β-Lactams Enhance Daptomycin Activity against Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium in In Vitro Pharmacokinetic/Pharmacodynamic Models

Enterococcus faecalis and Enterococcus faecium are frequently resistant to vancomycin and β-lactams. In enterococcal infections with reduced glycopeptide susceptibility, combination therapy is often administered. Our objective was to conduct pharmacokinetic/pharmacodynamic (PK/PD) models to evaluate β-lactam synergy with daptomycin (DAP) against resistant enterococci. One E. faecalis strain (R6981) and two E. faecium strains (R6370 and 8019) were evaluated. DAP MICs were obtained. All strains were evaluated for response to LL37, an antimicrobial peptide, in the presence and absence of ceftaroline (CPT), ertapenem (ERT), and ampicillin (AMP). After 96 h, in vitro models were run simulating 10 mg DAP/kg body weight/day, 600 mg CPT every 8 h (q8h), 2 g AMP q4h, and 1 g ERT q24h, both alone and in combination against all strains. DAP MICs were 2, 4, and 4 μg/ml for strains R6981, R6370, and 8019, respectively. PK/PD models demonstrated bactericidal activity with DAP-CPT, DAP-AMP, and DAP-ERT combinations against strain 8019 (P < 0.001 and log₁₀ CFU/ml reduction of >2 compared to any single agent). Against strains R6981 and R6370, the DAP-AMP combination demonstrated enhancement against R6370 but not R6981, while the combinations of DAP-CPT and DAP-ERT were bactericidal, demonstrated enhancement, and were statistically superior to all other regimens at 96 h (P < 0.001) against both strains. CPT, ERT, and AMP similarly augmented LL37 killing against strain 8019. In strains R6981 and R6370, CPT and ERT aided LL37 more than AMP (P < 0.001). Compared to DAP alone, combination regimens provide better killing and prevent resistance. Clinical research involving DAP combinations is warranted.     

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

Biofilm formation is an important virulence factor that allows bacteria to resist host responses and antibacterial agents. The aim of the study was to assess the in vitro activities of several antimicrobials alone or in combination against two Staphylococcus aureus isolates in a novel pharmacokinetic/pharmacodynamic (PK/PD) model of biofilm for 3 days. One methicillin-susceptible S. aureus strain (SH1000) and one methicillin-resistant S. aureus strain (N315) were evaluated in a modified biofilm reactor with polystyrene coupons. Simulated regimens included vancomycin (VAN) plus rifampin (RIF), moxifloxacin (MOX), and high doses (10 mg/kg of body weight/day) of daptomycin (DAP) alone or combined with RIF or clarithromycin (CLA). Against viable planktonic bacteria (PB) and biofilm-embedded bacteria (BB) of SH1000, neither DAP nor MOX alone was bactericidal. In contrast, the combination of DAP or MOX with CLA significantly increased the activity of the two agents against both PB and BB (P < 0.01), and DAP plus CLA reached the limit of detection at 72 h. Against PB of N315, DAP alone briefly achieved bactericidal activity at 24 h, whereas sustained bactericidal activity was observed at 32 h with VAN plus RIF. Overall, only a minimal reduction was observed with both regimens against BB (<2.8 log₁₀ CFU/ml). Finally, the combination of DAP and RIF was bactericidal against both PB and BB, achieving the limit of detection at 72 h. In conclusion, we developed a novel in vitro PK/PD model to assess the activities of antimicrobials against mature bacterial biofilm. Combinations of DAP or MOX with CLA were the most effective regimens and may represent promising options to treat persistent infections caused by S. aureus biofilms.Biofilms are complex bacterial communities embedded in a self-produced glycocalyx slime that protects the cells from environmental and antimicrobial threats. It has been reported that antimicrobial MICs of bacteria embedded in biofilms can be 10 to 1,000 times higher than those in a planktonic state (4). The reasons for this decreased susceptibility are not completely understood. However, to explain the diminished activity of antimicrobials against biofilm-embedded cells, poor penetration of the drug through the layers of the matrix as well as heterogeneity of the growth within the biofilm have been suggested (22, 28, 36).Because bacteria can produce biofilms on medical devices, biofilm-associated infections have a tremendous impact on the management of patient health. Staphylococcus aureus is a pathogen commonly associated with biofilm-related infections, such as osteomyelitis, prosthetic joint infections, endocarditis, and catheter-related infections (22). Various in vitro scenarios of biofilm formation have evaluated the activity of several antimicrobials and have reported different outcomes (13, 15, 31, 32).Daptomycin (DAP) is a newly available lipopeptide antibiotic with potent in vitro and in vivo bactericidal activities against S. aureus, including methicillin-resistant S. aureus (MRSA) (5, 33). Daptomycin has been already evaluated in different in vitro biofilm models, and conflicting results have been reported (15, 18, 32). For instance, with an in vitro model of catheter-related infection, LaPlante and Mermel found that daptomycin at 5 mg/ml was able to eradicate S. aureus biofilms, whereas lower concentrations of daptomycin (clinically irrelevant) did not eradicate biofilm-embedded cells (18). With a static microtiter plate model, Hajdu et al. did not find any reduction in the viable count of Staphylococcus epidermidis biofilm cells with doses of daptomycin up to 128 times the MIC (15). Potential explanations for the discrepancies in these results might be differences in the regimens tested and in the models used for the evaluations.Because there have been several reports about the safety and efficacy of doses of daptomycin up to 12 mg/kg of body weight/day (3, 9, 17, 21, 24), we designed the present study to evaluate a high-dose regimen of daptomycin (10 mg/kg/day) alone or in combination with other agents and compared its activity to that of vancomycin (VAN) for a MRSA isolate and to that of moxifloxacin (MOX) for a MSSA strain. Although there is no optimal therapy, these two drugs are considered useful by clinicians in the treatment of biofilm-associated infections caused by S. aureus. We investigated the effects of combination therapy, since association of vancomycin with other drugs, such as rifampin (RIF) and clarithromycin (CLA), has already been proved beneficial (11, 19, 25, 31). Finally, because most of the models developed so far present major limitations with regard to antibiotic pharmacokinetics simulations, we adapted a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of biofilm formation to assess the antimicrobial activities of these drugs.(A portion of this work was presented at the 20th European Conference of Clinical Microbiology and Infectious Diseases in Vienna, Austria, 10 to 13 April 2010, as poster P3153.)     

