1 β-lactams increase the antibacterial activity of daptomycin against

32 Pages·2012·0.96 MB·English

AAC Accepts, published online ahead of print on 17 September 2012
Antimicrob. Agents Chemother. doi:10.1128/AAC.01525-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 β-lactams increase the antibacterial activity of daptomycin against clinical MRSA strains and
2 prevent selection of DAP-resistant derivatives
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4 Shrenik Mehta, Christopher Singh, Konrad B. Plata, Palas K Chanda, Arundhati Paul, Sarah Riosa,
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5 Roberto R. Rosato and Adriana E. Rosato∗ o
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7 Department of Pathology and Genomic Medicine, Center for Molecular and Translational Human o
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8 Infectious Diseases Research, The Methodist Hospital Research Institute, Houston, TX t
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10 Corresponding author:
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11 The Methodist Hospital Research Institute o
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12 6670 Bertner Ave., Room R6-113, Houston, TX 77030 il
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13 Phone: [phone]
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14 Fax: [phone] g
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15 e-mail: [email protected]
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18 Running title: Daptomycin/β-lactams and DAP resistance in MRSA
19 Keywords: MRSA, daptomycin, see-saw effect, β-lactams
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21 ABSTRACT
22 Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important
23 pathogens both in health care and community-onset infections. Daptomycin (DAP) is a cyclic
24 anionic lipopeptide recommended for treatment of skin, bacteremia and right-side endocarditis
25 caused by MRSA. Resistance to DAP (DAPR) has been reported in MRSA and is mostly
26 accompanied with parallel decrease in oxacillin resistance, process known as “see-saw” effect. Our
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27 study provides evidence that the see-saw effect applies to other β-lactams and carbapenems of o
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28 clinical use including nafcillin (NAF), cefotaxime (CTX), amoxicillin-clavulanic (AMC) and lo
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29 imipenem (IMP) in heterogeneous DAPR-MRSA but not in MRSA expressing homogeneous β- d
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30 lactam resistance. The combination DAP/β-lactam in terms of antibacterial efficacy was evaluated in
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31 isogenic DAPS/R MRSA originally obtained from patients that failed DAP monotherapy. Both in- m

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32 vitro (MIC, synergy kill-curve) and in-vivo (wax worm model) approaches were used. In these
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33 models, DAP/β-lactam was proven highly synergistic against both heterogeneous and homogenous :/
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34 clinical DAPR-MRSA strains. Mechanistically, β-lactams induced a reduction in the cell net positive a
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35 surface charge reverting the increased repulsion provoked by DAP alone, effect that may favor the s
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36 binding of DAP to the cell surface. An ease of in-vitro mutant selection was observed when DAPS- .
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37 MRSA strains were exposed to DAP. Importantly, the association DAP/β-lactams prevented the g
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38 selection of DAPR variants. In summary, our data show that DAP/β-lactam combination may n

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39 significantly enhance both the in-vitro and in-vivo efficacy of anti-MRSA therapeutic options against p
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40 DAPR- infections, and represent an option in preventing DAPR selection in persistent or refractory il 9
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41 MRSA infections.
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43 INTRODUCTION
44 Methicillin–resistant Staphylococcus aureus (MRSA) has emerged as one of the most important
45 pathogens, both in hospital (HA-MRSA) and community-acquired infections (CA-MRSA) (30).
46 Resistance to β-lactam antibiotics is due to the acquisition of mecA, a gene encoding penicillin-
47 binding protein 2a (PBP-2a), a β-lactam-insensitive target enzyme that cross-links cell wall and D
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48 allows the cell to grow while the its usual cross-linking enzymes are bound and inactivated by β- n
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49 lactam antibiotics. Most strains of S. aureus also produce β-lactamase encoded by blaZ, which can d
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50 hydrolyze β-lactam antibiotics and render them inactive (18,49). Heterogeneous expression of β- f
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51 lactam resistance in MRSA strains is a characteristic of both HA-MRSA and CA-MRSA strains, by
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52 which only an small part of population expresses resistance to ≥10 μg of oxacillin per ml :
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53 (heterotypic resistance [HeR]), while in other isolates, most of the population expresses resistance to c
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54 a high level (homotypic resistance [HoR]) (7,13,14,43). The cyclic anionic lipopeptide antibiotic m
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55 daptomycin (DAP) is a produced by Streptomyces roseosporus (4), and is recommended for rg
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56 treatment of skin and skin structure infections, bacteremia and right-side endocarditis caused by n

