Generic Vancomycin Products Fail In Vivo despite Being Pharmaceutical Equivalents of the Innovator

Generic versions of intravenous antibiotics are not required to demonstrate therapeutic equivalence with the innovator because therapeutic equivalence is assumed from pharmaceutical equivalence. To test such assumptions, we studied three generic versions of vancomycin in simultaneous experiments with the innovator and determined the concentration and potency of the active pharmaceutical ingredient by microbiological assay, single-dose pharmacokinetics in infected mice, antibacterial effect by broth microdilution and time-kill curves (TKC), and pharmacodynamics against two wild-type strains of Staphylococcus aureus by using the neutropenic mouse thigh infection model. The main outcome measure was the comparison of magnitudes and patterns of in vivo efficacy between generic products and the innovator. Except for one product exhibiting slightly greater concentration, vancomycin generics were undistinguishable from the innovator based on concentration and potency, protein binding, in vitro antibacterial effect determined by minimal inhibitory or bactericidal concentrations and TKC, and serum pharmacokinetics. Despite such similarities, all generic products failed in vivo to kill S. aureus, while the innovator displayed the expected bactericidal efficacy: maximum antibacterial effect (E_max) (95% confidence interval [CI]) was 2.04 (1.89 to 2.19), 2.59 (2.21 to 2.98), and 3.48 (2.92 to 4.04) versus 5.65 (5.52 to 5.78) log₁₀ CFU/g for three generics and the innovator product, respectively (P < 0.0001, any comparison). Nonlinear regression analysis suggests that generic versions of vancomycin contain inhibitory and stimulatory principles within their formulations that cause agonistic-antagonistic actions responsible for in vivo failure. In conclusion, pharmaceutical equivalence does not imply therapeutic equivalence for vancomycin.The World Health Organization (WHO) and all drug regulatory agencies (DRA) support commercialization of generic medicines because they control costs and are irreplaceable therapeutic options in countries lacking the innovator product (10, 41). WHO defines two products as therapeutically equivalent “if they are pharmaceutically equivalent and, after administration in the same molar dose, their effects with respect to both efficacy and safety are essentially the same, as determined from appropriate bioequivalence, pharmacodynamic, clinical, or in vitro studies” (41). Parenteral formulations, however, are not required to demonstrate therapeutic equivalence because it “may be considered self-evident” (41).Such assumptions have never been challenged, but there are reasons to do so for parenteral antimicrobials. First, many antibacterials are secreted in nature by microorganisms, and industrial production of the active pharmaceutical ingredient (API) involves complex processes for biosynthesis, purification, and manufacture, hard to replicate even for the designer (22). Second, two molecules may look similar without being identical, displaying different biological effects (2). Third, makers of generic drugs do not necessarily know the nature, composition, and pharmacological interactions of excipients employed by the innovator to avoid polymorphs of the API (33). Fourth, while most medicines interact with the host only, antimicrobials also confront the invader organism, a dynamic triangle with numerous possibilities of biologic variation (3, 11, 17). Thus, mixing the exactitude of chemistry with the variability of biology could generate unpredictable effects in seriously sick patients, but differences between the generic and the innovator might pass unnoticed among the complexity of infectious diseases in which death is one of the expected outcomes.Vancomycin (VAN) is a fermentation product of Amycolatopsis orientalis, an actinomycete discovered in 1955 in a dirt sample sent from Borneo to scientists at Eli Lilly (24, 27). Infusion reactions were common initially, but technology led the innovator to a safer product (8). Differences in composition are well known (36) and even advertised (Baxter promotional material; Baxter, Bogota, Colombia), but DRA worldwide support commercialization of vancomycin generics based on scant in vitro data claiming unaltered efficacy (9). After 50 years of unparalleled performance of vancomycin against Gram-positive pathogens, in vitro susceptibility has certainly decreased, and nowadays more than 20 clinical studies blame vancomycin for ineffectiveness and claim success for new, very expensive replacements (15). Without exception, all these studies fail to mention the manufacturer of the vancomycin products involved, despite the fact that most hospitals around the globe prefer generics. The present study was designed to fill the gap in evidence regarding in vivo efficacy of vancomycin generics compared with the innovator, spanned from November 2002 to November 2009, and allowed experimentation before and after Eli Lilly sold its brand name for the drug along with the secrets of manufacture (32). The null hypothesis was the assumption made by WHO and DRA, i.e., that pharmaceutical equivalence of vancomycin generics predicts their therapeutic equivalence with the innovator. Our data reject such a hypothesis.     