Daptomycin versus vancomycin in a methicillin-resistant Staphylococcus aureus endophthalmitis rabbit model: bactericidal effect, safety, and ocular pharmacokinetics

Staphylococcus aureus is a frequent cause of acute endophthalmitis, and infection with this virulent bacterium is often associated with a poor visual outcome. In this study, we investigated the bactericidal efficacy and the safety of intravitreal daptomycin (DAP), a lipopeptide antibiotic with broad-spectrum activity against Gram-positive bacteria, compared with those of intravitreal vancomycin (VAN) in a methicillin-resistant S. aureus endophthalmitis rabbit model. The pharmacokinetics and pharmacodynamics of daptomycin in the infected eyes were also studied. Rabbits were randomly divided into three treatment groups (n = 8) and one untreated group (n = 4), to compare the effect of single intravitreal injections of 0.2 mg and 1 mg of daptomycin (DAP 0.2 and DAP 1 groups, respectively) with that of 1 mg of intravitreal vancomycin (VAN 1 group). Vitreal aspirates were regularly collected and grading of ocular inflammation was regularly performed until euthanasia on day 7. In the DAP 0.2 group, 62.5% of the eyes were sterilized and the mean bacterial count presented a reduction of 1 log unit. In the DAP 1 and VAN 1 groups, the infection was eradicated (100% and 87.5% of eyes sterilized, respectively), with a 4-log-unit reduction of the mean bacterial count. The bactericidal efficacy in the DAP 1 group was not inferior to that in the VAN 1 group and was superior to that of the other regimens in limiting the ocular inflammation and preserving the architecture of the ocular structures (P < 0.05). The elimination half-life (t(1/2β)) of daptomycin was independent of the administered dose (38.8 ± 16.5 h and 40.9 ± 6.7 h, respectively, for the DAP 0.2 and DAP 1 groups) and was significantly longer than the t(1/2β) of vancomycin (20.5 ± 2.0 h for the VAN 1 group) (P < 0.05). This antibiotic could therefore be considered for the treatment of intraocular infections caused by Gram-positive bacteria.     

Ceftaroline-Fosamil Efficacy against Methicillin-Resistant Staphylococcus aureus in a Rabbit Prosthetic Joint Infection Model

Ceftaroline (CPT), the active metabolite of the prodrug ceftaroline-fosamil (CPT-F), demonstrates in vitro bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and is effective in rabbit models of difficult-to-treat MRSA endocarditis and acute osteomyelitis. However, its in vivo efficacy in a prosthetic joint infection (PJI) model is unknown. Using a MRSA-infected knee PJI model in rabbits, the efficacies of CPT-F or vancomycin (VAN) alone and combined with rifampin (RIF) were compared. After each partial knee replacement with a silicone implant that fit into the tibial intramedullary canal was performed, 5 × 10⁷ MRSA CFU (MICs of 0.38, 0.006, and 1 mg/liter for CPT, RIF, and VAN, respectively) was injected into the knee. Infected animals were randomly assigned to receive no treatment (controls) or CPT-F (60 mg/kg of body weight intramuscularly [i.m.]), VAN (60 mg/kg i.m.), CPT-F plus RIF (10 mg/kg i.m.), or VAN plus RIF starting 7 days postinoculation and lasting for 7 days. Surviving bacteria in crushed tibias were counted 3 days after ending treatment. Although the in vivo mean log₁₀ CFU/g of CPT-treated (3.0 ± 0.9, n = 12) and VAN-treated (3.5 ± 1.1, n = 12) crushed bones was significantly lower than those of controls (5.6 ± 1.1, n = 14) (P < 0.001), neither treatment fully sterilized the bones (3/12 were sterile with each treatment). The mean log₁₀ CFU/g values for the antibiotics in combination with RIF were 1.9 ± 0.5 (12/14 were sterile) for CPT-F and 1.9 ± 0.5 (12/14 were sterile) for VAN. In this MRSA PJI model, the efficacies of CPT-F and VAN did not differ; thus, CPT appears to be a promising antimicrobial agent for the treatment of MRSA PJIs.     