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57 MRSA, as well as patients with prolonged MRSA bacteremia (>7 days) which are at high risk for r
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58 metastatic complications and death (2). DAP mechanism of action is based on its Ca+2-dependent , 2
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59 insertion into the bacterial cell membrane producing in turn its depolarization followed by the 9

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60 extrusion of potassium ions, arrest of macromoles synthesis and cell death (3). A number of gene g
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61 mutations have been found to be associated with DAP resistance including those in mprF, the two- s
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62 component system YycFG and RNA polymerase subunits RpoB/C (17). Both changes in membrane
63 fluidity (27,28) and cell wall thickness (46) were also shown to affect S.aureus DAP susceptibility.
64 DAP-resistant (DAPR) S. aureus strains remain rare. However, when encountered, they present a
65 treatment challenge as optimal therapy is still undefined (34). Recent guidelines from the Infectious
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66 Diseases Society of America for recommended DAPR MRSA treatment options remain limited to a
67 small number of options (if the strain is still DAP-susceptible [DAPS]), such as linezolid,
68 quinuspristin-dalfopristin, trimethroprim-sulfamethoxazole, and telavancin (10,23,40).
69 Unfortunately, these compounds are typically bacteriostatic or may be limited by safety concerns
70 (40). Since DAPR MRSA infections occur most commonly in patients with complicated, deep-seated
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71 infections such as osteomyelitis, septic arthritis, or endocarditis, the optimal therapy should be an
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72 agent that is both bactericidal and relatively safe. Interestingly, we and others have observed that lo
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73 resistance to DAP sensitizes MRSA to oxacillin (OXA), a process known as a “see-saw” effect d

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74 (26,47). Similarly, the “see-saw” effect has been observed in some in-vitro-selected vancomycin- m

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75 intermediate S. aureus (VISA) strains in which gradual increases of vancomycin (VAN) minimum tt
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76 inhibitory concentrations (MICs) are accompanied by parallel decreases in the levels of β-lactam /a
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77 resistance (38,39). a
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78 In the present study, we demonstrate that combinations of DAP with OXA (in-vitro) and rg
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79 nafcillin (NAF) (in-vivo) display very potent synergistic interactions against both HeR and HoR n

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80 clinical MRSA strains. Furthermore, we also show that the DAP-mediated “see-saw” effect is r
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81 observed with other β-lactams with clinical applications including amoxicillin-clavulanic (AMC), , 2
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82 cefotaxime (CTX) and imipenem (IMP). Mechanistically, we found that co-incubation of DAP with 9

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83 OXA, AMC, CTX or IMP induced a significant reduction in the cell net positive surface charge, g
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84 reverting DAP-induced increase, which in turn may favor the binding of DAP to the cell surface.In s
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85 addition, our study provides evidence that the DAP/β-lactam combination may also be used to
86 prevent the emergence of DAP resistance during therapy. We expect that the information gleaned
87 from our studies will represent an important contribution for the treatment of MRSA infections.
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88 MATERIALS AND METHODS
89 Bacterial strains. All of the strains used in this study and are listed in Table 1 and were previously
90 reported (26). Pulsed-field gel electrophoresis was previously determined to reliably identify the
91 clonality of the isogenic strains (5,26,42).
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92 Materials and media. Trypticase Soy Agar with 5% sheep blood (BBL, Sparks, MD) and Mueller- o
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93 Hinton (MH) agar (BBL Microbiology System, Cockeysville, MD) with and without additives lo
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94 (Sigma, St. Louis, MO; United States Biochemicals, Cleveland, OH) were used for subculture and e
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95 maintenance of S. aureus strains. o
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96 Antibiotics. Standard reference powders [OXA, NAF, CTX, AMC, IMP and VAN) were obtained p
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97 from Sigma-Aldrich, St. Louis, MO. DAP was provided by Cubist Pharmaceuticals (Lexington, a
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98 MA). Antimicrobial susceptibility to OXA, NAF, CTX, AMC and VAN were determined according s
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99 to the guidelines of the Clinical and Laboratory Standards Institute (29). DAP MICs were r
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100 determined by Etest (AB Biodisk, Solna, Sweden). n

