificial sputum media for 72 h. Bacterial biofilms were viewed at 2000x (A) and 7000x (B).
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FIG 2.
Vancomycin (VAN) time-kill curves versus planktonic methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) at standard inoculum (~106 colony-forming units/mL). Dotted line indicates lower limit of detection.
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FIG 3. Vancomycin (VAN) time-kill curves versus planktonic methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) at high inoculum (~108 colony-forming units/mL). Dotted line indicates lower limit of detection.
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FIG 4.
Vancomycin (VAN) time-kill curves versus biofilm methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) following 72 h cultivation in artificial sputum media. Dotted line indicates lower limit of detection.
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A
B B
C
D
A
C
D
FIG 5. Population analysis profiles of methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) before (T0; purple solid line) and 24 h after (T24;blue dotted line) exposure to vancomycin 100 µg/mL in high inoculum (~108 colony-forming units/mL) time-kill assays.
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Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum Nicholas S. Britt , Daniel S. Hazlett , Rebecca T. Horvat , Rachael M. Liesman , Molly E. Steed PII: DOI: Reference:
S0924-8579(20)30037-6 https://doi.org/10.1016/j.ijantimicag.2020.105898 ANTAGE 105898
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
9 August 2019 4 January 2020
Please cite this article as: Nicholas S. Britt , Daniel S. Hazlett , Rebecca T. Horvat , Rachael M. Liesman , Molly E. Steed , Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum, International Journal of Antimicrobial Agents (2020), doi: https://doi.org/10.1016/j.ijantimicag.2020.105898
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HIGHLIGHTS
Vancomycin demonstrated poor activity against planktonic MRSA from CF sputum Vancomycin activity was reduced in biofilms cultivated in CF physiologic media Selection of resistant subpopulations can occur even at high vancomycin exposures Vancomycin monotherapy appears unlikely to eradicate MRSA from the CF lung
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Activity of Pulmonary Vancomycin Exposures versus Planktonic and Biofilm Methicillin-Resistant Staphylococcus aureus Isolated from Cystic Fibrosis Sputum
Nicholas S. Britt,a-b# Daniel S. Hazlett,a Rebecca T. Horvat,c Rachael M. Liesman,c Molly E. Steed,a
Department of Pharmacy Practice, University of Kansas School of Pharmacy, Lawrence, Kansas, USAa; Department of Internal Medicine, Division of Infectious Diseases, University of Kansas School of Medicine, Kansas City, Kansas, USA b; Department of Pathology and Laboratory Medicine, University of Kansas School of Medicine, Kansas City, Kansas, USAc
Running head: Pulmonary vancomycin MRSA activity in CF
Keywords: methicillin-resistant Staphylococcus aureus; cystic fibrosis; biofilms; multidrug-resistance; antibiotic tolerance; vancomycin
# Address correspondence to
Nicholas S. Britt, [email protected]. 3901 Rainbow Blvd, Mailstop 4047 University of Kansas Medical Center Kansas City, KS 66160-7231 Tel: (913) 588-5391; Fax: (913) 588-2355
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ABSTRACT Vancomycin is commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection in patients with cystic fibrosis (CF) lung disease; however, there are limited data to support the in vitro activity of this agent against MRSA isolated from CF sputum. The primary objective of this study was to evaluate the activity of vancomycin at pulmonary concentrations (intravenous and inhaled) against 4 clinical MRSA CF sputum isolates in planktonic and biofilm time-kill (TK) experiments. Vancomycin minimum inhibitory concentrations (MICs) were determined for these isolates at standard inoculum (SI; ~106 colony-forming units [CFU]/mL) and high inoculum (HI; ~108 CFU/mL), and in biofilms cultivated using physiologic media recapitulating the microenvironment of the CF lung. Vancomycin concentrations of 10, 25, 100, and 275 µg/mL were evaluated in TK experiments against planktonic MRSA at varying inocula and versus biofilm MRSA. Vancomycin MICs increased from 0.5 µg/mL at SI to 8-16 µg/mL when tested at HI. Vancomycin MICs were further increased to 16-32 µg/mL in biofilm studies. In TK experiments, vancomycin displayed bactericidal activity (≥ 3 log10 killing at 24 h) against 1/4 and 0/4 planktonic MRSA isolates at SI and HI, respectively. Against MRSA biofilms, vancomycin was bactericidal against 0/4 isolates. Based on these findings, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of patients with CF, even at high concentrations similar to those observed with inhaled therapy. Novel vancomycin formulations with enhanced biofilm penetration or combination therapy with other potentially synergistic agents should be explored.