Inoculum Effects of Ceftobiprole,Daptomycin, Linezolid,and Vancomycin with Staphylococcus aureus and Streptococcus pneumoniae at Inocula of 105 and 107 CFU Injected into Opposite Thighs of Neutropenic Mice

Reduced bactericidal efficacy at a high inoculum is known as the inoculum effect (IE). We used neutropenic mice to compare the IEs of ceftobiprole (CFB), daptomycin (DAP), linezolid (LZD), and vancomycin (VAN) against 6 to 9 strains of Staphylococcus aureus and 4 strains of Streptococcus pneumoniae at 2 inocula in opposite thighs of the same mice. Neutropenic mice had 10^4.5 to 10^5.7 CFU/thigh (low inoculum [LI]) in one thigh and 10^6.4 to 10^7.2 CFU/thigh (high inoculum [HI]) in the opposite thigh when treated for 24 h with subcutaneous (s.c.) doses every 12 h of DAP at 0.024 to 100 mg/kg of body weight and LZD at 0.313 to 320 mg/kg and s.c. doses every 6 h of CFB at 0.003 to 160 mg/kg and VAN at 0.049 to 800 mg/kg. Dose-response data were analyzed by a maximum effect (E_max) model using nonlinear regression. Static doses for each drug and at each inoculum were calculated, and the difference between HI and LI (IE index) gave the magnitude of IE for each drug-organism combination. Mean (range) IE indexes of S. aureus were 2.9 (1.7 to 4.6) for CFB, 4.1 (2.6 to 9.3) for DAP, 4.6 (1.7 to 7.1) for LZD, and 10.1 (6.3 to 20.3) for VAN. In S. pneumoniae, the IE indexes were 2.5 (1.3 to 3.3) for CFB, 2.0 (1.6 to 2.8) for DAP, 1.9 (1.7 to 2.2) for LZD, and 1.5 (0.8 to 3.2) for VAN; these values were similar for all drugs. In S. aureus, the IE was much larger with VAN than with CFB, DAM, and LZD (P < 0.05). An in vivo time course of vancomycin activity showed initiation of killing at 4- to 16-fold-higher doses at HI than at LI despite similar initial growth of controls.     

Analysis of synergy between beta-lactams and anti-methicillin-resistant Staphylococcus aureus agents from the standpoint of strain characteristics and binding action

In light of the increasing number of clinical cases resistant to traditional monotherapies and the lack of novel antimicrobial agents, combination therapy is an appealing solution for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the efficacy of anti-MRSA agents, such as vancomycin (VAN), daptomycin (DAP), and linezolid (LZD), in conjunction with 13 beta-lactams and non-beta-lactams. We assessed the in vitro activities of the various combinations against 40 MRSA strains based on the maximum synergistic effect (MSE), an index calculated from the MIC change with a combination agent. Nearly all the anti-MRSA agents, which were combined with beta-lactams as well as VAN and DAP, showed a synergistic effect with arbekacin. VAN also exhibited varying degrees of synergy depending on the type of beta-lactam, whereas DAP and LZD showed similar synergy with different beta-lactams. These effects were confirmed by antibiotic kill curves, except for the apparent interaction between LZD and beta-lactams. The MSE results were analyzed according to strain characteristics including susceptibility to combination agents, staphylococcal cassette chromosome mec type, and presence of the blaZ gene; however, no obvious correlations were observed. In a fluorescence binding assay, the fluorescence intensity of boron-dipyrromethene (BODIPY)-VAN decreased, whereas that of BODIPY-DAP increased in combination with a beta-lactam agent. These findings suggest that beta-lactam combinations are promising treatment options for MRSA infections and that the type of beta-lactam combined with VAN is important for the synergistic effect.     

Telavancin Demonstrates Activity against Methicillin-Resistant Staphylococcus aureus Isolates with Reduced Susceptibility to Vancomycin,Daptomycin, and Linezolid in Broth Microdilution MIC and One-Compartment Pharmacokinetic/Pharmacodynamic Models