Generic vancomycin enriches resistant subpopulations of Staphylococcus aureus after exposure in a neutropenic mouse thigh infection model

Previous studies have shown that "bioequivalent" generic products of vancomycin are less effective in vivo against Staphylococcus aureus than the innovator compound. Considering that suboptimal bactericidal effect has been associated with emergence of resistance, we aimed to assess in vivo the impact of exposure to innovator and generic products of vancomycin on S. aureus susceptibility. A clinical methicillin-resistant S. aureus (MRSA) strain from a liver transplant patient with persistent bacteremia was used for which MIC, minimum bactericidal concentration (MBC), and autolytic properties were determined. Susceptibility was also assessed by determining a population analysis profile (PAP) with vancomycin concentrations from 0 to 5 mg/liter. ICR neutropenic mice were inoculated in each thigh with ～7.0 log(10) CFU. Treatment with the different vancomycin products (innovator and three generics; 1,200 mg/kg of body weight/day every 3 h) started 2 h later while the control group received sterile saline. After 24 h, mice were euthanized, and the thigh homogenates were plated. Recovered colonies were reinoculated to new groups of animals, and the exposure-recovery process was repeated until 12 cycles were completed. The evolution of resistance was assessed by PAP after cycles 5, 10, 11, and 12. The initial isolate displayed reduced autolysis and higher resistance frequencies than S. aureus ATCC 29213 but without vancomycin-intermediate S. aureus (VISA) subpopulations. After 12 cycles, innovator vancomycin had significantly reduced resistant subpopulations at 1, 2, and 3 mg/liter, while the generic products had enriched them progressively by orders of magnitude. The great capacity of generic vancomycin to select for less susceptible organisms raises concerns about the role of therapeutic inequivalence of any antimicrobial on the epidemiology of resistance worldwide.     

Therapeutic equivalence requires pharmaceutical, pharmacokinetic, and pharmacodynamic identities: true bioequivalence of a generic product of intravenous metronidazole

Animal models of infection have been used to demonstrate the therapeutic failure of "bioequivalent" generic products, but their applicability for this purpose requires the accurate identification of those products that are truly bioequivalent. Here, we present data comparing one intravenous generic product of metronidazole with the innovator product in a neutropenic mouse thigh anaerobic infection model. Simultaneous experiments allowed comparisons (generic versus innovator) of potency and the concentration of the active pharmaceutical ingredient (API), analytical chemistry (liquid chromatography/mass spectrometry [LC/MS]), in vitro susceptibility testing, single-dose serum pharmacokinetics (PK) in infected mice, and in vivo pharmacodynamics (PD) against Bacteroides fragilis ATCC 25825 in synergy with Escherichia coli SIG-1 in the neutropenic mouse thigh anaerobic infection model. The Hill dose-response model followed by curve-fitting analysis was used to calculate and compare primary and secondary PD parameters. The generic and the innovator products were identical in terms of the concentration and potency of the API, chromatographic and spectrographic profiles, MIC and minimal bactericidal concentrations (MBC) (2.0 mg/liter), and mouse PK. We found no differences between products in bacteriostatic doses (BD) (15 to 22 mg/kg of body weight per day) or the doses needed to kill 1 log (1LKD) (21 to 29 mg/kg per day) or 2 logs (2LKD) (28 to 54 mg/kg per day) of B. fragilis under dosing schedules of every 12 h (q12h), q8h, or q6h. The area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) was the best PD index to predict the antibacterial efficacy of metronidazole (adjusted coefficient of determination [AdjR(2)] = 84.6%), and its magnitude to reach bacteriostasis in vivo (56.6 ± 5.17 h) or to kill the first (90.8 ± 9.78 h) and second (155.5 ± 22.2 h) logs was the same for both products. Animal models of infection allow a thorough demonstration of the therapeutic equivalence of generic antimicrobials.     

Stability of meropenem and effect of 1 beta-methyl substitution on its stability in the presence of renal dehydropeptidase I.

The stability of meropenem in the presence of renal dehydropeptidase I (DHP-I) varied extremely with the animal source of the enzyme. Meropenem, compared with imipenem, was rather easily hydrolyzed by DHP-Is from mice, rabbits, and monkeys, while it showed a higher resistance to guinea pig and beagle dog DHP-Is. In addition, meropenem was four times more resistant than imipenem to human DHP-I. The 1 beta-methyl substituent on carbapenems, i.e., meropenem and 1 beta-methyl imipenem, made them considerably more resistant to mouse and swine DHP-Is than the 1-unsubstituted derivatives are.     