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101 Comparison of relative net cell surface charge. Net cell surface charge was determined in DAP- il
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102 R/S (CB1634/CB1631) strains exposed to DAP, OXA and DAP/OXA by quantifying the association 2
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103 of the highly cationic molecule cytochrome c (pI 10; Sigma) to the staphylococcal surface. The
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104 amount of cytochrome c remaining in the postcentrifugation supernatant after a 10-min binding g
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105 interaction with S. aureus cells was quantified spectrophotometrically at an optical density at 530 nm t
106 (OD ). The more unbound cytochrome c is detected in the supernatant, the more a positive charge
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107 existed on the bacterial cell surface. Strains SA113 and SA113 null mutant dltA were used as
108 controls (dltA positive and negative), respectively (32).
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109 Selection of DAPR n-vitro mutants from DAPS strains. To analyze spontaneous DAP resistance,
110 large inoculums (109 CFU/ml) of DAPS strains CB1631 and CB5011 (Table 1) were serially diluted
111 in MH agar/Ca2+ 50 μg/mL with increasing concentrations of DAP (1x, 2x, 4x, and 8x DAP MICs
112 values). The plates were then incubated for 48 h at 35 °C before the colonies were enumerated.
113 Colonies isolated on DAP-containing agar plates were re-tested for DAP MIC values. For
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114 progressive DAP resistance selection, bacteria (107 CFU/ml) were exposed in MH broth/Ca2+ o
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115 50 μg/mL to stepwise, two-fold increasing concentrations of DAP alone for 7 consecutive days (12). lo
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116 After 24 h incubation at 37°C, the tubes displaying the highest antibiotic concentration and still d

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117 showing turbidity were used to inoculate a new series of tubes containing antibiotic dilutions. The m

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118 stability of DAPR derivatives was assessed by serial passage of the organisms on antibiotic-free tt
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119 medium for 5 consecutive days. a
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120 Synergy time-kill curves. Bactericidal synergy assays (at 0, 2, 4, 6, 8, and 24 h) for DAP and β- sm
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121 lactams OXA, AMC, and IMP were performed using MH broth/50 μg/mL Ca2+ with an initial rg
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122 inoculum of 1x106 CFU/ml at ½ MICs (based on individual strain E-Test data shown in Table 2), as n

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123 previously described (19). A minimum of two independent experiments were run for each DAP/β- r
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124 lactam combination. 2
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125 Treatment of infected Galleria mellonella (“wax” worm) larvae with DAP and β-lactams. b
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126 Groups of Galleria mellonella larvae (10/group) were inoculated with 10μl of a bacterial suspension ue
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127 of CB1634, CB5012 and CB1631 containing 1.5x106 CFU/ml as previously described (9, 31)
128 injected into the last left proleg and incubated for two hours with wood chips at 37C. All larvae
129 were confirmed to be alive at 2 h post-inoculation (here designated 0 h). Then, the first treatment
130 doses of DAP (10mg/kg) and NAF (5mg/kg) as recommended for clinical use (1,35) were
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131 administered in PBS into the right hind most proleg, and reincubated for 24h at 37C. In addition,
132 one group of larvae that had been inoculated with live bacteria received PBS only as treatment.
133 Repeat treatment doses of DAP, NAF, or PBS were given at 24 and 48 h. In addition, the uninfected
134 control group received PBS treatments to control for multiple injections. G. mellonella larvae
135 possess an immune system with reasonable homology to vertebrates, containing a digestive tract,
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136 loosely organized muscular system, biosynthetic fat body and hemolymph that, analogous to blood,
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137 transports nutrients, hemocytes and immune molecules. At least two of the six subsets of hemocytes lo
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138 described in G. mellonella larvae are capable of phagocytosis (22). Numerous enzymatic cascades d

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139 akin to complement fixation and blood coagulation occur in the hemolymph, resulting in hemolymph m

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140 clotting and melanin production, key defense mechanisms against invading microbes. These tissue tt
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141 types are similar to those encountered by S. aureus during invasive infections in humans. a
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142 DNA manipulation and sequencing. Chromosomal DNA was prepared by using a Qiagen genomic m
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143 DNA preparation kit (Qiagen, Inc. Valencia, CA) according to the manufacturer's directions. rg
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144 Sequencing of all PCR amplification products was performed by the Nucleic Acid Research Facility n

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145 at GENEWIZ (South Plainfield, NJ). Sequence analysis of mprF from in-vitro-selected mutants from r
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146 DAPS clinical isolates was performed by using mprF primers as previously described (26). ,
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147 Consensus sequences were assembled from both orientations with DNASTAR Advance 10 software 9