Methicillin-resistant Staphylococcus aureus (MRSA) isolates have arisen with reduced susceptibility to several anti-MRSA agents. Telavancin (TLV), a novel anti-MRSA agent, retains low MICs against these organisms. Our objective was to determine the MICs for TLV, daptomycin (DAP), vancomycin (VAN), and linezolid (LZD) against daptomycin-nonsusceptible (DNS) S. aureus, vancomycin-intermediate S. aureus (VISA), heteroresistant VISA (hVISA), and linezolid-resistant (LZD^r) S. aureus. We also evaluated these agents against each phenotype in pharmacokinetic/pharmacodynamic (PK/PD) models. Seventy DNS, 100 VISA, 180 hVISA, and 25 LZD^r MRSA isolates were randomly selected from our library and tested to determine their MICs against TLV, DAP, VAN, and LZD via broth microdilution and a Trek panel. Four isolates were randomly selected for 168-h in vitro models to evaluate treatment with TLV at 10 mg/kg of body weight/day, DAP at 10 mg/kg/day, VAN at 1 g every 12 h (q12h), and LZD at 600 mg q12h. The MIC_50/90 for TLV, DAP, VAN, and LZD against 70 DNS S. aureus isolates were 0.06/0.125 μg/ml, 2/4 μg/ml, 1/2 μg/ml, and 2/2 μg/ml, respectively. Against 100 VISA isolates, the MIC_50/90 were 0.06/0.125 μg/ml, 1/1 μg/ml, 4/8 μg/ml, and 1/2 μg/ml, respectively. Against 170 hVISA isolates, the MIC_50/90 were 0.06/0.125 μg/ml, 0.5/1 μg/ml, 1/2 μg/ml, and 1/2 μg/ml, respectively. Against 25 LZD^r isolates, the MIC_50/90 were 0.03/0.06 μg/ml, 1/1 μg/ml, 2/2 μg/ml, and 8/8 μg/ml, respectively. The TLV MIC was >0.125 μg/ml for 10/365 (2.7%) isolates. In PK/PD models, TLV was universally bactericidal at 168 h and statistically superior to all antibiotics against DNS S. aureus strain R2334. These data further establish the potency of TLV against resistant MRSA. The model data demonstrate in vitro bactericidal activity of TLV against hVISA, VISA, DNS S. aureus, and LZD^r S. aureus strains. Further clinical research is warranted.     

Evaluation of Ceftaroline Alone and in Combination against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Daptomycin and Vancomycin in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Annually, medical device infections are associated with >250,000 catheter-associated bloodstream infections (CLABSI), with up to 25% mortality. Staphylococcus aureus, a primary pathogen in these infections, is capable of biofilm production, allowing organism persistence in harsh environments, offering antimicrobial protection. With increases in S. aureus isolates with reduced susceptibility to current agents, ceftaroline (CPT) offers a therapeutic alternative. Therefore, we evaluated whether CPT would have a role against biofilm-producing methicillin-resistant S. aureus (MRSA), including those with decreased susceptibilities to alternative agents. In this study, we investigated CPT activity alone or combined with daptomycin (DAP) or rifampin (RIF) against 3 clinical biofilm-producing MRSA strains in an in vitro biofilm pharmacokinetic/pharmacodynamic (PK/PD) model. Simulated antimicrobial regimens were as follows: 600 mg of CPT every 8 h (q8h) (free maximum concentration of drug [fC_max], 17.04 mg/liter; elimination half-life [t_1/2], 2.66 h), 12 mg/kg of body weight/day of DAP (fC_max, 14.7 mg/liter; t_1/2, 8 h), and 450 mg of RIF q12h (fC_max, 3.5 mg/liter; t_1/2, 3.4 h), CPT plus DAP, and CPT plus RIF. Samples were obtained and plated to determine colony counts. Differences in log₁₀ CFU/cm² were evaluated by analysis of variance with Tukey''s post hoc test. The strains were CPT and vancomycin susceptible and DAP nonsusceptible (DNS). CPT displayed activity throughout the experiment. DAP demonstrated initial activity with regrowth at 24 h in all strains. RIF was comparable to the drug-free control, and little benefit was observed when combined with CPT. CPT plus DAP displayed potent activity, with an average log₁₀ CFU/cm² reduction of 3.33 ± 1.01 from baseline. CPT demonstrated activity against biofilm-producing DNS MRSA. CPT plus DAP displayed therapeutic enhancement over monotherapy, providing a potential option for difficult-to-treat medical device infections.     

Detection of synergy using the combination of polymyxin B with either meropenem or rifampin against carbapenemase-producing Klebsiella pneumoniae

Polymyxin B (PB) plus meropenem (MER) or rifampin (RIF) was tested by Etest® method and time-kill assay (TKA) against 14 genetically unique clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae. PB + MER: Etest, 43% synergy; TKA, 64% synergy. Concordance between methods was 79%. For PB + RIF: Etest, 21% synergy; TKA, 100% synergy. Concordance between methods was 21%.     