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1. INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive genetic disorder associated with persistent lung infection [1, 2]. Staphylococcus aureus is commonly isolated from individuals with CF and infections due to methicillin-resistant S. aureus (MRSA) have increased in recent years [3-5]. A longitudinal study of patients with CF by Dasenbrook et al. found an association between MRSA infection and more rapid decline in lung function than those without MRSA [6]. More importantly, persistence of MRSA in the respiratory tract is associated with an increased risk of death compared to transient or cleared MRSA infection [7]. Intravenous vancomycin is a recommended treatment for symptomatic culturepositive MRSA in CF patients [8]. However, vancomycin only distributes into the lungs at roughly 50% of serum concentrations [9]. The ability of vancomycin to penetrate the thick mucus and biofilm characteristic of the CF lung in order to reach efficacious concentrations is also unclear. Inhaled vancomycin presents an attractive alternative to intravenous therapy due to the ability to deliver increased concentrations of the antibiotic to the site of infection while minimizing toxicity from systemic exposure. The antibacterial activity of these pulmonary vancomycin concentrations, however, has not been fully assessed. Therefore, the objective of this study was to evaluate the in vitro activity of pulmonary vancomycin exposures (intravenous and inhaled) against MRSA isolates from the sputum of patients with CF lung disease.
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2. MATERIALS AND METHODS 2.1. Bacterial Strains. Four consecutive clinical MRSA sputum isolates (CF005, CF006, CF007, and CF009) from 4 separate adult patients with CF were collected from the University of Kansas Hospital clinical microbiology laboratory (Kansas City, Kansas, USA) and frozen at -80oC prior to subsequent analysis. Methicillin resistance was confirmed by polymerase chain reaction for the mecA gene. ATCC® 25923 was used as the control strain for susceptibility and bactericidal activity testing. Mu3 (ATCC® 700698) was used as the reference strain for hVISA determination [10]. 2.2. Antimicrobials. Vancomycin powder was commercially purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fresh vancomycin stock solutions were prepared daily. 2.3. Media. Cation-adjusted Mueller-Hinton broth (CAMHB; Becton-Dickinson, Sparks, MD, USA) was used as media for susceptibility testing and time-kill experiments. Brain heart infusion agar (BHIA; Becton-Dickinson, Sparks, MD, USA) was used for all population susceptibility analyses. Tryptic soy agar (TSA; Becton-Dickinson, Sparks, MD, USA) was used for all other colony counts. In addition to conventional CAMHB, we performed susceptibility testing and biofilm time-kill experiments using artificial sputum media (ASM), a physiologic culture media developed to recapitulate the microenvironment of the CF lung [11, 12]. This media contains amino acids, mucin, and exogenous DNA, and has been validated as a biofilm model for CF sputum [13]. Artificial sputum media was prepared according to a previously published protocol and stored refrigerated at 4oC for up to 30 days [14].
