Nafcillin enhances innate immune-mediated killing of methicillin-resistant Staphylococcus aureus
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- Sakoulas, G., Okumura, C.Y., Thienphrapa, W. et al. J Mol Med (2014) 92: 139. doi:10.1007/s00109-013-1100-7
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Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated “sensitization” to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy.
Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia.
Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides.
Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane.
Nafcillin enhances killing of MRSA by neutrophils.
Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.
KeywordsMRSAInnate immunityBeta-lactamsNafcillinHost defense peptides
The treatment of bacterial infections has become increasingly challenging due to continued emergence of antibiotic resistance. Emblematic of this problem are strains of methicillin-resistant Staphylococcus aureus (MRSA) that have reached epidemic proportions in many countries [1–3]. In the United States, S. aureus is the most common cause of hospital- and community-associated bacterial infections of the bloodstream, skin and soft tissue, and other sites, with MRSA strains comprising a large majority in many locales [4, 5]. Clinical outcomes of patients with invasive MRSA infections are significantly worse than those with methicillin-susceptible isolates, including longer hospitalizations and higher mortality (20–30 %) despite modern therapeutic interventions [6, 7]. Both hospital- and community-associated MRSA may exhibit broad resistance to multiple classes of antibiotics [3, 5], and a recent upward “creep” in vancomycin minimum inhibitory concentrations (MICs) among MRSA isolates is associated with even higher treatment failure rates [8, 9].
The ability of MRSA to produce severe invasive disease, frequently in otherwise healthy individuals, reflects multiple virulence mechanisms for host tissue invasion and immune evasion [10–12]. Normal innate antistaphylococcal immunity depends significantly upon cationic host defense peptides (HDPs) such as cathelicidins and defensins, produced by epithelial cells (e.g., skin keratinocytes) and phagocytes (e.g., neutrophils), to provide a critical first line of defense against invasive infection [13, 14]. S. aureus partially counteracts HDP killing through mechanisms such as modification of cell envelope charge and hydrophobicity, expression of drug efflux pumps, HDP-binding proteins or proteases, or modulation of cell membrane order (fluidity–rigidity) [15–18]. Notably, recent surveys have found that MRSA strains are significantly more resistant to human cathelicidin LL-37 compared to methicillin-sensitive strains [19, 20] perhaps contributing to their enhanced survival in vivo during skin or bloodstream infections.
Optimal combination antibiotic therapy for MRSA bacteremia has not been established . We recently documented a salutary clinical experience in seven patients with refractory MRSA bacteremia, using a combination of daptomycin plus the anti-staphylococcal β-lactams, nafcillin or oxacillin . In that report, we demonstrated a net reduction in the in vitro bacterial surface charge induced by diverse anti-staphylococcal β-lactams, with an associated enhancement of daptomycin binding to the staphylococcal envelope . Although the native daptomycin molecule is an anionic lipopeptide, it complexes in vivo with divalent calcium (Ca2+) to become positively charged  and target bacterial membranes in a fashion similar to cationic HDPs [24, 25]. Indeed, in recent analyses of MRSA strains that develop resistance to daptomycin during the course of therapy, concomitant resistance to cationic HDPs has been noted [26, 27]. We hypothesized that anti-staphylococcal β-lactams might also sensitize MRSA to enhanced clearance by innate HDPs and the host cells that deploy them.
In the current study, we explore the capacity of the commonly prescribed β-lactam agent nafcillin to sensitize a cadre of MRSA strains to killing by cathelicidin and other prototypical HDPs. The relevance of these outcomes is extended to ex vivo analyses of whole blood, neutrophil, and keratinocyte killing, and the effect of nafcillin exposure on MRSA survival is examined in a murine challenge model. For this foremost of contemporary bacterial pathogens, the combined results highlight potential utilities of a safe existing antibiotic otherwise dismissed by standard antimicrobial testing.