Garenoxacin (BMS-284756) and moxifloxacin in experimental meningitis caused by vancomycin-tolerant pneumococci

The emergence of multidrug-resistant strains of Streptococcus pneumoniae drives the development and evaluation of new antipneumococcal agents, especially for the treatment of bacterial meningitis. The aims of the present study were to assess the antibacterial effectiveness of two new quinolones, garenoxacin (BMS; BMS-284756) and moxifloxacin (MOX) in experimental meningitis caused by a vancomycin (VAN)-tolerant S. pneumoniae strain and to compare the results with those obtained by therapy with VAN and ceftriaxone (CRO) in combination. Meningitis was induced in young male New Zealand White rabbits by intracisternal inoculation of a VAN-tolerant pneumococcal strain (strain Tupelo) from a child with meningitis. Sixteen hours after inoculation, therapy was given by intravenous administration of BMS at 20 mg/kg of body weight, followed 5 h later by administration at a dosage of 10 mg/kg (n = 9 animals) or MOX as two doses of 20 mg/kg every 5 h (n = 8 animals). For comparison, we studied the following groups: (i) animals treated with VAN (20 mg/kg every 5 h, three doses) and CRO (125 mg/kg, one dose) (n = 9), (ii) animals infected with a VAN-tolerant strain but not treated (n = 8), (iii) animals infected with a VAN-tolerant pneumococcus isolated from the nasopharynx of a carrier and treated with BMS (n = 8), and (iv) animals infected with a cephalosporin-resistant type 6B S. pneumoniae strain and treated with BMS (n = 6). The MICs of penicillin, CRO, VAN, BMS, and MOX for the Tupelo strain were 2, 1, 0.5, 0.06, and 0.03 micro g/ml, respectively. The rates of killing of strain Tupelo (the change in the log(10) number of CFU per milliliter per hour) in cerebrospinal fluid at 5 h were -0.70 +/- 0.35, -0.61 +/- 0.44, and -0.49 +/- 0.36 for BMS, MOX, and VAN-CRO, respectively. Therapy with BMS and MOX was as effective as therapy with VAN-CRO against VAN-tolerant pneumococcal meningitis in rabbits.     

Occurrence of extended-spectrum beta-lactamases among chromosomal AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens in Korea and investigation of screening criteria

We assessed the occurrence and screening criterion for extended-spectrum beta-lactamases (ESBLs) among AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens. The 413 isolates (158 E. cloacae, 126 C. freundii, and 129 S. marcescens) isolated from 11 clinical laboratories in Korea were investigated. ESBL production was confirmed by double-disk synergy test and inhibitor-potentiated diffusion test using ceftazidime (CAZ), cefotaxime (CTX), aztreonam (AZT), and cefepime (FEP) with or without clavulanic acid. One hundred seven isolates (25.9%) were as ESBL producers. Of them, resistance was transferred by conjugation in 82 isolates. In transconjugants, structural genes for CTX-M (53.7%), TEM (46.3%), SHV (29.3%) were found. To evaluate the ESBL screening minimum inhibitory concentration (MIC) criteria, MICs for cefuroxime, CAZ, CTX, AZT, and FEP were determined and cutoff value was selected using receiver operator characteristic curve. The FEP MIC > or = 1 microg/mL had the highest sensitivity (95.3%), specificity (82.7%), and positive (65.8%) and negative predictive values (98.3%).     

In vitro activities of piperacillin against beta-lactamase-negative ampicillin-resistant Haemophilus influenzae

The in vitro activities of piperacillin (PIP) against beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae were compared with those of cefotaxime (CTX) and ceftriaxone (CRO), and the potency of PIP as therapy for meningitis caused by BLNAR is also discussed. PIP showed good activity (MIC at which 90% of strains are inhibited, 0.25 micro g/ml) against 69 BLNAR strains, and its activity was comparable to that of CRO and superior to that of CTX. No significant correlation was observed between the MICs of PIP and CTX or CRO or AMP, whereas a high correlation was observed between the MICs of CTX and CRO. In the killing study, PIP showed potent bactericidal activity compared with those of CTX and CRO. By microscopic examination, PIP caused the formation of a spindle and short filamentous cells with bulges and induced cell lysis in BLNAR strains, while treatment with CTX and CRO resulted in the formation of large, spherical cells without any obvious lysis. The affinity of Bocillin FL, a fluorescent penicillin used for determination of the 50% inhibitory concentration (IC(50)s) for penicillin-binding proteins (PBPs), to PBPs 3a and 3b of BLNAR strains was drastically decreased compared with that to an AMP-susceptible strain (ATCC 33391). In the case of the BLNAR strains, the IC(50)s for PBPs 1a, 1b, and 2 were similar to those for the PBPs of ATCC 33391. Since the affinity of binding to PBPs 3a and 3b of the BLNAR strains decreased drastically, the second targets among the PBPs were PBP 2 for PIP, PBP1 (1a and 1b) for CTX and CRO. In conclusion, PIP showed excellent activities against BLNAR strains in a manner different from those of cephem antibiotics, suggesting that it could be a candidate therapeutic agent for the treatment of meningitis caused by BLNAR strains.     