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2.4. Planktonic susceptibility testing. Vancomycin minimum inhibitory concentrations (MICs) were determined by broth microdilution by testing concentrations of 0.125-64 μg/mL at a standard inoculum of approximately 1 × 106 colony-forming units (CFU)/mL (acceptable range, 5.5-6.5 log10 CFU/mL) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [15]. Vancomycin MIC ≤ 2 μg/mL were considered susceptible, per CLSI. All samples were incubated at 35oC for 24 h under aerobic conditions and read by visual inspection. High inoculum MIC testing was performed by broth microdilution at a starting inoculum of approximately 1 × 108 CFU/mL
(acceptable
range,
7.5-8.5
log10
CFU/mL)
by
testing
vancomycin
concentrations of 2–1,024 μg/mL. Following 24 h of incubation at 35 oC under aerobic conditions, bacterial growth was visualized by addition of 20 μL of a 0.5 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue; Sigma-Aldrich, St. Louis, MO, USA) to each well, as previously described [16]. After an additional 20 min of incubation at 35oC, a color change from pale yellow to blue indicated the presence of viable bacterial cells [16]. All testing was performed in duplicate to ensure reproducibility. 2.5. Biofilm susceptibility testing. For biofilm susceptibility testing, bacterial suspension (0.5 McFarland standard) was added to ASM in 2 mL flat-bottom culture plates and biofilms were cultivated for 72 h at 35oC under aerobic conditions and continuous shaking at 75 rpm. Following biofilm conditioning, vancomycin was added at concentrations of 2-1,024 μg/mL and plates were incubated for an additional 24 h at 35oC under aerobic conditions. Biofilms were disrupted by addition of cellulase and a series of manual pipetting and vortex steps to displace any viable bacterial cells from
6
mature biofilm structures prior to MIC determination [14]. Bacterial viability was assessed by the addition of MTT and MICs determined as the lowest vancomycin concentration without a visible color change from pale yellow to blue in the corresponding well. All susceptibility testing was performed in at least duplicate to ensure reproducibility. 2.6. Minimum bactericidal concentration (MBC) determination. Planktonic vancomycin MBCs were determined at standard and high inoculum following the broth microdilution method recommended by CLSI guidelines for bactericidal activity testing [17]. Vancomycin tolerance was defined as an MBC/MIC ratio ≥ 32 per CLSI guidelines [17]. For MBC determination, a 100 μL aliquot of all wells corresponding to vancomycin concentrations above the MIC was plated onto TSA, allowed to visibly dry at room temperature, and cross-streaked using a sterile cotton-tipped swab to account for antibiotic carryover effect common to vancomycin MBC assays [17]. Vancomycin MBC was defined as the lowest concentration with ≥ 99.9% killing (as compared to initial measured inoculum) after 24 h incubation at 35oC. The maximum evaluated concentration was 1,024 μg/mL in high inoculum assays. 2.8. Vancomycin time-kill assays. Time-kill assays of planktonic MRSA were performed at standard inoculum and high inoculum. We evaluated intravenous vancomycin pulmonary concentrations approximating the Cmedian (10 μg/mL) and Cmax (25 μg/mL) from a previous study of lung epithelial lining fluid [9]. To evaluate the activity of pulmonary concentrations observed with inhaled vancomycin, we tested the approximate Cmedian (100 μg/mL) and Cmax (275 μg/mL) obtained from published results of a Phase I trial of inhalable vancomycin [18]. For time-kill assays versus planktonic
7
MRSA, experiments were performed in 2 mL of CAMHB in flat-bottom cell culture plates. A 100 μL sample at each concentration was removed from incubation (35 oC) at 0, 4, 8, and 24 h and serially diluted 1:10 in cold 0.9% sodium chloride. Appropriate dilutions were drop plated on TSA and colonies were counted by visual inspection after 24 h incubation at 35oC. Mature MRSA biofilms were cultivated for time-kill assays by calibrating bacterial suspensions to a 0.5 McFarland standard in ASM and incubating for 72 h at 35 oC under aerobic conditions and continuous shaking at 75 rpm. Following this conditioning phase, biofilms were treated with vancomycin at concentrations of 100 μg/mL and 275 μg/mL. Vancomycin killing was determined by sampling at 0, 4, 8, and 24 h. For each sample, biofilms were disrupted by adding cellulase and shaking at 150 rpm for 1 h, followed by cycles of manual pipetting and vortex until no biofilm was apparent by visual inspection. Dilutions were drop plated on TSA for final colony count determination after 24 h incubation at 35oC. Time-kill curves were constructed by plotting mean colony counts (log10 CFU/mL) using GraphPad Prism (version 6.04; GraphPad Software, Inc., La Jolla, CA, USA). Bactericidal activity of vancomycin was defined as a ≥ 3 log 10 CFU/mL reduction in viable bacteria after 24 h in comparison with the starting inoculum. Bacteriostatic activity was defined as a < 3 log10 CFU/mL reduction in viable bacteria with an absence of growth in comparison with the starting inoculum. All time-kill assays were performed in triplicate. 2.9. Population analysis profiles. To determine the effect of pulmonary vancomycin exposure on the population susceptibility of MRSA isolates to vancomycin, population analysis profiles (PAPs) were performed before (T 0) and after 24 h exposure