Daptomycin-susceptible USA100 MRSA D592 and its isogenic daptomycin-nonsusceptible, vancomycin-intermediate (VISA) strain, D712, were from the case study and described previously . Additional well-characterized MRSA strains studied were ATCC33591, TCH1516 (USA300), UAMS 1182 (USA300), Sanger 252 (USA200), VRSA-MI and VRSA-PA (exhibiting high-level vanA-mediated vancomycin resistance), and VISA-NJ HIP5836, obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), http://www.narsa.net/control/member/repositories.
Antimicrobial peptide killing assays
Cationic host defense peptides differing in anatomic and host source, molecular mass, net charge at pH 7.5, and proposed mechanism(s) of action were studied. Human cathelicidin LL-37 (net charge +6 at pH 7.5) and the murine cathelidicin mCRAMP (net charge +6 at pH 7.5) were purchased from AnaSpec, Inc. (Fremont, CA, USA); human neutrophil α-defensin-1, hNP-1 (net charge +3 at pH 7.5), was purchased from Peptide International (Louisville, KY, USA); and RP-1 (a synthetic 18 amino acid congener modeled after the α-helical microbicidal domain of the platelet factor IV family of peptides; net charge +8 at pH 7.5) was synthesized and authenticated as previously detailed . The concentrations of HDPs utilized in the formal killing assays were based on extensive pilot studies and represented levels that did not rapidly kill the starting inoculum in the absence of antibiotic pre-sensitization.
Bacteria were grown to stationary phase (14–16 h) at 37 °C with shaking in 5 mL of antibiotic-free LB broth or LB containing varying sub-inhibitory concentrations of the antibiotic of interest. Beta-lactams were purchased from Sigma-Aldrich (St. Louis, MO, USA), while anti-MRSA antibiotics vancomycin, linezolid, and daptomycin were purchased from the University of California, San Diego hospital pharmacy. For antibiotics with in vitro activity against MRSA and used clinically to treat MRSA infections, a concentration of 0.25× MIC was used. Bacteria were pelleted by centrifugation for 10 min, washed in 5 mL sterile phosphate-buffered saline (PBS), and resuspended to an OD600 nm of 0.5, approximating 108 cfu/mL. Bacteria were then serially diluted to ∼104 cfu/mL using RPMI + 5 % LB media and deployed in specific antimicrobial peptide killing assays at a final density of 103 cfu/mL in 100 μL in RPMI + 5 % LB. Details of these HDP killing assays have been previously published . At time 0 and after selected exposure time points, 10-μL samples were plated on tryptic soy agar plates and colonies enumerated after 24 h. HDP exposure times were optimized, based on a number of pilot studies as either 60 min (LL-37; mCRAMP) or 90 min (RP-1; hNP-1). The mean percentage survival (±SD) was quantified and expressed in relation to cfus at time 0.
Cathelicidin LL-37 binding studies
MRSA Sanger252 was grown to OD600 nm 0.6–0.8 in phenol-free RPMI media with and without nafcillin 5 or 10 mg/L to which was then added rhodamine-labeled LL-37 (Phoenix Pharmaceuticals, Burlingame, CA, USA) at 1 μM (5 mg/L). After 30 min at 37 °C with shaking at 200 rpm, cells were washed three times with phenol-free RPMI. The final wash contained 1 mg/L DAPI (4′,6-diamidino-2-phenylindole) nucleic acid stain and the cells were visualized microscopically .
Whole blood killing assays
Blood from healthy donors was collected using 50 μg/mL lepirudin (Refludan; Schering, Kenilworth, NJ, USA) as anti-coagulant to preserve complement activity . MRSA strains 592, 712, and Sanger 252 were grown to log phase in either 20 μg/mL nafcillin or media alone, and 104 cfu was incubated in a total volume of 400 μL blood, rotating at 37 °C. After 60 min, 25 μL was removed and blood was lysed in water. Remaining bacteria were enumerated on Todd–Hewitt agar (THA) plates and survival was calculated as the percentage of the initial inoculum. Experiments were performed using blood from at least three individual donors. These studies were approved by the University of California San Diego Human Research Protections Program.