A new carbapenem drug dosage metric for carbapenem usage and correlation with carbapenem resistance of Pseudomonas aeruginosa

The emergence and dissemination of antimicrobial resistance is a worldwide problem. Inappropriate antimicrobial use contributes to this resistance, and several metrics of drug usage have been used to monitor their consumption and rational use. We examined several existing drug metrics, and developed a new one, dose/duration-density (D/d²), for a the best correlation between carbapenem usage and carbapenem resistance of Pseudomonas aeruginosa. The annual changes of antimicrobial use density (AUD), days of therapy (DOT), daily dose (DD) and D/d² for meropenem, imipenem and total carbapenems was analyzed for a correlation with carbapenem susceptibility of P. aeruginosa from 2006 through 2015 at a university hospital. The substitution of meropenem for imipenem usage, and an approximate 10% increase in carbapenem susceptibility of P. aeruginosa occurred over the study period. There were significant correlations of the meropenem susceptibility of P. aeruginosa and meropenem usage as measured by the meropenem DD, of imipenem susceptibility and imipenem AUD and DOT, and overall carbapenem susceptibility and imipenem DOT. The D/d² for meropenem, imipenem and total carbapenems had significant correlations with individual and all carbapenem susceptibility of P. aeruginosa. These D/d² is the best single carbapenem use metric for correlating carbapenem usage with P. aeruginosa resistance. Further studies are warranted to consider the value of D/d² for other antimicrobials and bacteria.     

Pharmacodynamic Evaluation of the Activities of Six Parenteral Vancomycin Products Available in the United States

A recent report found that generic parenteral vancomycin products may not have in vivo efficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similar in vitro microbiological activities and murine serum pharmacokinetics. We compared the in vitro and in vivo activities of six of the parenteral vancomycin products available in the United States. The in vitro assessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. The in vivo studies included dose-ranging experiments with the 6 vancomycin products for three isolates of Staphylococcus aureus in a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to any in vitro evaluation. Inhibitory sigmoid maximal bacterial kill (E_max) modeling of the relationship between vancomycin dosage and the killing of the bacteria in mice in vivo yielded similar E_max and EC₅₀ (drug exposure driving one-half E_max) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in the in vitro or in vivo activities among the six vancomycin products evaluated.     

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.     

In vivo dynamics of carbapenem-resistant Pseudomonas aeruginosa selection after suboptimal dosing

We have previously demonstrated Pseudomonas aeruginosa resistance selection because of suboptimal carbapenem exposures in an in vitro infection model, but the in vivo relevance of the observations is not well established. In this study, we examined the impact of carbapenem exposures on resistance selection using a neutropenic murine pneumonia model. Neutropenic mice were infected with approximately 10⁶ CFU of P. aeruginosa intratracheally. Ten animals each were treated with 400 or 50 mg/kg of meropenem intraperitoneally or placebo every 8 h, given 2 h after infection for 2 to 4 days. Quantitative assessment of bacterial burden in lung tissues was performed at baseline, upon death, or at the end of experiment. Meropenem (400 mg/kg) offered a significant survival benefit, but selective amplification of the OprD⁻ mutant population in lung tissue was observed in 20% to 30% of the animals. Our data suggested that suboptimal meropenem exposures might facilitate in vivo selection of resistance in a heterogeneous P. aeruginosa population.     

Impact of susceptibility profiles of Gram-negative bacteria before and after the introduction of ertapenem at a medical center in northern Taiwan from 2004 to 2010