8
(T24) to vancomycin 100 μg/mL in high inoculum time-kill assays, as previously described [10]. This concentration was chosen to best simulate the vancomycin concentration achieved for the longest duration of the dosing interval following administration of inhaled vancomycin [18]. A bacterial suspension of approximately 1 × 108 CFU/mL was plated on freshly prepared BHIA plates containing 0, 0.5, 1, 2, 3, 4, and 6 mg/L of vancomycin. After 48 h incubation at 35 oC, colony counts were determined by visual inspection (lower limit of detection 2 log10 CFU/mL). Colony counts were plotted against increasing concentrations of vancomycin and the resultant area under the curve (AUC) was calculated by the trapezoidal method. A shift in the population susceptibility profile was evaluated by calculating the ratio of the before and after PAP AUC (T24:T0 PAP AUC ratio). Any strain with an AUC ratio ≥ 0.9 times that of Mu3 was considered hVISA, as previously defined [10]. 2.10. Scanning electron microscopy (SEM). To confirm mature MRSA biofilm formation in ASM, SEM experiments were performed. Bacterial biofilms were cultivated in 72 h of ASM in a 2 mL flat-bottom cell culture plate, gently transferred to a microcentrifuge tube, and centrifuged at 10,000 rpm for approximately 30 sec to form a bacterial pellet. Following centrifugation, the supernatant was gently removed, and the sample was fixed with 2% glutaraldehyde in 0.1M cacodylate buffer. The primary fixative was then rinsed twice for 10 min with 0.1M cacodylate buffer. Samples were post-fixed in 1% osmium tetroxide in 1% cacodylate buffer for 30 min, thrice rinsed with sterile
deionized
water,
and
dehydrated
stepwise
using
increasing
ethanol
concentrations. Samples were dried using bone dry carbon dioxide in an EMS 850 critical point drier, mounted and sputter coated with gold/palladium using an
9
EMS/Quorum 150 R ES sputter coater. Final preparations were viewed using a Hitachi S-2700 SEM and captured as .TIFF files using the Quartz PCI system. 2.11. Statistical analysis. Quantitative changes in the CFU/mL between tested vancomycin concentrations were compared for each strain at 24 h by two-way analysis of variance with Tukey’s post-hoc test. A P-value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS (version 20.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 6.04; GraphPad Software, Inc., La Jolla, CA, USA) software.
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3. RESULTS All MRSA sputum isolates were susceptible to vancomycin at standard inoculum when tested using conventional culture media (Table 1). Three of the 4 strains (CF006, CF007, and CF009) met the threshold for vancomycin tolerance (minimum bactericidal concentration [MBC]/minimum inhibitory concentration [MIC] ratio ≥ 32). When tested at high inoculum, vancomycin MICs increased from 0.5 μg/mL to 8-16 μg/mL, consistent with the inoculum effect. Additionally, vancomycin MBC values increased beyond the maximum evaluated concentration of 1,024 μg/mL. Vancomycin MIC and MBC did not differ when tested in artificial sputum media (ASM) at either standard or high inoculum, suggesting that drug was stable in this physiologic media. Vancomycin MICs further increased to 16-32 μg/mL against biofilm MRSA cultivated in ASM. Mature MRSA biofilm formation was confirmed by scanning electron microscopy (SEM; Figure 1). In standard inoculum time-kill experiments, vancomycin was bactericidal at all concentrations against only 1 of the 4 planktonic MRSA isolates (CF005; Figure 2A-D). Against strain CF009, vancomycin was bacteriostatic at all concentrations except 275 μg/mL (Figure 2D). For all other isolates and concentrations, vancomycin was bacteriostatic with no significant dose-response relationship (P > 0.5; Figure 2A-D). In high inoculum time-kill experiments versus planktonic MRSA, bacterial growth was observed at a vancomycin concentration of 10 μg/mL for 3 of 4 strains, consistent with the MIC shifts observed in high inoculum susceptibility testing (Figure 3B-D). As expected based on the results of high inoculum MBC determinations, vancomycin 25, 100, and 275 µg/mL was bacteriostatic against all 4 strains analyzed (Figure 3A-D).
