Pharmacokinetic/Pharmacodynamic Index Linked to In Vivo Efficacy of the Ampicillin-Ceftriaxone Combination against Enterococcus faecalis.

Combination therapy with ampicillin plus ceftriaxone (AMP+CRO) is the first-line therapy for treating severe infections due to Enterococcus faecalis. However, the pharmacokinetic/pharmacodynamic (PK/PD) index linked to the in vivo efficacy of the combination is not yet defined, hindering dose optimization in the clinic. Because classical PK/PD indices are not directly applicable to antimicrobial combinations, two novel indices were tested in the optimized murine model of infection by E. faecalis to delineate the potentiation of AMP by CRO: the time above the CRO threshold (T>threshold) and the time above the AMP instantaneous MIC (T>MICi). The potential clinical relevance was evaluated by simulating human doses of AMP and CRO. Hill's equation fitted well the exposure-response data in terms of T>threshold, with a CRO threshold of 1 mg/L. The required exposures were 46%, 49%, and 52% for stasis and 1- and 2-log10 killing, respectively. Human ceftriaxone doses of 2 g every 12 h (q12h) would reach the target in >90% of strains with thresholds ≤64 mg/L. The AMP T>MICi index also fitted well, and the required exposures were 37%, 41%, and 46% for stasis and 1- and 2-log10 killing, respectively. In humans, the addition of CRO would allow use of lower AMP doses to reach the same T>MICi and to treat strains with higher MICs. This is the first report of the PK/PD indices and required magnitudes linked to AMP+CRO against E. faecalis; these results can be used as the basis to guide the design of clinical trials to improve combined therapy against enterococci.

Impact on resistance of the use of therapeutically equivalent generics: the case of ciprofloxacin.

Therapeutic nonequivalence of generic antibiotics may lead to treatment failure and enrichment of resistance. However, there has been no demonstration that an equivalent generic displays the same resistance selection profile as the innovator drug. We aimed to test this hypothesis with five generic versions of ciprofloxacin by assessing their pharmaceutical equivalence with microbiological assays and their efficacy against Pseudomonas aeruginosa PAO1 in the neutropenic murine thigh infection model. One equivalent generic was selected for analysis by high-pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS), to confirm chemical identity, and resistance selection experiments in a hollow-fiber (HF) system simulating two clinical dosing regimens. Total and resistant populations were measured, and the MICs of the resistant cells with and without an efflux pump inhibitor were determined. LC-MS/MS found no differences between products, and the innovator and the generic selected resistance with the same magnitude and mechanism after 7 days of treatment in the HF system, supporting the fact that a generic with demonstrated equivalence in vivo is also equivalent regarding resistance selection.

An optimized mouse thigh infection model for enterococci and its impact on antimicrobial pharmacodynamics.

Negligible in vivo growth of enterococci and high-level dispersion of data have led to inaccurate estimations of antibiotic pharmacodynamics (PD). Here we improved an in vivo model apt for PD studies by optimizing the in vitro culture conditions for enterococci. The PD of vancomycin (VAN), ampicillin-sulbactam (SAM), and piperacillin-tazobactam (TZP) against enterococci were determined in vivo, comparing the following different conditions of inoculum preparation: aerobiosis, aerobiosis plus mucin, and anaerobiosis plus mucin. Drug exposure was expressed as the ratio of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC/MIC) (VAN) or the time in a 24-h period that the drug concentration for the free, unbound fraction exceeded the MIC under steady-state pharmacokinetic conditions (fT(>MIC)) (SAM and TZP) and linked to the change in log10 CFU/thigh. Only anaerobiosis plus mucin enhanced the in vivo growth, yielding significant PD parameters with all antibiotics. In conclusion, robust in vivo growth of enterococci was crucial for better determining the PD of tested antibacterial agents, and this was achieved by optimizing the procedure for preparing the inoculum.

A strain-independent method to induce progressive and lethal pneumococcal pneumonia in neutropenic mice.