This retrospective observational study evaluated the impact of antimicrobial consumption on antimicrobial susceptibility among aerobic Gram-negative bacteria after introducing ertapenem to the formulary of a teaching hospital (1130 beds) in northern Taiwan. Data on consumption of various antimicrobial agents, expressed as defined daily dose/1000 patient-days (DDD/1000 PD), were collected retrospectively from hospital pharmacy records 2 years before and 5 years after the introduction of ertapenem (October 2005). During the study period, the consumption of ampicillin and aminoglycosides decreased significantly. In contrast, the consumption of cefoxitin, ceftazidime, cefpirome, piperacillin–tazobactam, carbapenems (ertapenem, imipenem, and meropenem), and fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) increased significantly over time. There was a significant increase in the rate of susceptibility of Escherichia coli to ampicillin, cefotaxime, ceftazidime, piperacillin–tazobactam, cefpirome, amikacin, and levofloxacin; an increase in the rate of susceptibility of Klebsiella pneumoniae to ceftazidime, cefepime, cefpirome, piperacillin–tazobactam, meropenem, levofloxacin, and amikacin; a significant decrease in the rate of susceptibility of Pseudomonas aeruginosa to meropenem; and a significant decrease in the rate of susceptibility of Acinetobacter baumannii to ceftazidime, carbapenems, ciprofloxacin, and levofloxacin. The rate of antibiotic susceptibility to ertapenem of extended spectrum β-lactamase producers, including E. coli and K. pneumoniae, remained stable. Usage of ertapenem was found to be negatively and significantly associated with the susceptibility rates of P. aeruginosa to meropenem and gentamicin. Significantly negative correlations were noted between the use of ertapenem and the rates of susceptibility of A. baumannii to ceftazidime, piperacillin–tazobactam, carbapenems (imipenem and meropenem), ciprofloxacin, and levofloxacin.     

Antimicrobial susceptibility of common pathogens isolated from postoperative intra-abdominal infections in Japan

The principle of empirical therapy for patients with intra-abdominal infections (IAI) should include antibiotics with activity against Enterobacteriaceae and Bacteroides fragilis group species. Coverage of Pseudomonas aeruginosa, Enterobacter cloacae, and Enterococcus faecalis is also recommended for hospital-associated IAI. A nationwide survey was conducted to investigate the antimicrobial susceptibility of pathogens isolated from postoperative IAI. All 504 isolates were collected at 26 institutions and referred to a central laboratory for susceptibility testing. Lower susceptibility rates to ciprofloxacin and cefepime were demonstrated in Escherichia coli. Among E. coli, 24.1% of strains produced extended-spectrum β-lactamase (ESBL). Carbapenems, piperacillin/tazobactam, cephamycins/oxacephem, aminoglycosides, and tigecycline had high activity against E. coli, including ESBL-producing isolates. Among E. cloacae, low susceptibility rates to ceftazidime were demonstrated, whereas cefepime retained its activity. P. aeruginosa revealed high susceptibility rates to all antimicrobials tested except for imipenem. Among B. fragilis group species, low levels of susceptibility were observed for cefoxitin, moxifloxacin, and clindamycin, and high susceptibility rates were observed for piperacillin/tazobactam, meropenem, and metronidazole. Ampicillin, piperacillin, and glycopeptides had good activity against E. faecalis. Imipenem had the highest activity against E. faecalis among carbapenems. In conclusion, we suggested the empirical use of antimicrobials with the specific intent of covering the main organisms isolated from postoperative IAI. Piperacillin/tazobactam, meropenem, or doripenem, are appropriate in critically ill patients. Combination therapy of cefepime (aztreonam in patients with β-lactam allergy) plus metronidazole plus glycopeptides, imipenem/cilastatin or cephamycins/oxacephem plus ciprofloxacin plus metronidazole are potential therapeutic options.     

An Immunocompromised Child with Bloodstream Infection Caused by Two Escherichia coli Strains,One Harboring NDM-5 and the Other Harboring OXA-48-Like Carbapenemase

We describe a 16-year-old neutropenic patient from the Middle East with bloodstream infection caused by two carbapenemase-producing Escherichia coli isolates that we characterized by whole-genome sequencing. While one displayed meropenem resistance and was bla_NDM positive, the other demonstrated meropenem susceptibility yet harbored bla_OXA181 (which encodes a bla_OXA48-like enzyme). This report highlights the challenge of laboratory detection of bla_OXA48-like enzymes and the clinical implications of genotypic resistance detection in carbapenemase-producing Enterobacteriaceae.     

The Resistome of Pseudomonas aeruginosa in Relationship to Phenotypic Susceptibility

Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa, obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. β-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa. This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.     

Antibiotic resistance surveillance over a 4-year period (2000–2003) in Turkey: results of the MYSTIC Program