Characterizing vancomycin-resistant Enterococcus strains with various mechanisms of daptomycin resistance developed in an in vitro pharmacokinetic/pharmacodynamic model

Two daptomycin (DAP) regimens were evaluated in a pharmacokinetic/pharmacodynamic (PK/PD) model, and the mutants recovered were examined for changes in phenotypic characteristics. Three Enterococcus faecium strains (vancomycin-resistant Enterococcus [VRE] ATCC 51559, VRE 12311, and VRE SF 12047) were utilized in a 7-day, 1-compartment in vitro PK/PD model. The simulated dosing regimens were DAP at 6 mg/kg/day (free C(max) [fC(max)] = 7.9 μg/ml, half-life [t(1/2)] = 8 h) and DAP at 10 mg/kg/day (fC(max) = 13.17 μg/ml, t(1/2) = 8 h). Samples were plated daily on Mueller-Hinton agar containing DAP at 16 μg/ml and 50 mg/liter Ca(2+) to assess the emergence of DAP resistance. For each strain, the mutant with the highest DAP MIC was then evaluated for changes in relative surface charge, cell wall thickness, and cytoplasmic membrane depolarization induced by DAP. The initial DAP MICs were 4 μg/ml for all 3 strains. A dose-dependent response and regrowth were observed for DAP 6 mg/kg/day and DAP 10 mg/kg/day against all 3 strains. Mutants of VRE ATCC 51559 (MIC = 128 and 64 μg/ml) and VRE 12311 (MIC = 256 and 32 μg/ml) were recovered from the DAP 6 mg and DAP 10 mg regimen, respectively. For VRE SF 12047, a mutant (MIC = 64 μg/ml) was recovered from the DAP 6 mg model. All mutants displayed an increase in relative surface charge compared to those of their respective parent strains. The DAP-resistant mutants displayed a 43 to 58% increase in cell wall thickness (P < 0.0001), while DAP membrane depolarization decreased by 53 to 65% compared to that of the susceptible strains. VRE with DAP resistance displayed increased surface charge, increased cell wall thickness, and decreased depolarization induced by DAP, consistent with previous observations in Staphylococcus aureus with reduced DAP susceptibility. Further characterization of DAP-resistant VRE is warranted.     

A Novel Extended-Spectrum TEM-Type β-Lactamase (TEM-52) Associated with Decreased Susceptibility to Moxalactam in Klebsiella pneumoniae

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum β-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from bla_TEM-1 by three mutations leading to the following amino acid substitutions: Glu₁₀₄→Lys, Met₁₈₂→Thr, and Gly₂₃₈→Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated bla_TEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (K_i, 0.16 μM) was 10-fold higher than that of TEM-3 (K_i, 1.9 μM). Allelic replacement analysis revealed that the combination of Lys₁₀₄, Thr₁₈₂, and Ser₂₃₈ was responsible for the increase in the MICs of MOX for the TEM-52 producers.     

Comparison of Six Generic Vancomycin Products for Treatment of Methicillin-Resistant Staphylococcus aureus Experimental Endocarditis in Rabbits

Concerns have recently emerged about the potency and the quality of generic vancomycin (VAN) products approved for use in humans, based on experiments in a neutropenic mouse thigh infection model. However, other animal models may be more appropriate to decipher the bactericidal activities of VAN generics in vivo and to predict their efficacy in humans. We aimed to compare the bactericidal activities of six generic VAN products currently used in France (Mylan and Sandoz), Spain (Hospira), Switzerland (Teva), and the United States (Akorn-Strides and American Pharmaceutical Products [APP]) in a rabbit model of aortic valve endocarditis induced by 8 × 10⁷ CFU of methicillin-resistant Staphylococcus aureus (MRSA) strain COL (VAN MIC, 1.5 μg/ml). In vitro, there were no significant differences in the time-kill curve studies performed with the six generic VAN products. Ten rabbits in each group were treated with intravenous (i.v.) VAN, 60 mg/kg of body weight twice a day (b.i.d.) for 4 days. Mean peak serum VAN levels, measured 45 min after the last injection, ranged from 35.5 (APP) to 45.9 μg/ml (Teva). Mean trough serum VAN levels, measured 12 h after the last injection, ranged from 2.3 (Hospira) to 9.2 (APP) μg/ml. All generic VAN products were superior to controls (no treatment) in terms of residual organisms in vegetations (P < 0.02 for each comparison) and in the spleen (P < 0.005 for each comparison). Pairwise comparisons of generic VAN products found no significant differences. In conclusion, a stringent MRSA endocarditis model found no significant differences in the bactericidal activities of six generic VAN products currently used in Europe and America.     