BACKGROUND: Experimental models of pneumonia with penicillin non-susceptible Streptococcus pneumoniae (PNSSP) are hard to reproduce because the majority of strains with clinical relevance (like serotypes 6B, 9 V and 19 F) have low murine virulence. By optimization of culture and inoculum conditions of PNSSP (using porcine mucin), our aim was to develop a suitable, reliable and reproducible pneumonia mouse model for anti-infective pharmacology research. RESULTS: Seven PNSSP strains, including serotypes 6B, 9 V, 14 and 19 F were included. Strain INS-E611 displayed the highest murine virulence and was chosen to validate the lung model. Nose-instilled pneumococci grew between 2.1 and 2.5 log10 CFU/g of lung in 24 hours when an optimized culture of bacterial cells was used, but animals were all alive and recovered of infection after 36 h. In contrast, inoculum supplementation with mucin led to 100% mortality related to a successful lung infection confirmed by histopathology. These findings were reproduced with all seven PNSSP strains in neutropenic mice. Immunocompetent animals cleared all strains spontaneously. CONCLUSIONS: This pneumonia model produces a progressive and uniformly fatal lung infection with diverse serotypes of PNSSP independently of their intrinsic murine virulence.

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.

Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs.

Timely and accurate diagnostics are essential to fight the COVID-19 pandemic, but no test satisfies both conditions. Dogs can scent-identify the unique odors of volatile organic compounds generated during infection by interrogating specimens or, ideally, the body of a patient. After training 6 dogs to detect SARS-CoV-2 by scent in human respiratory secretions (in vitro diagnosis), we retrained 5 of them to search and find the infection by scenting the patient directly (in vivo screening). Then, efficacy trials were designed to compare the diagnostic performance of the dogs against that of the rRT-PCR in 848 human subjects: 269 hospitalized patients (COVID-19 prevalence 30.1%), 259 hospital staff (prevalence 2.7%), and 320 government employees (prevalence 1.25%). The limit of detection in vitro was lower than 10-12 copies ssRNA/mL. During in vivo efficacy experiments, our 5 dogs detected 92 COVID-19 positive patients among the 848 study subjects. The alert (lying down) was immediate, with 95.2% accuracy and high sensitivity (95.9%; 95% C.I. 93.6-97.4), specificity (95.1%; 94.4-95.8), positive predictive value (69.7%; 65.9-73.2), and negative predictive value (99.5%; 99.2-99.7) in relation to rRT-PCR. Seventy-five days after finishing in vivo efficacy experiments, a real-life study (in vivo effectiveness) was executed among the riders of the Metro System of Medellin, deploying the human-canine teams without previous training or announcement. Three dogs were used to examine the scent of 550 volunteers who agreed to participate, both in test with canines and in rRT-PCR testing. Negative predictive value remained at 99.0% (95% C.I. 98.3-99.4), but positive predictive value dropped to 28.2% (95% C.I. 21.1-36.7). Canine scent-detection in vivo is a highly accurate screening test for COVID-19, and it detects more than 99% of infected individuals independent of key variables, such as disease prevalence, time post-exposure, or presence of symptoms. Additional training is required to teach the dogs to ignore odoriferous contamination under real-life conditions.

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 (Emax) (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.

In vitro and in vivo comparison of the anti-staphylococcal efficacy of generic products and the innovator of oxacillin.

BACKGROUND: Oxacillin continues to be an important agent in the treatment of staphylococcal infections; many generic products are available and the only requirement for their approval is demonstration of pharmaceutical equivalence. We tested the assumption that pharmaceutical equivalence predicts therapeutic equivalence by comparing 11 generics with the innovator product in terms of concentration of the active pharmaceutical ingredient (API), minimal inhibitory (MIC) and bactericidal concentrations (MBC), and antibacterial efficacy in the neutropenic mouse thigh infection model. METHODS: The API in each product was measured by a validated microbiological assay and compared by slope (potency) and intercept (concentration) analysis of linear regressions. MIC and MBC were determined by broth microdilution according to Clinical and Laboratory Standard Institute (CLSI) guidelines. For in vivo efficacy, neutropenic ICR mice were inoculated with a clinical strain of Staphylococcus aureus. The animals had 4.14 +/- 0.18 log10 CFU/thigh when treatment started. Groups of 10 mice per product received a total dose ranging from 2.93 to 750 mg/kg per day administered q1h. Sigmoidal dose-response curves were generated by nonlinear regression fitted to Hill equation to compute maximum effect (Emax), slope (N), and the effective dose reaching 50% of the Emax (ED50). Based on these results, bacteriostatic dose (BD) and dose needed to kill the first log of bacteria (1LKD) were also determined. RESULTS: 4 generic products failed pharmaceutical equivalence due to significant differences in potency; however, all products were undistinguishable from the innovator in terms of MIC and MBC. Independently of their status with respect to pharmaceutical equivalence or in vitro activity, all generics failed therapeutic equivalence in vivo, displaying significantly lower Emax and requiring greater BD and 1LKD, or fitting to a non-sigmoidal model. CONCLUSIONS: Pharmaceutical or in vitro equivalence did not entail therapeutic equivalence for oxacillin generic products, indicating that criteria for approval deserve review to include evaluation of in vivo efficacy.