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a global study that provides antimicrobial susceptibility data in centers prescribing meropenem. The activity of meropenem and 7 broad-spectrum antimicrobials have been examined against 5208 bacterial isolates from 9 Turkish centers between 2000 and 2003. Cumulative susceptibility rates against all species of Enterobacteriaceae combined were ranked as follows: meropenem (99.3%), imipenem (97.6%), cefepime (80.0%), piperacillin–tazobactam (73.6%), ceftazidime (70.3%), ciprofloxacin (70.1%), cefotaxime (66.9%), and tobramycin (67.2%). The production of extended-spectrum β-lactamases (ESBLs) was detected in 48.7% of Klebsiella pneumoniae and in 19.5% of Escherichia coli isolates. Of ESBL producing K. pneumoniae isolates, 75.7% were resistant to tobramycin, 40.3% to ciprofloxacin, and 48.3% to piperacillin–tazobactam. Only piperacillin/tazobactam and carbapenems were active against more than 50% of Pseudomonas aeruginosa at the National Committee for Clinical Laboratory Standards-susceptible breakpoint, and the carbapenems were the most active compounds against Acinetobacter spp. These data confirm the continued potency of meropenem against Enterobacteriaceae in units where it is actively being prescribed.     

Development of resistance in wild-type and hypermutable Pseudomonas aeruginosa strains exposed to clinical pharmacokinetic profiles of meropenem and ceftazidime simulated in vitro

In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.     

Bacterial Resistance: A Worldwide Problem

The therapeutic crisis produced by emerging antimicrobial resistances has compromised the chemotherapy of hospitalized patients with serious infections. For the most prevalent resistance problems, meropenem, a new carbapenem, appears to provide a potency and spectrum for: 1) extended-spectrum β-lactamase-producing Enterobacteriaceae; 2) Bush-Jacoby-Merdeiros group 1 enzyme-producing ceftazidime-resistant Enterobacter spp., Citrobacter freundii, and some Serratia spp.; 3) ceftazidime- and imipenem-resistant Pseudomonas aeruginosa; and 4) some Streptococcus spp. with elevated penicillin MICs. Documented in vitro study results using 1997 Gram-negative blood stream infection isolates indicate a wider spectrum and a two- to fourfold greater potency for meropenem compared with imipenem. This was especially true for P. aeruginosa where 93.4% of strains were susceptible to meropenem (84.1% for imipenem). Also among over 30,000 reported in vitro meropenem results from the United States and Europe, 90.6% of Gram-positive cocci and 99.1% of anaerobes were inhibited at ≤4 μg/ml. Over 90% of ceftazidime-resistant blood stream infection strains were meropenem susceptible, a rate greater than those of imipenem, ciprofloxacin, and gentamicin. As the clinical utility of many contemporary antimicrobial agents is challenged by emerging resistance, the carbapenems (meropenem, imipenem) appear positioned for a greater role in the treatment of infections in hospitalized patients.     

Structural Changes and Differentially Expressed Genes in Pseudomonas aeruginosa Exposed to Meropenem-Ciprofloxacin Combination

The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa. The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.     

Efficacies of calcium–EDTA in combination with imipenem in a murine model of sepsis caused by Escherichia coli with NDM-1 β-lactamase

We evaluated the efficacy of ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium calcium salt (Ca-EDTA), as an inhibitor for New Delhi metallo-β-lactamase-1 (NDM-1) in vitro antibiotic susceptibility and in a mouse model of sepsis caused by Escherichia coli. Ca-EDTA drastically reduced the MICs of carbapenems for all NDM-producing bacteria [imipenem (IPM) ≤1–2 μg/ml; meropenem (MEPM) ≤1–4 µg/ml]. In the neutropenic murine model of sepsis, the bacterial burden was further reduced by combination therapy using imipenem/cilastatin sodium (IPM/CS) and Ca-EDTA to 2.3 × 10³ CFU/liver, compared with 2.9 × 10⁴ CFU/liver for IPM/CS alone. These data demonstrated the possibility of Ca-EDTA for clinical applications. In our understanding, this is the first report examining the effect of Ca-EDTA on a mouse sepsis model caused by NDM-1-producing bacteria.     

In vitro activity of doripenem, a carbapenem for the treatment of challenging infections caused by gram-negative bacteria, against recent clinical isolates from the United States