Comparative Prevalence of Acute Kidney Injury in Chinese Patients Receiving Vancomycin with Concurrent β-Lactam Antibiotics: A Retrospective Cohort Study

PurposeThe combination of vancomycin and piperacillin/tazobactam (VAN + PTZ) provides a broad spectrum of activity against multiple pathogens. However, a major issue in previous research concerned significant nephrotoxicity associated with this drug combination, and most studies have been conducted in American and European countries, with no similar data available from China. Therefore, this study evaluated the nephrotoxic effects of VAN + PTZ in a large-scale Chinese cohort to determine the prevalence of acute kidney injury (AKI) in this population by comparing PTZ and vancomycin monotherapies and the combined use of vancomycin and β-lactam antibiotics.MethodsThis retrospective cohort study identified adult patients who received vancomycin either as monotherapy or in combination with PTZ or carbapenem (VAN + CAR) for at least 48 hours at Jiangsu Province Hospital from January 1, 2017, to December 31, 2018. Patients were also evaluated for the development of AKI, defined according to the Kidney Disease Improving Global Outcome criteria. Duration of vancomycin exposure, steady-state trough vancomycin concentrations, and other risk factors for AKI were assessed. A Bayesian network meta-analysis was conducted to validate our results and comparatively evaluate the nephrotoxicity of β-lactam antibiotics in combination with vancomycin.FindingsIn all, 752 patients were included in the present study. The prevalence of AKI was higher in the VAN + PTZ group than in the VAN and VAN + CAR groups (15.2% vs 4.0% and 6.0%, respectively). After adjustment for confounding factors, VAN + PTZ was still related to AKI (odds ratio [OR] = 4.37; 95% CI, 1.65–11.59; P = 0.003). The network meta-analysis indicated that VAN + PTZ was associated with a significantly higher risk for AKI than was VAN (OR = 3.23; 95% CI, 2.50–4.35), PTZ (OR = 2.86; 95% CI, 1.92–4.12), VAN + cefepime (FEP) (OR = 2.37; 95% CI, 1.80–3.19), or VAN + CAR (OR = 2.28; 95% CI, 1.64–3.21). However, there was no significant difference with respect to AKI prevalence among the VAN, PTZ, VAN + FEP, and VAN + CAR groups.ImplicationsThe prevalence of AKI was higher with VAN + PTZ therapy than with VAN or PTZ monotherapy or with the concurrent use of VAN and FEP or CAR in our study. Clinicians should adequately assess renal function and consider this differential risk for nephrotoxicity when choosing empiric antibiotics in hospitalized patients to minimize the rates of AKI.     

Update of dalbavancin spectrum and potency in the USA: report from the SENTRY Antimicrobial Surveillance Program (2011)

Dalbavancin (DAL) is an investigational lipoglycopeptide with a prolonged serum half-life allowing once weekly dosing. DAL potency was assessed in the 2011 SENTRY Antimicrobial Surveillance Program among 1555 isolates sampled from all 9 US Census regions. Monitored Gram-positive cocci included Staphylococcus aureus (SA; 1,036/50.4% MRSA), coagulase-negative staphylococci (CoNS; 115), Enterococcus faecalis (25), E. faecium (31), Streptococcus pyogenes (155), Streptococcus agalactiae (153), and viridans group streptococci (VGS; 40). All susceptibility (S) testing used Clinical and Laboratory Standards Institute reference broth microdilution methods and interpretations. DAL (MIC_50/90, 0.06/0.06 μg/mL) was 8- and 16-fold more active than daptomycin (DAP) and vancomycin (VAN), respectively against SA, with MSSA and MRSA having the same MIC₉₀ results. CoNS was slightly more DAL-S (MIC₅₀, ≤0.03μg/mL). The highest staphylococcal DAL MIC was only 0.25 μg/mL. β-Haemolytic streptococci (βHS) and VGS had DAL MIC results ranging from ≤0.03 to 0.25 μg/mL (MIC₉₀, 0.06–0.12 μg/mL), and only enterococci showed elevated DAL MIC results. VanA phenotype–resistant E. faecalis or E. faecium had DAL MIC values at ≥1 μg/mL; VanB strains were DAL-S (MIC, ≤0.25 μg/mL). All cited DAL quantitative values were consistent with earlier surveillance data (2006–2009), without evidence of MIC creep. In conclusion, year 2011 SENTRY Program data for DAL documents sustained potent activity against SA, CoNS, βHS, VGS, and VAN-S enterococci, which averaged 4- to 32-fold greater than VAN, DAP, or linezolid.     

Cefotaxime and desacetylcefotaxime in neonates and children: a review of microbiologic, pharmacokinetic, and clinical experience

Over the past 5 yr, we have conducted two clinical and two pharmacokinetic investigations of cefotaxime (CTX) and desacetylcefotaxime (dCTX) in neonates, infants, and children. A total of 50 children with culture-proven bacterial meningitis were randomized to receive either 200 mg/kg/day of CTX (n = 23, mean age 24.4 mo) or standard doses of ampicillin (AMP) and chloramphenicol succinate (CAPS; n = 27, mean age 16.6 mo). Results were similar between the CTX and Amp/CAPS groups for clinical/microbiological cures (100% versus 96%, respectively) and for survival without sequelae (78% vs. 77%, respectively). All bacterial isolates were sensitive to CTX, and the comparison of the MIC/MBC values for CTX to the CSF bactericidal titers suggested antimicrobial activity for dCTX. In a second clinical trial, 20 infants (1 wk-3 mo) were treated with 200 mg/kg/day of CTX for Gram-negative enteric bacillary meningitis. Cultures of CSF obtained 24 hr after the initiation of treatment were sterile in all subjects. Survival and complication rates of 95% and 21%, respectively, were observed. This compared favorably to previously published experiences with alternate treatment regimens for Gram-negative meningitis in the newborn. In both meningitis studies, the safety profile for CTX was excellent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model