Neutrophil killing assays
Neutrophils were freshly isolated from the blood of healthy donors using PolyMorphPrep-kit (Fresenius Kabi) and erythrocytes lysed with sterile H2O as previously described . Organisms prepared identically to those used in the whole blood killing assays above were grown to log phase in 20 μg/mL (D592 and D712) or 2 μg/mL (Sanger 252) nafcillin, 0.5 μg/mL vancomycin, 0.1 μg/mL ceftaroline or media alone (untreated), and incubated at a multiplicity of infection (MOI; MRSA/neutrophil ratio) = 1, with 5 × 105 neutrophils in RPMI containing 2 % (vol/vol) of 70 °C heat-inactivated FBS . After 90 min, incubation at 37 °C + 5 % CO2, cells were lysed with 0.025 % Triton X-100, and the total number of remaining bacteria was enumerated on THA plates. Survival was calculated as the mean percentage (±SD) of the initial inoculum. Where indicated, in parallel studies, neutrophils were treated with 10 μg/mL diphenyleneiodonium chloride (DPI; Sigma) versus vehicle control (DMSO) for 1 h prior (in order to block the respiratory burst), or 5 μg/mL cytochalasin D (cyt D; Sigma) versus vehicle control (DMSO) for 10 min prior to the addition of bacteria (in order to block phagocytosis); survival was calculated as the mean percentage (±SD) of the bacterial growth control, grown under the same conditions without neutrophils. Experiments were performed using blood from at least three individual donors. These studies were approved by the University of California San Diego Human Research Protections Program.
In order to assess the impacts of nafcillin pretreatment on neutrophil phagocytic capacity, neutrophils prepared as described above were resuspended to 106 cells/mL in Opti-MEM culture medium (Invitrogen, Grand Island, NY, USA) containing 1 mg/mL pHrodo S. aureus BioParticles Conjugates (Invitrogen) prepared according to the manufacturer’s instructions. Samples were prepared with 0, 5, or 20 μg/mL nafcillin. Each sample was aliquoted 100 μL in triplicate to 96-well plates (105 cells/well); the plates were centrifuged 350×g for 5 min and incubated at 37 °C with 5 % CO2. Fluorescence was measured every 15–30 min at 544 nm excitation/590 nm emission.
H2O2 sensitivity assays
To quantify the impact of β-lactam pre-exposures on subsequent resistance to oxidative stress, MRSA Sanger 252 was grown overnight to stationary phase (12–16 h) in LB broth with or without sublethal nafcillin. Cells were pelleted, washed in PBS, and resuspended in PBS containing 1.5 % H2O2. After 90 min, cells were diluted and plated on THA plates, incubated overnight, and viable cfus enumerated to determine mean percent of surviving bacteria (±SD). A minimum of three independent assays was carried out.
HaCaT keratinocyte killing assay
We used HaCaT keratinocyte cells to examine the effects of nafcillin on the cathelicidin-mediated anti-staphylococcal activity of keratinocytes. HaCaT keratinocyte cells passaged in the absence of antibiotics were seeded 2.5 × 105 onto 24-well tissue culture plates. After 24 h, cells were washed with 1× PBS and 450 μL RPMI + 2 % 70 °C heat-inactivated FBS was added to each well. MRSA Sanger 252 was grown to log phase (OD600 nm 0.5) in LB broth, resuspended in PBS to OD600 nm 0.4, and diluted in PBS such that 50 μL was added to each well [this represented 2.5 × 105 cfu (MOI = 1.0) or 2.5 × 104 cfu (MOI = 0.1)]. Bacteria were centrifuged onto cells at 350×g for 10 min and incubated at 37 °C in humidified 5 % CO2 incubator for 2 h. At the end of the incubation, 50 μL 0.25 % Triton X-100 was added to each well, cells were removed with a cell scraper, resuspended in the media, diluted 1:10–10,000, and 25 μL plated onto THA plates for bacterial enumeration. Experiments were performed twice in triplicate, and results were expressed as the ratio of bacterial cfu in HaCaT/bacterial cfu in cell-free control.