A new pharmacodynamic approach to study antibiotic combinations against enterococci in vivo: Application to ampicillin plus ceftriaxone.

The combination of ampicillin (AMP) and ceftriaxone (CRO) is considered synergistic against Enterococcus faecalis based on in vitro tests and the rabbit endocarditis model, however, in vitro assays are limited by the use of fixed antibiotic concentrations and the rabbit model by poor bacterial growth, high variability, and the use of point dose-effect estimations, that may lead to inaccurate assessment of antibiotic combinations and hinder optimal translation. Here, we tested AMP+CRO against two strains of E. faecalis and one of E. faecium in an optimized mouse thigh infection model that yields high bacterial growth and allows to define the complete dose-response relationship. By fitting Hill's sigmoid model and estimating the parameters maximal effect (Emax) and effective dose 50 (ED50), the following interactions were defined: synergism (Emax increase ≥2 log10 CFU/g), antagonism (Emax reduction ≥1 log10 CFU/g) and potentiation (ED50 reduction ≥50% without changes in Emax). AMP monotherapy was effective against the three strains, yielding valid dose-response curves in terms of dose and the index fT>MIC. CRO monotherapy showed no effect. The combination AMP+CRO against E. faecalis led to potentiation (59-81% ED50 reduction) and not synergism (no changes in Emax). Against E. faecium, the combination was indifferent. The optimized mouse infection model allowed to obtain the complete dose-response curve of AMP+CRO and to define its interaction based on pharmacodynamic parameter changes. Integrating these results with the pharmacokinetics will allow to derive the PK/PD index bound to the activity of the combination, essential for proper translation to the clinic.

Application of microbiological assay to determine pharmaceutical equivalence of generic intravenous antibiotics.

BACKGROUND: Demonstration of equivalent amounts of the same active pharmaceutical ingredient (API) between generic and innovator products (pharmaceutical equivalence) is a basic requirement of regulatory agencies for intravenous generic drugs prior to clinical use, and constitutes the pivotal point to assume therapeutic equivalence. Physicochemical methods are preferred instead of biological assays to determine concentration of drugs in biological fluids, but it does not permit direct quantification of potency. Here, we report a microbiological assay using large plates designed to determine potency and concentration of pharmaceutical-grade antibiotics for injection and a statistical method to assess the in vitro equivalence of generic products with respect to the innovator. METHODS: The assay is based on the concentration-dependent variation of the inhibitory effect of antibiotics on reference bacteria (B. subtilis ATCC 6633, S. aureus ATCC 6538p and S. epidermidis ATCC 12228) in a seeded agar (Difco Antibiotic Media), producing a concentration-response linear relationship with two parameters: y-intercept (concentration) and slope (potency). We compared the parameters of 22 generic products (amikacin 4, gentamicin 15, and vancomycin 3 products) against the innovator and the reference powder by Overall Test for Coincidence of the Regression Lines (Graphpad Prism 5.0). RESULTS: The validation method yielded excellent results for linearity (r(2) > or = 0.98), precision (intra-assay variation < or = 11%; inter-assay variation < or = 10%), accuracy, and specificity tests according to international pharmacopoeial requirements. Except for one generic of vancomycin that had 25% more API (P(y-intercept) = 0.001), the pharmaceutical equivalence was demonstrated in 21 generics with undistinguishable slopes and intercepts (P > 0.66). Potency estimates were 99.8 to 100.5, 99.7 to 100.2 and 98.5 to 99.9% for generic products of amikacin, gentamicin and vancomycin, respectively. CONCLUSION: The proposed method allows rapid, cost-saving, precise, and accurate determination of pharmaceutical equivalence of drugs in pharmaceutical dosage-form, and may be used as a technique for testing generic antibiotics prior to their approval for human use.