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Vancomycin time-kill curves versus biofilm-embedded MRSA are displayed in Figure 4. Vancomycin concentrations of 10 μg/mL and 25 μg/mL were not tested because they were subinhibitory against the majority of strains. In an ASM biofilm timekill model, vancomycin was bacteriostatic at concentrations of 100 μg/mL and 275 μg/mL against all strains tested and there were no differences in antibacterial activity between these concentrations (P > 0.5). The results of T0 and T24 vancomycin population analysis profiles (PAPs) are depicted in Figure 5. None of the strains analyzed met criteria for heterogenous vancomycin intermediate S. aureus (hVISA) classification by population analysis (AUCtest/AUCMu3 ≥ 0.9) before or after vancomycin exposure. However, a right-shift in the population susceptibility profile to vancomycin was noted in 3 of the 4 strains (Figure 5A-C). 4. DISCUSSION In this report, we describe the pharmacodynamic (PD) effects of vancomycin pulmonary concentrations against MRSA isolated from the sputum of adult CF patients. Generally, vancomycin demonstrated poor activity against these strains, particularly at high bacterial inoculum and in biofilms mimicking those which would be observed in the CF lung. We showed that vancomycin susceptibility is lost against these strains with increasing bacterial density and that vancomycin is minimally active even at high concentrations that would be observed with inhaled therapy. Lack of activity may be further explained by the fact that most of the strains tested were tolerant to vancomycin at standard inoculum and all strains were vancomycin tolerant at high inoculum. Among vancomycin tolerant strains, increased
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exposure to vancomycin may result in collateral inhibition of murein hydrolases, resulting in a loss of autolytic activity [19]. The lack of vancomycin bactericidal activity we observed does not appear to be due to a slow-growing phenotype, as a ~2 log10 CFU/mL growth was consistently observed after only 4 h in antibiotic-free media. To our knowledge, there are no data on the prevalence of vancomycin tolerance among MRSA isolated from the sputum of individuals with CF, however, we previously reported vancomycin tolerance to be present in 26.7% of MRSA bloodstream isolates at our institution [20]. The only patient-specific factor associated with this phenotype was previous exposure to vancomycin, which is frequently used to treat pulmonary MRSA among CF patients [20, 21]. A contributing factor to the high rate of vancomycin tolerance we observed in these clinical strains may also be chronic co-colonization with Pseudomonas
aeruginosa.
Previous
research
demonstrates
that
S.
aureus
downregulates penicillin-binding protein 4 in response to quorum sensing molecules released from P. aeruginosa, resulting in vancomycin tolerance [22]. Specifically, P. aeruginosa has been shown to “protect” S. aureus planktonic and biofilm populations from vancomycin through exogenous release of 2-n-heptyl-4-hydroxyquinolone N-oxide (HQNO) and siderophores without small-colony variant reversion [23]. This is of high clinical significance given the near universal prevalence of P. aeruginosa and MRSA cocolonization in patients with CF. The present research suggests that the vancomycin tolerant phenotype may persist even in monoculture, resulting in poor vancomycin activity. To our knowledge, this was the first study to evaluate the antibacterial activity of clinically achievable vancomycin concentrations versus MRSA biofilms mimicking those
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observed in patients with CF. Previous studies of vancomycin activity and penetration of biofilm-embedded MRSA have used models in which cells are cultivated on abiotic surfaces, such as polystyrene pegs or coupons, which are not representative of biofilm formation in patients with CF [23]. Nonetheless, these models have consistently shown vancomycin to have poor activity against biofilm-producing S. aureus. [24-27]. This is consistent with the findings of the current study, in which ASM biofilm-embedded MRSA isolates demonstrated reduced in vitro susceptibility to vancomycin. All strains tested were highly sensitive to changing bacterial density, as increasing the inoculum from 106 to 108 CFU/mL resulted in a profound loss of vancomycin susceptibility (16- to 32-fold MIC increase). This is consistent with previous in vitro analyses of the inoculum effect on vancomycin activity versus MRSA [16]. Even more pronounced was the decrease in bactericidal activity with vancomycin MBCs increasing to >1,024 μg/mL for all 4 strains. Clinically, CF lung disease is characterized by a large overall bacterial density (~1010 CFU/mL), of which the composition varies significantly between patients and over an individual’s lifetime [28, 29]. Bacterial density of MRSA in any single CF patient is difficult to estimate in the clinical setting, as bacteria are often embedded in complex polymicrobial mucus biofilms. For this reason, we tested vancomycin activity against varying MRSA inocula (standard and high) and modes of growth (planktonic and biofilm). We found vancomycin activity against MRSA isolated from CF sputum was consistently poor across multiple inocula and was further decreased against biofilm-entrenched MRSA characteristic of CF lung disease. We further evaluated the influence of pulmonary concentrations of inhaled vancomycin on the corresponding population susceptibility profiles. The observed right-