Animal model of MRSA cutaneous infection
We used a murine subcutaneous infection model in order to translate our in vitro and ex vivo studies above to in vivo relevance. For ex vivo antimicrobial exposure studies in these investigations, MRSA Sanger 252 was grown overnight to stationary phase in 40 mL of antibiotic-free LB or LB containing sublethal nafcillin (5 μg/mL) or vancomycin (0.25 μg/mL). Bacteria were then washed in 40 mL PBS, and resuspended in 2 mL of PBS + 2 mL of Cytodex beads (1 μg/mL), yielding approximately 1010 cfu/mL. Bacterial inocula were confirmed for bacteria grown in the presence or absence of antibiotic, with less than 2-fold variability in colony counts and identical OD600 nm. We point out that we have repeatedly observed slightly lower CFU/OD600 nm ratios for nafcillin-grown MRSA due to increases in cellular aggregation by microscopy. Then, 0.1 mL of the bacterial suspension was injected subcutaneously (sc) into 8-week-old (∼25 g) female CD1 mice, and developing lesion sizes were manually measured for 3 days and expressed as square millimeters. For in vivo antimicrobial exposure studies, MRSA Sanger 252 grown in antibiotic-free LB broth was prepared and injected sc as above into female CD1 mice that received either sterile PBS or nafcillin 2.5 mg (100 mg/kg) sc, dosed 8, 4, and 0.5 h before bacterial injection, 4 h after the bacterial injection, and every 12 h thereafter for 3 days. Lesion sizes were recorded on days 2–4 after bacterial injection; animals were sacrificed and their lesions harvested and quantitatively cultured for enumeration of surviving cfu (±SD) at day 4. All animal studies were performed under protocols that were reviewed and approved by the UCSD Institutional Animal Use and Care Committee. All animal work in this investigation was performed in accordance with national and local guidelines that are in place to maximize humane animal treatment.
Data were analyzed using GraphPad Prism 5.03 (GraphPad Software). Analyses were performed using either the nonparametric Kruskal–Wallis, one-way ANOVA with Tukey’s post-test multiple comparison modification or the unpaired Student’s t test where appropriate. Correlation analysis was performed using Spearman’s rho where indicated. P values <0.05 were considered statistically significant.
Nafcillin and other β-lactam antibiotics sensitize MRSA to killing by LL-37 and other mammalian HDPs
To explore potential host factors in the dramatic clinical response of the case study, we tested how exposure to nafcillin affected the activity of human cathelicidin LL-37 against the patient’s original (strain D592, daptomycin- and vancomycin-susceptible) and the day 21 (strain D712, daptomycin-nonsusceptible and vancomycin-intermediate-resistant) MRSA isolates. The concentration of LL-37 can be severely increased at sites of inflammation, and levels up to 25 μg/mL have been reported in bronchoalveolar lavage fluid from infants with pulmonary infections  and cystic fibrosis patients , and much higher levels (over 1 mg/mL) have been reported from inflammatory skin lesions (e.g., psoriasis) . A variety of different heat-killed Gram-positive and Gram-negative bacterial species induced production of 1 to 8 g/mL of LL-37 from freshly isolated human neutrophils upon 24 h in vitro exposure .
Nafcillin sensitizes MRSA to killing by human whole blood, neutrophils, and keratinocytes
Cathelicidin LL-37 exerts its anti-staphylococcal activity within the phagolysosome , and the effect of nafcillin sensitization of MRSA to killing by LL-37 and other neutrophil HDPs (e.g., hNP-1) likely plays a major role in neutrophil intracellular killing. Notably, nafcillin sensitization to neutrophil killing was preserved even when oxidative burst function was blocked pharmacologically by diphenyleneiodonium (DPI) (Fig. 4d); furthermore, growth in sublethal nafcillin did not increase the susceptibility of MRSA to hydrogen peroxide killing (Fig. 4f). Keratinocytes also produce abundant cathelicidin HDP in response to injury or bacterial infection, contributing in a critical fashion to the cutaneous innate immune barrier [14, 43]. Prior growth in sublethal nafcillin increased the susceptibility of MRSA to killing by human keratinocyte cell line HaCaT (Fig. 4g).