OBJECTIVES: To extend the mutant selection window (MSW) hypothesis to include antibiotics in addition to fluoroquinolones, the pharmacodynamics of daptomycin (DAP) and vancomycin (VAN) and their ability to prevent the selection of resistant Staphylococcus aureus were studied in an in vitro model that simulates antibiotic concentrations below the MIC, between the MIC and the mutant prevention concentration (MPC), and above the MPC. METHODS: Two clinical isolates of S. aureus, S. aureus 866 (MIC(DAP) 0.35, MIC(VAN) 0.7, MPC(DAP) 1.1, MPC(VAN) 2.4 mg/L) and S. aureus 10 (MIC(DAP) 1.1, MIC(VAN) 1.3, MPC(DAP) 5.5, MPC(VAN) 11 mg/L), were exposed for five consecutive days to once-daily daptomycin (half-life 9 h) and twice-daily vancomycin (half-life 6 h) at the ratio of 24 h area under the concentration-time curve (AUC24) to MIC that varied over a 16- to 30-fold range. The cumulative antimicrobial effect was expressed by its intensity (I(E)). Changes in susceptibility and numbers of surviving organisms on agar plates containing 2x and 4x MIC of daptomycin or vancomycin were monitored daily. RESULTS: The I(E)-log AUC24/MIC plots were bacterial strain- and antibiotic-independent. This allowed combination of data obtained with both antibiotics and both organisms. Based on the sigmoid relationship between I(E) and the AUC24/MIC (r2 = 0.9), the antistaphylococcal effect of the therapeutic doses of daptomycin (4 and 6 mg/kg) against a hypothetical S. aureus with MIC equal to the MIC90 (AUC24/MIC90 380 and 570 h, respectively) was predicted to be similar to the effect of two 1 g doses of vancomycin given at a 12 h interval (AUC24/MIC90 200 h). AUC24/MIC relationships of the final-to-initial MIC ratio and logarithm of the ratio of maximal-to-initial numbers of organisms resistant to 2x and 4x MIC of daptomycin or vancomycin were bell-shaped and bacterial strain- and antibiotic-independent. Based on these relationships, an AUC24/MIC ratio that protects against the selection of resistant mutants was predicted at > or = 200 h. This protective value is less than the AUC24/MIC90s provided by the 4 mg/kg dose and considerably less than the 6 mg/kg dose of daptomycin, but it is close to the AUC24/MIC90 provided by two 1 g doses of vancomycin. CONCLUSIONS: These findings support the MSW hypothesis and suggest comparable antistaphylococcal effects of clinically achievable AUC24/MIC90s of daptomycin and vancomycin but slightly better prevention against the selection of resistant S. aureus by daptomycin.     

Activities of new macrolides and fluoroquinolones against Mycobacterium ulcerans infection in mice

Mice infected in the left hind footpad with 5 log(10) acid-fast bacilli of Mycobacterium ulcerans were divided into an untreated control group and 17 treatment groups that received one of the following regimens for 4 weeks (all doses in milligrams per kilogram): 100 mg of azithromycin (AZM), 100 mg of clarithromycin (CLR), or 50 mg of AZM for a duration of 5 days a week (daily), three times a week, or once weekly. In addition, the following regimens were administered daily: 100 mg of telithromycin (TLM), sparfloxacin (SPX), or moxifloxacin (MOX); 200 mg of levofloxacin (LVX); 100 mg of streptomycin (STR) or amikacin (AMK); 10 mg of rifampin (RIF); and the combination of 10 mg of RIF and 100 mg of AMK (RIF+AMK). After completion of treatment, mice were observed for 30 weeks. The effectiveness of treatment regimens was assessed in terms of the delay in median time to footpad swelling in treated mice compared with that in the untreated controls. Clear-cut bactericidal activity, i.e., an observed delay in footpad swelling that exceeded the period of treatment, was observed in the STR-, AMK-, and RIF+AMK-treated mice. However, all mice treated with either AMK or STR alone had swollen footpads before the end of the 30-week observation period, suggesting regrowth of M. ulcerans. In contrast, 50% of the mice treated with the RIF+AMK combination exhibited no lesion even after 30 weeks, suggesting cure. The remaining regimens could be assigned to one of three groups: (i) no activity (50 mg of AZM, 100 mg of AZM thrice weekly, TLM, and LVX); (ii) bacteriostatic activity, i.e., a delay in footpad swelling shorter than the 4-week treatment duration (100 mg of AZM daily or once weekly, CLR thrice or once weekly, and MOX); or (iii) weak bactericidal activity (CLR daily and SPX). The RIF+AMK combination and possibly RIF+STR warrant further study for the treatment of M. ulcerans infection in humans.