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shift in the T24:T0 PAP AUC ratio suggests that selection of MRSA subpopulations with reduced vancomycin susceptibility can occur even at high vancomycin exposures. The concentration-dependent nature of population susceptibility shifts has been previously demonstrated by Rose and colleagues, with the largest shifts observed at lower vancomycin AUC/MIC ratios [30]. Therefore, although we observed a significant rightshift in the population susceptibility profile after only 24 h of vancomycin 100 μg/mL exposure, this shift may be even more pronounced at lower vancomycin concentrations relative to the MIC, such as those that would be observed with intravenous vancomycin, or with prolonged vancomycin exposures. The clinical implications of our findings are further supported by a recent doubleblind, randomized, placebo-controlled trial that investigated the addition of inhaled vancomycin therapy to an intensive MRSA eradication protocol that included oral trimethoprim-sulfamethoxazole or doxycycline combined with oral rifampin and MRSA decolonization with nasal mupirocin and chlorhexidine gluconate cleanses [32]. The addition of inhaled vancomycin to this multimodal strategy did not result in significant differences in MRSA eradication rates nor improved lung function [32]. Furthermore, 28% of inhaled vancomycin-treated patients experienced bronchospasm despite pretreatment with albuterol, requiring discontinuation with the trial [32]. Novel vancomycin formulations with enhanced biofilm penetration and tolerability, such as those currently in development (ClinicalTrials.gov identifier: NCT03181932), or prolonged suppressive combination therapy with other potentially synergistic agents will likely be required to optimize outcomes in these patients.
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The current study was strengthened by its use of MRSA strains isolated from the sputum of “real-world” patients with CF. Additionally, clinical pulmonary concentrations from CF patients treated with both intravenous and inhalable vancomycin were evaluated to enhance external validity and clinical relevance. Our study also had several noteworthy limitations. First, the use of a 24 h time-kill methodology did not allow for replication of the true vancomycin concentration-time profile observed clinically. We attempted to partially mitigate this by using multiple concentrations available in published pharmacokinetic (PK) data of intravenous and inhaled vancomycin [18]. However, due to the methodology used, replication of our findings in animal or in vitro PK/PD models over a longer time period of vancomycin exposure and against additional isolates is warranted. Estimated pulmonary concentrations from intravenous vancomycin were derived from epithelial lining fluid ELF from healthy volunteers, as this data is not yet available in the CF population. Vancomycin PD targets in patients with CF are no different than those without CF; however, patients with CF may require more aggressive dosing due to increased clearance. It is well known that there is a large inter-patient variability in vancomycin exposures observed in the clinic, further complicating experimental design. Ultimately, we chose to evaluate vancomycin concentrations which we believe are representative of clinically achievable exposures in CF patients. An additional point to consider is that correlation of antibiotic susceptibility and in vitro activity with clinical response is problematic in the management of CF pulmonary infections. The CF lung is characterized by thick mucus and most bacterial communities persist firmly entrenched in biofilms, which may limit the clinical applicability of standard planktonic susceptibility testing [31]. We attempted to
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overcome this by employing an ASM biofilm TK model mimicking the biochemical conditions of the CF lung. However, the clinical applicability of biofilm antibiotic susceptibility testing in CF is still uncertain and additional data on clinical outcomes associated with intravenous and inhaled vancomycin in this vulnerable patient population is needed [31]. Only clinical strains from CF sputum were evaluated in this study and it is unclear whether the effects observed are unique to CF isolates or the growth modalities tested. Furthermore, we analyzed only a single isolated MRSA phenotype from each patient included in the study. Clinically, multiple MRSA phenotypes are likely to exist in a complex polymicrobial environment that may affect vancomycin activity. 5. CONCLUSIONS In summary, vancomycin demonstrated minimal in vitro activity against MRSA strains isolated from CF sputum, particularly at high inoculum and in biofilm. While inhaled vancomycin represents an achievement in the armamentarium to treat respiratory MRSA infections, the results of this study highlight the uniqueness and resilience of organisms isolated from CF patients. Inhaled vancomycin affords the advantage of an improved nephrotoxicity profile and higher concentrations localized at the site of infection compared to intravenous therapy. Based on the results of this study, however, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of CF patients.