Nafcillin modifies MRSA pathogenesis in a murine subcutaneous infection model
For the past several decades, clinical medicine has observed the emergence of multidrug-resistant pathogens of increasing concern [44–46]. Alarm over antimicrobial resistance among Gram-negative bacteria centers on the paucity of novel compounds with activity against increasingly common Acinetobacter baumanii and carbapenem-resistant Enterobacteriaciae. For Gram-positive bacteria in general, and for MRSA in particular, the concern is less centered on the lack of novel antibiotics, but rather on the millions of infections caused in both healthcare and community settings, with increasing adverse consequences individually to patients and to the healthcare system overall. Despite the fact that the U.S. Food and Drug Administration has approved five new antibiotics for treatment of MRSA infection in the last 12 years, data consistently show increased mortality of MRSA bacteremia compared to methicillin-sensitive S. aureus (MSSA) bacteremia [6, 7]. Invasive MRSA infection was recently estimated to account for more deaths per year in the USA than HIV/AIDS . In infections caused by MRSA, clinicians have had no choice but to resort to “second or third tier” antimicrobials, in particular vancomycin, bemoaning extensive data and common appreciation of the superiority of β-lactam antibiotics over glycopeptides and other classes as anti-staphylococcal agents for MSSA [48, 49].
In the current study, we have shown that nafcillin, at concentrations far below MIC, sensitize MRSA to killing by human cathelicidin LL-37 and other cationic HDPs, in association with increased LL-37 binding to the MRSA cell envelope. Nafcillin-mediated sensitization of MRSA was subsequently observed in killing assays where enhanced HDP killing is anticipated, such as whole blood, neutrophils, and keratinocytes. This effect of sensitization to LL-37-mediated killing, while induced at various degrees by all other β-lactams tested, was not induced by sub-MIC concentrations of other commonly prescribed non-β-lactam, anti-MRSA antibiotics, in particular vancomycin, consistent with prior observations . Along these lines, we recently reported that addition of the β-lactam ampicillin to ongoing daptomycin therapy led to rapid clearance of a protracted bacteremia caused by ampicillin- and vancomycin-resistant Enterococcus faecium and potentiated killing of the isolate by LL-37 and other cationic HDPs . Thus, it appears that β-lactams, unlike glycopeptides, may exert their in vivo anti-staphylococcal (and anti-enterococcal) activity in two ways: (a) directly against susceptible organisms, and (b) indirectly by boosting the pharmacodynamic effects of endogenous antimicrobial HDPs against both non-susceptible and susceptible organisms. This may at least partially explain the clinical superiority of β-lactams over vancomycin against MSSA and underscores the importance of prioritizing β-lactam therapy over glycopeptide therapy in serious MSSA infections .
While detailed mechanism of this “sensitizing” effect is still unknown, it has been hypothesized that β-lactams may prompt the release of lipoteichoic acid (LTA) from the cell envelope, which could either increase cell wall autolysin activity or reduce substrate availability for dlt-mediated LTA D-alanylation. A lack of D-alanylated LTA may in turn enhance relative net negative envelope charge and thus increase susceptibility to killing by cationic daptomycin and HDPs . Critical events defining β-lactam and HDP interaction may also lie in the divisome complex. Daptomycin insertion into the Gram-positive membrane results in patches of membrane defects triggering recruitment of cell division proteins such as divIVA to these sites . Pharmacological inhibition of the ClpXP protease that regulates the cell division protein FtsZ increases S. aureus susceptibility to daptomycin and LL-37 , while inhibition of FtsZ directly can restore methicillin susceptibility to MRSA .