Nontherapeutic equivalence of a generic product of imipenem-cilastatin is caused more by chemical instability of the active pharmaceutical ingredient (imipenem) than by its substandard amount of cilastatin.

BACKGROUND: We demonstrated therapeutic nonequivalence of "bioequivalent" generics for meropenem, but there is no data with generics of other carbapenems. METHODS: One generic product of imipenem-cilastatin was compared with the innovator in terms of in vitro susceptibility testing, pharmaceutical equivalence, pharmacokinetic (PK) and pharmacodynamic (PD) equivalence in the neutropenic mouse thigh, lung and brain infection models. Both pharmaceutical forms were then subjected to analytical chemistry assays (LC/MS). RESULTS AND CONCLUSION: The generic product had 30% lower concentration of cilastatin compared with the innovator of imipenem-cilastatin. Regarding the active pharmaceutical ingredient (imipenem), we found no differences in MIC, MBC, concentration or potency or AUC, confirming equivalence in terms of in vitro activity. However, the generic failed therapeutic equivalence in all three animal models. Its Emax against S. aureus in the thigh model was consistently lower, killing from 0.1 to 7.3 million less microorganisms per gram in 24 hours than the innovator (P = 0.003). Against K. pneumoniae in the lung model, the generic exhibited a conspicuous Eagle effect fitting a Gaussian equation instead of the expected sigmoid curve of the Hill model. In the brain infection model with P. aeruginosa, the generic failed when bacterial growth was >4 log10 CFU/g in 24 hours, but not if it was less than 2.5 log10 CFU/g. These large differences in the PD profile cannot be explained by the lower concentration of cilastatin, and rather suggested a failure attributable to the imipenem constituent of the generic product. Analytical chemistry assays confirmed that, besides having 30% less cilastatin, the generic imipenem was more acidic, less stable, and exhibited four different degradation masses that were absent in the innovator.

Etiologic diagnosis of chronic osteomyelitis: a prospective study.

BACKGROUND: Although bone specimens were established 25 years ago as the gold standard for etiologic diagnosis of chronic osteomyelitis, recent studies suggest that nonbone specimens are as accurate as bone to identify the causative agent. We examined concordance rates between cultures from nonbone and bone specimens in 100 patients. METHODS: Prospective study conducted at Hospital Universitario San Vicente de Paul, a 750-bed university-based hospital located in Medellín, Colombia. We included patients with chronic osteomyelitis who had been free of antibiotic therapy for at least 48 hours, excluding those with diabetic foot and decubitus ulcers. At least 1 nonbone and 1 bone specimen were taken from each individual and subjected to complete microbiologic analysis. RESULTS: Bone cultures allowed agent identification in 94% of cases, including anaerobic bacteria in 14%. Cultures of nonbone and bone specimens gave identical results in 30% of patients, with slightly better concordance in chronic osteomyelitis caused by Staphylococcus aureus (42%) than by all other bacterial species (22%). However, statistical concordance determined by the Cohen kappa statistic was less than 0 (-0.0092+/-0.0324), indicating that the observed concordance was no better than that expected by chance alone (P>.99). CONCLUSIONS: Appropriate diagnosis and therapy of chronic osteomyelitis require microbiologic cultures of the infected bone. Nonbone specimens are not valid for this purpose.

In vivo pharmacodynamics of piperacillin/tazobactam: implications for antimicrobial efficacy and resistance suppression with innovator and generic products.