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ACKNOWLEDGMENTS This study was presented in part at the 55th Interscience Conference on Antimicrobial Agents and Chemotherapy in San Diego, CA, USA. This study was classified as nonhuman subjects research and ethical approval was not required per institutional policy We would like to acknowledge Pat St. John and Larysa Stroganova at the KUMC EMRL for technical assistance, as well as Mallory Schroeder and Daniel Younggren (University of Kansas School of Pharmacy) for data collection.
DECLARATIONS Funding: NSB was supported in part by Clinical and Translational Science Award TL1 TR000120-03 from the National Institutes of Health (NIH). Electron microscopy was performed at the University of Kansas Medical Center Electron Microscopy Research Laboratory (KUMC EMRL), which is supported in part by NIH grant P20GM104936. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Competing Interests: All authors report no relevant conflicts of interests Ethical Approval: Not required
Contributions NSB and MES conceived and designed the study, oversaw all experiments, and analyzed and interpreted resulting data. DSH performed experiments and acquired data. RTH and RML assisted with data interpretation. All authors contributed to drafting the manuscript and have approved the final version of the article.
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TABLE 1. Microbiological characteristics of 4 clinical methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates a Standard media Physiologic media (CAMHB) (ASM) VAN Biofilm VAN SI MIC VAN HI MIC VAN SI MIC VAN HI MIC Isolate MIC (MBC) b (MBC) c (MBC) b (MBC) c (MBC) d 0.5 8 0.5 8 16 CF005 (1) (> 1,024) (1) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF006 (16) (> 1,024) (16) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF007 (32) (> 1,024) (32) (> 1,024) (> 1,024) 0.5 16 0.5 16 32 CF009 (16) (> 1,024) (16) (> 1,024) (> 1,024) CAMHB, cation-adjusted Mueller-Hinton broth; ASM, artificial sputum media; VAN, vancomycin; SI, standard inoculum; HI, high inoculum; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration a All units in µg/mL b ~106 colony-forming units (CFU)/mL c ~108 CFU/mL d Determined following 72 h biofilm conditioning in ASM
A
B
FIG 1. Representative scanning electron microscopy (SEM) images of biofilm methicillin-resistant Staphylococcus aureus strain CF007 cultivated in artificial sputum media for 72 h. Bacterial biofilms were viewed at 2000x (A) and 7000x (B).
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FIG 2.
Vancomycin (VAN) time-kill curves versus planktonic methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) at standard inoculum (~106 colony-forming units/mL). Dotted line indicates lower limit of detection.
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FIG 3. Vancomycin (VAN) time-kill curves versus planktonic methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) at high inoculum (~108 colony-forming units/mL). Dotted line indicates lower limit of detection.
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FIG 4.
Vancomycin (VAN) time-kill curves versus biofilm methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) following 72 h cultivation in artificial sputum media. Dotted line indicates lower limit of detection.
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A
B B
C
D
A
C
D
FIG 5. Population analysis profiles of methicillin-resistant Staphylococcus aureus cystic fibrosis sputum isolates CF005 (A), CF006 (B), CF007 (C), and CF009 (D) before (T0; purple solid line) and 24 h after (T24;blue dotted line) exposure to vancomycin 100 µg/mL in high inoculum (~108 colony-forming units/mL) time-kill assays.
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