These current data, along with our previously published case series , suggest significant potential therapeutic benefit of using β-lactams as adjunctive MRSA therapy in combination with daptomycin or other possibly anti-MRSA antibiotics. In addition, these data suggest that the choice of surgical antibiotic prophylaxis in patients who are colonized with MRSA deserves further scrutiny. While clinicians have frequently used vancomycin prophylaxis in this setting, our present data, particularly in the animal model, support the notion that β-lactams may more effective in preventing the establishment of an MRSA infection than in vitro susceptibility metrics might suggest .
While there have been extensive reporting of drug–drug interactions for synergies and antagonisms between traditional antibiotics, few such studies have explored such interactions between conventional with endogenous antibiotics (i.e., HDPs). An early report by Yeaman et al.  suggested that platelet microbicidal proteins could synergize with and prolong the post-antibiotic effect of cell-wall-acting antibiotics against S. aureus. Conversely, Kristian et al. demonstrated that various bacteriostatic antibiotics (e.g., erythromycin, tetracycline, etc.) acted antagonistically to impair killing of Gram-positive and Gram-negative bacteria by LL-37 and other HDPs, as well as by serum and whole blood .
As our previous case series outlined, the addition of an anti-staphylococcal β-lactam to daptomycin therapy in refractory MRSA bacteremia resulted in rapid bacteremia clearance . We have continued to employ this combination successfully using cefazolin and ceftaroline in combination with daptomycin for refractory MRSA and MSSA bacteremias as well as refractory methicillin-resistant Staphylococcus epidermidis bacteremia in cases of infected left ventricular assist devices that cannot be removed (unpublished data). We originally attributed the benefit of this regimen to direct synergy between β-lactams and daptomycin against MRSA, a phenomenon recently confirmed by others and shown to prevent selection of daptomycin-resistant derivatives . Moreover, recent clinical data suggests that for patients with underlying renal insufficiency and endovascular S. aureus infections, the addition of β-lactam to daptomycin may improve outcome compared to daptomycin monotherapy . The current study provides an additional explanation for the clinical efficacy findings, i.e., the potential for boosting HDP-mediated and/or neutrophil-mediated killing of MRSA by nafcillin. Our findings indicate that β-lactam antibiotics deemed ineffective by conventional susceptibility testing could in fact have significant pharmacodynamic interactions with endogenous HDPs that translate to clinical utility.
One potential caution to this approach is that in vitro subinhibitory nafcillin can increase expression of S. aureus exotoxins, including toxic shock syndrome toxin-1, Panton–Valentine leukocidin, and α-hemolysin . However, it is encouraging that concomitant β-lactams did not negatively influence clinical outcome in cases of MRSA bacteremia derived from soft tissue infection . Larger scale prospective controlled clinical studies are needed to formally evaluate the clinical benefit of adjunctive β-lactam therapy and the optimal combination therapies for MRSA bacteremia.
We thank Anna Salvioni for assistance with peroxide assays. This research was supported by National Institutes of Health grants HD071600 (GS), AI057153 (VN), AI052453 (VN), R01GM073898 (JP), AI39108 (ASB), AI39001 (MRY), and AI48031 (MRY). CYO was supported through the UCSD/SDSU IRACDA Postdoctoral Fellowship Program (GM06852). No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflicts of interest
GS has received speaking honoraria from Cubist, Forest, and Pfizer Pharmaceuticals, consulting fees from Cubist and Forest Pharmaceuticals, and research grant support from Forest Pharmaceuticals. ASB has current research grants from Cubist Pharmaceuticals and Trius Therapeutics, and has received speaking honoraria from Cubist Pharmaceuticals. MRY is a founder of NovaDigm Therapeutics, Inc., and has participated in research programs supported in part by grants from Cubist Pharmaceuticals. VN was on the Scientific Advisory Board of Trius Therapeutics.