Recent studies have shown that the pharmacodynamic (PD) index driving the efficacy of ß-lactam/ß-lactamase inhibitor combinations such as ceftazidime/avibactam and ceftolozane/tazobactam is the percentage of time the free inhibitor concentration is above a threshold (fT>threshold). However, data with piperacillin/tazobactam (TZP) are scarce. Here we aimed to assess the relationship between fT>threshold and TZP antibacterial efficacy by a population pharmacokinetic study in mice and dose-effect experiments in a neutropenic murine thigh infection model with two isogenic strains of Escherichia coli differentially expressing TEM-1 ß-lactamase. We also explored the dynamics of resistance selection with the innovator and a non-equivalent generic, extrapolated the results to the clinic by Monte Carlo simulation of standard TZP doses, and estimated the economic impact of generic-selected resistance. The fT>threshold index described well the efficacy of TZP versus E. coli, with threshold values from 0.5 mg/L to 2 mg/L and mean exposures of 42% for stasis and 56% for 1 log10 kill. The non-equivalent generic required a longer exposure (fT>threshold 33%) to suppress resistance compared with the innovator (fT>threshold 22%), leading to a higher frequency of resistance selection in the clinical simulation (16% of patients with the generic vs. 1% with the innovator). Finally, we estimated that use of TZP generics in a scenario of 25% therapeutic non-equivalence would result in extra expenses approaching US$1 billion per year in the USA owing to selection of resistant micro-organisms, greatly offsetting the savings gained from generic substitution and further emphasising the need for demonstrated and not assumed therapeutic equivalence.

Antifungal pharmacodynamics: Latin America's perspective.

The current increment of invasive fungal infections and the availability of new broad-spectrum antifungal agents has increased the use of these agents by non-expert practitioners, without an impact on mortality. To improve efficacy while minimizing prescription errors and to reduce the high monetary cost to the health systems, the principles of pharmacokinetics (PK) and pharmacodynamics (PD) are necessary. A systematic review of the PD of antifungals agents was performed aiming at the practicing physician without expertise in this field. The initial section of this review focuses on the general concepts of antimicrobial PD. In vitro studies, fungal susceptibility and antifungal serum concentrations are related with different doses and dosing schedules, determining the PD indices and the magnitude required to obtain a specific outcome. Herein the PD of the most used antifungal drug classes in Latin America (polyenes, azoles, and echinocandins) is discussed.

Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases.

BACKGROUND: For its low cost and ease of handling, the mouse remains the preferred experimental animal for preclinical tests. To avoid the interaction of the animal immune system, in vivo antibiotic pharmacodynamic studies often employ cyclophosphamide (CPM) to induce neutropenia. Although high doses (350-450 mg/kg) are still used and their effects on mouse leukocytes have been described, a lower dose (250 mg/kg) is widely preferred today, but the characteristics and applicability of this approach in outbred mice have not been determined. METHODS: Fifteen female ICR mice were injected intraperitoneally with 150 and 100 mg/kg of CPM on days 1 and 4, respectively. Blood samples (approximately 160 microL) were drawn from the retro-orbital sinus of each mouse on days 1, 4, 5, 6, 7 and 11. Leukocytes were counted manually and the number of granulocytes was based on microscopic examination of Wright-stained smears. The impact of neutropenia induced by this method was then determined with a variety of pathogens in three different murine models of human infections: pneumonia (Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus), meningoencephalitis (S. pneumoniae), and the thigh model (S. aureus, Escherichia coli, Bacteroides fragilis). RESULTS: The basal count of leukocytes was within the normal range for outbred mice. On day 4, there was an 84% reduction in total white blood cells, and by day 5 the leukopenia reached its nadir (370 +/- 84 cells/mm3). Profound neutropenia (< or =10 neutrophils/mm3) was demonstrated at day 4 and persisted through days 5 and 6. Lymphocytes and monocytes had a 92% and 96% decline between days 1 and 5, respectively. Leukocytes recovered completely by day 11. Mice immunosupressed under this protocol displayed clinical and microbiological patterns of progressive and lethal infectious diseases after inoculation in different organs with diverse human pathogens. CONCLUSION: A CPM total dose of 250 mg/kg is sufficient to induce profound and sustained neutropenia (<10 neutrophils/mm3) at least during 3 days in outbred mice, is simpler than previously described methods, and allows successful induction of infection in a variety of experimental models.