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148 for Windows (InforMax, Bethesda, MD). S. aureus N315 (accession # BA000018) was used as a
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149 positive control. s
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150 Statistical analyses. Statistical tests were performed using SPSS v17.0 for Windows (SPSS Inc.,
151 Chicago, IL, USA). The survival data were plotted using the Kaplan–Meier method.
152 RESULTS
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153 In-vitro susceptibility to β-lactams. MICs of several β-lactams were determined in clinical MRSA
154 isogenic strains recently described (26) that were obtained from patients with S. aureus infections
155 before and after treatment with DAP. As summarized in Table 1, the DAPR-mediated “see-saw”
156 effect to OXA susceptibility we previously reported in heterogeneous DAPR-CB5012 and -CB1634
157 MRSA strains (26) was also observed with other β-lactams including NAF, CTX, AMC, and IMP
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158 (Table 1). In contrast, homogeneous MRSA DAPR-CB5014 and -CB5036 strains displayed no DAP- w
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159 mediated “see-saw” effect with MIC values that remained unchanged between DAPS and DAPR loa
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160 strains. No gain on resistance to VAN was observed with MICs range from 1 to 2 μg/mL for the d

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161 DAPS/DAPR strains, respectively. Thus, these results showed that the DAP-mediated “see-saw” m

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162 effect previously described in these sets of clinical MRSA strains could be extended to other β- ttp
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163 lactams as well. a
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164 In-vitro Activity of DAP/β-lactam Combination Against DAPR MRSA Strains. Previous m
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165 observations (10,40,47), together with our results showing that the DAP-mediated “see-saw” effect
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166 may apply not only to OXA (26) but also to other β-lactams (Table 1), led us to investigate whether
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167 co-administration of DAP and β-lactams may provide a novel and effective approach against DAPR il
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168 MRSA infections. The interactions between DAP and several β-lactams currently used in clinical 0
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169 therapeutics were analyzed in DAPR strains by using synergy time-kill curves. Clinical b
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170 heterogeneous DAPR-CB1634 and DAPR -CB5012 MRSA strains (Fig. 1 and Suppl. Fig. 1, u
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171 respectively) were used to determine the in-vitro efficacy of DAP in combination with OXA, IMP,
172 AMC and CTX. In vitro synergy-kill experiments were performed at 0, 2, 4, 6, 8, and 24 h using MH
173 broth with an initial inoculum of 1x106 CFU/ml in the presence of ½ MICs (Table 1) of DAP and
174 each of the β-lactams. The size of the inoculum was determined by matching bacterial counts
175 commonly achieved in all target tissues of animals with experimental infective endocarditis (44,45).
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176 At these concentrations, DAP or β-lactams OXA, IMP or AMC added alone displayed at most a
177 delayed growth during the first hours of incubation without significant bactericidal effects (Fig. 1
178 and Suppl. Fig. 1). In contrast, when administered together, the combination of DAP/β-lactams was
179 highly synergistic as demonstrated by cell killing at 24 h ≥ 5 log CFU vs. single agents and the initial
180 inoculum (Fig. 1 and Suppl. Fig. 1). These results suggested that the DAP/β-lactam combination
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181 may have a major impact as an anti-infective alternative. w
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182 Analysis of DAP/β-lactam effects in homogeneous β-lactam resistant DAPR-CB5014 and - de
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183 CB5036 strains. Among the group of strains under investigation, two of them (DAPR-CB5036 and - fro
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184 CB5014) did not display the DAP-mediated “see-saw” effect, i.e. their MICs to OXA remained the h
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185 same (≥ 512) when compared to their isogenic DAPS counterparts (Table 1 and (26)). These strains :/
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186 may pose a different challenge both from a mechanistic point of view and, more importantly, in the c
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187 clinical environment. Considering their MIC profiles to OXA, we may have anticipated no DAP/β- m
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188 lactam interactions. However, it is plausible to hypothesize that such a mechanism may have not /

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189 been revealed by phenotypic analyses in the absence of DAP pressure, as previously shown in the A
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190 context of VAN+OXA combination therapies in VRSA strains (16). To test this hypothesis, il
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191 overnight culture of strains DAPR-CB5014 (Figure 2A) and CB5036 (data not shown) were grown in 2
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192 the presence of sub-lethal concentrations of DAP (¼-½ MICs) with 50μg/mL Ca2+, after which the
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193 adjusted inoculum was plated onto MH agar containing DAP (¼ - ½ of MICs, 0.5-1μg/ml, gu
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194 respectively). OXA Etest strips were placed on the plates and incubated for 24 h, after which a t
195 pronounced decrease of the MIC to OXA was observed, i.e., from 512 μg/ml to 1.5/0.64 μg/ml,
196 DAP 0.5/1 μg/ml, respectively (Fig.2A). Similar results were obtained with strain DAPR- CB5036
197 (data not shown). To further investigate these interactions, in vitro synergy kill curves (0, 2, 4, 6, 8
198 and 24 h) of DAPR-CB5014 (Fig. 2B) and -CB5036 (data not shown) were performed using MH
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