Doripenem, a 1β-methylcarbapenem, is a broad-spectrum antibiotic approved for the treatment of complicated urinary tract and complicated intra-abdominal infections. An indication for hospital-acquired pneumonia including ventilator-associated pneumonia is pending. The current study examined the activity of doripenem against recent clinical isolates for the purposes of its ongoing clinical development and future longitudinal analysis. Doripenem and comparators were tested against 12,581 U.S. clinical isolates collected between 2005 and 2006 including isolates of Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pneumoniae, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. MICs (μg/ml) were established by broth microdilution. By MIC₉₀, doripenem was comparable to imipenem and meropenem in activity against S. aureus (methicillin susceptible, 0.06; resistant, 8) and S. pneumoniae (penicillin susceptible, ≤0.015; resistant, 1). Against ceftazidime-susceptible Enterobacteriaceae, the MIC₉₀ of doripenem (0.12) was comparable to that of meropenem (0.12) and superior to that of imipenem (2), though susceptibility of isolates exceeded 99% for all evaluated carbapenems. The activity of doripenem was not notably altered against ceftazidime-nonsusceptible or extended-spectrum β-lactamase screen-positive Enterobacteriaceae. Doripenem was the most potent carbapenem tested against P. aeruginosa (MIC₉₀/% susceptibility [%S]: ceftazidime susceptible = 2/92%S, nonsusceptible = 16/61%S; imipenem susceptible = 1/98.5%S, nonsusceptible = 8/56%S). Against imipenem-susceptible Acinetobacter spp., doripenem (MIC₉₀ = 2, 89.1%S) was twice as active by MIC₉₀ as were imipenem and meropenem. Overall, doripenem potency was comparable to those of meropenem and imipenem against gram-positive cocci and doripenem was equal or superior in activity to meropenem and imipenem against Enterobacteriaceae, including β-lactam-nonsusceptible isolates. Doripenem was the most active carbapenem tested against P. aeruginosa regardless of β-lactam resistance.     

Comparative In Vitro and In Vivo Efficacies of Human Simulated Doses of Ceftazidime and Ceftazidime-Avibactam against Pseudomonas aeruginosa

The combination of ceftazidime and avibactam possesses potent activity against resistant Gram-negative pathogens, including Pseudomonas aeruginosa. We compared the efficacies of human simulated doses of ceftazidime and ceftazidime-avibactam using a hollow-fiber system and neutropenic and immunocompetent murine thigh infection models. Twenty-seven clinical P. aeruginosa isolates with ceftazidime MICs of 8 to 128 mg/liter and ceftazidime-avibactam MICs of 4 to 32 mg/liter were utilized in neutropenic mouse studies; 15 of the isolates were also evaluated in immunocompetent mice. Six isolates were studied in both the hollow-fiber system and the neutropenic mouse. In both systems, the free drug concentration-time profile seen in humans given 2 g of ceftazidime every 8 h (2-h infusion), with or without avibactam at 500 mg every 8 h (2-h infusion), was evaluated. In vivo activity was pharmacodynamically predictable based on the MIC. Ceftazidime decreased bacterial densities by ≥0.5 log unit for 10/27 isolates, while ceftazidime-avibactam did so for 22/27 isolates. In immunocompetent animals, enhancements in activity were seen for both drugs, with ceftazidime achieving reductions of ≥0.3 log unit for 10/15 isolates, whereas ceftazidime-avibactam did so against all 15 isolates. In vitro, ceftazidime resulted in regrowth by 24 h against all isolates, while ceftazidime-avibactam achieved stasis or better against 4/7 isolates. Mutants with elevated ceftazidime-avibactam MICs appeared after 24 h from 3/7 isolates studied in vitro; however, no resistant mutants were detected in vivo. Against this highly ceftazidime-nonsusceptible population of P. aeruginosa, treatment with human simulated doses of ceftazidime-avibactam resulted in pharmacodynamically predictable activity, particularly in vivo, against isolates with MICs of ≤16 mg/liter, and this represents a potential new option to combat these difficult-to-treat pathogens.     

Differences between meropenem and imipenem disk to detect carbapenemase in gram-negative bacilli using simplified carbapenem inactivation method

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health. In the present study, we compared the difference between meropenem and imipenem disk for detecting carbapenemase-producing gram-negative bacilli using simplified carbapenem inactivation method (sCIM). 106 Enterobacteriaceae, including 74 CPE, 17 Pseudomonas aeruginosa including 10 carbapenemase-producing isolates and 36 Acinetobacter baumannii including 20 carbapenem-resistant isolates preserved in our laboratory were tested. Based on sCIM method, the test bacteria were tested with both meropenem and imipenem disk, respectively. In Enterobacteriaceae, the usage of both meropenem and imipenem disk showed high concordance (99.1%). Meropenem disk cannot identify positive isolates among the 10 P. aeruginosa and 20 A. baumannii isolates due to low carbapenem hydrolytic ability of the carbapenemase produced by these strains. Thus, meropenem disk was found to be similar to imipenem disk, presenting high specificity and sensitivity in the detection of carbapenemase in Enterobacteriaceae, but it cannot be used for the detection of carbapenemase in P. aeruginosa and A. baumannii.