Impact on Bacterial Resistance of Therapeutically Nonequivalent Generics: The Case of Piperacillin-Tazobactam.

Previous studies have demonstrated that pharmaceutical equivalence and pharmacokinetic equivalence of generic antibiotics are necessary but not sufficient conditions to guarantee therapeutic equivalence (better called pharmacodynamic equivalence). In addition, there is scientific evidence suggesting a direct link between pharmacodynamic nonequivalence of generic vancomycin and promotion of resistance in Staphylococcus aureus. To find out if even subtle deviations from the expected pharmacodynamic behavior with respect to the innovator could favor resistance, we studied a generic product of piperacillin-tazobactam characterized by pharmaceutical and pharmacokinetic equivalence but a faulty fit of Hill's Emax sigmoid model that could be interpreted as pharmacodynamic nonequivalence. We determined the impact in vivo of this generic product on the resistance of a mixed Escherichia coli population composed of ∼99% susceptible cells (ATCC 35218 strain) and a ∼1% isogenic resistant subpopulation that overproduces TEM-1 ß-lactamase. After only 24 hours of treatment in the neutropenic murine thigh infection model, the generic amplified the resistant subpopulation up to 20-times compared with the innovator, following an inverted-U dose-response relationship. These findings highlight the critical role of therapeutic nonequivalence of generic antibiotics as a key factor contributing to the global problem of bacterial resistance.

Optimization of culture conditions to obtain maximal growth of penicillin-resistant Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae, particularly penicillin-resistant strains (PRSP), constitute one of the most important causes of serious infections worldwide. It is a fastidious microorganism with exquisite nutritional and environmental requirements to grow, a characteristic that prevents the development of useful animal models to study the biology of the microorganism. This study was designed to determine optimal conditions for culture and growth of PRSP. RESULTS: We developed a simple and reproducible method for culture of diverse strains of PRSP representing several invasive serotypes of clinical and epidemiological importance in Colombia. Application of this 3-step culture protocol consistently produced more than 9 log10 CFU/ml of viable cells in the middle part of the logarithmic phase of their growth curve. CONCLUSION: A controlled inoculum size grown in 3 successive steps in supplemented agar and broth under 5% CO2 atmosphere, with pH adjustment and specific incubation times, allowed production of great numbers of PRSP without untimely activation of autolysis mechanisms.

[Vancomycin-resistant Staphylococcus aureus]. / Staphylococcus aureus resistente a vancomicina.

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.

Relevance of various animal models of human infections to establish therapeutic equivalence of a generic product of piperacillin/tazobactam.

After demonstrating with diverse intravenous antibacterials that pharmaceutical equivalence (PE) does not predict therapeutic equivalence, we tested a single generic product of piperacillin/tazobactam (TZP) in terms of PE, pharmacokinetics and in vitro/vivo pharmacodynamics against several pathogens in neutropenic mouse thigh, lung and brain infection models. A generic product was compared head-to-head against the innovator. PE was evaluated by microbiological assay. Single-dose serum pharmacokinetics were determined in infected mice, and the MIC/MBC were determined by broth microdilution. In vivo experiments were done in a blind fashion. Reproducibility was tested on different days using different infecting organisms and animal models. Neutropenic MPF mice were infected in the thighs with Staphylococcus aureus GRP-0057 or Pseudomonas aeruginosa PA01 and in the lungs or brain with Klebsiella pneumoniae ATCC 10031. Treatment started 2h (thigh and brain) or 14 h (lung) after infection and was administered every 3h over 24h (thigh and lung) or 48 h (brain). Both products exhibited the same MIC/MBC against each strain, yielded overlaid curves in the microbiological assay (P>0.21) and were bioequivalent (IC90 83-117% for AUC test/reference ratio). In vivo, the generic product and innovator were again undistinguishable in all models and against the different bacterial pathogens involved. The relevance of these neutropenic murine models of infection was established by demonstrating their accuracy to predict the biological response following simultaneous treatment with a generic product or the innovator of TZP. Therapeutic equivalence of the generic product was proved in every model and against different pathogens.