The sub-MIC selective window decreases along the digestive tract: determination of the minimal selective concentration of oxytetracycline in sterilised intestinal contents.

Introduction: The administration of antibiotics can expose the digestive microbiota of humans and animals to sub-inhibitory concentrations, potentially favouring the selection of resistant bacteria. The minimal selective concentration (MSC) is a key indicator to understand this process. The MSC is defined as the lowest concentration of an antibiotic that promotes the growth of a resistant strain over a susceptible isogenic strain. It represents the lower limit of the sub-minimal inhibitory concentration (MIC) selective window, where resistant mutants can be selected. Previous studies focused on determining the MSC under standard culture conditions, whereas our research aimed to determine the MSC in a model that approximates in vivo conditions. Methods: We investigated the MSC of oxytetracycline (OTC) in Mueller-Hinton broth (MHB) and sterilised intestinal contents (SIC) from the jejunum, caecum and rectum (faeces) of pigs, using two isogenic strains of Escherichia coli (one susceptible and one resistant to OTC). Additionally, the MIC of OTC against the susceptible strain was determined to assess the upper limit of the sub-MIC selective window. Results: Our study took a novel approach, and the results indicated that MIC and MSC values were lower in MHB than in SIC. In the latter, these values varied depending on the intestinal segment, with distal compartments exhibiting higher MIC and MSC values. Moreover, the sub-MIC selective window of OTC in SIC narrowed from the jejunum to the rectum, with a significantly closer MSC to MIC in faecal SIC. Discussion: The results suggest that OTC binds to digestive contents, reducing the fraction of free OTC. However, binding alone does not fully explain our results, and interactions between bacteria and intestinal contents may play a role. Furthermore, our findings provide initial estimates of low concentrations facilitating resistance selection in the gut. Finally, this research enhances the understanding of antimicrobial resistance selection, emphasising the intricate interplay between antibiotics and intestinal content composition in assessing the risk of resistance development in the gut.

Predicted efficacy and tolerance of different dosage regimens of benzylpenicillin in horses based on a pharmacokinetic study with three IM formulations and one IV formulation.

Introduction: Benzylpenicillin (BP) is a first-line antibiotic in horses but there are discrepancies between manufacturers and literature recommendations regarding dosing regimen. Objectives of this study were to evaluate pharmacokinetics and local tolerance of four different formulations of BP in adult horses, and to suggest optimized dosing regimen according to the formulation. Methods: A cross-over design was used in 3 phases for the intramuscular injection of three different products: procaine BP alone, procaine BP/ benzathine BP combination or penethamate hydriodide were administered IM in the gluteal muscles of 6 horses for 3 days. Single IV administration of sodium BP was performed to the same horses with a dose of 22,000 IU BP/kg bwt 39 weeks after last IM injection. BP plasma concentrations were determined by UPLC assay coupled with mass spectrometry and a PK/PD analysis was conducted to predict the efficacy of various dosing regimens by estimating values of the fT>MIC index for different minimum inhibitory concentrations (MIC). Tolerance at the site of IM injection was monitored by creatine kinase activity quantified with a validated chemistry system and clinical scorings. Results and discussion: Except one neurological reaction following one administration of penethamate hydriodide, the tolerance was good. Procaine BP alone, procaine BP/benzathine BP combination or penethamate hydriodide intramuscular administrations at a dosage of 22,000 IU BP/kg bwt q24h for 5 days would yield plasma concentrations that should be effective against bacteria with MIC of ≤0.256, 0.125 or 0.064 mg/L respectively. Of all the tested treatments, the use of a sodium BP by IV Constant Rate Infusion (CRI) for 10 hours a day was deemed to be the most efficient. All the formulations tested in this study are adequate to treat infections with susceptible Streptococcus equi.

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

Antimicrobial combinations against Helicobacter pylori including benzoxadiazol-based flavodoxin inhibitors: in vitro characterization.

IMPORTANCE: The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

Pharmacokinetic-pharmacodynamic cutoff values for benzylpenicillin in horses to support the establishment of clinical breakpoints for benzylpenicillin antimicrobial susceptibility testing in horses.

Introduction: The aim of this international project was to establish a species-specific Clinical Breakpoint for interpretation of Antimicrobial Susceptibility Testing of benzylpenicillin (BP) in horses. Methods: A population pharmacokinetic model of BP disposition was developed to compute PK/PD cutoff values of BP for different formulations that are commonly used in equine medicine around the world (France, Sweden, USA and Japan). Investigated substances were potassium BP, sodium BP, procaine BP, a combination of procaine BP and benzathine BP and penethamate, a prodrug of BP. Data were collected from 40 horses that provided 63 rich profiles of BP corresponding to a total of 1022 individual BP plasma concentrations. Results: A 3-compartment disposition model was selected. For each of these formulations, the PK/PD cutoff was estimated for different dosage regimens using Monte Carlo simulations. The fAUC/MIC or fT>MIC were calculated with a free BP fraction set at 0.4. For fAUC/MIC, a target value of 72 h (for a 72h treatment) was considered. For fT>MIC, efficacy was assumed when free plasma concentrations were above the explored MIC (0.0625-2 mg/L) for 30 or 40 % of the dosing interval. For continuous infusion, a fT>MIC of 90 % was considered. It was shown that a PK/PD cutoff of 0.25 mg/L can be achieved in 90 % of horses with routine regimen (typically 22,000 IU/kg or 12.4 mg/kg per day) with IM procaine BP once a day (France, Japan, Sweden but not USA1) and with IM sodium BP at 14.07 mg/kg, twice a day or IV sodium BP infusion of 12.4 mg/kg per day. In contrast, penethamate and the combination of procaine BP and benzathine BP were unable to achieve this PK/PD cutoff not even an MIC of 0.125 mg/L. Discussion: The PK/PD cutoff of 0.25 mg/L is one dilution lower than the clinical breakpoint released by the CLSI (0.5 mg/ L). From our simulations, the CLSI clinical breakpoint can be achieved with IM procaine BP twice a day at 22,000 IU i.e. 12.4 mg/kg.

Biased computation of probability of target attainment for antimicrobial drugs.

The medical literature is replete with articles in which there is confusion between "free concentration" and "unbound fraction" (fu ), which is the ratio of free to total plasma concentration. The lack of clarity in distinguishing between these two terms has led to biased computations, erroneous interpretations, and misleading recommendations. The problems are highlighted in this paper, taking the example of calculation of Probability of Target Attainment (PTA). This metric is used to propose pharmacokinetic/pharmacodynamic (PK/PD) breakpoints required for the interpretation of Antimicrobial Susceptibility Testing. Based on Monte Carlo simulations of the PK/PD index, area under the unbound concentration time curve/minimum inhibitory concentration (fAUC/MIC), computation of PTA from total plasma concentrations scaled by fu ineluctably leads to biased estimates. The bias is greater if the variability associated with fu is added, instead of removing it during this scaling. The explanation for the bias is that total plasma drug concentrations are intrinsically more variable than the corresponding free concentrations. This is due to the variability of antimicrobial binding for total, but not for free plasma concentrations. In consequence, the greater variability always leads to underestimation of the PK/PD cutoff (i.e., the critical MIC that is guaranteed for a given percentile of the population). A further consequence is an increase in calculated dosage required to attain the targeted quantile. This erroneous approach, of using free antimicrobial drug fraction, is not limited to the derivation of PK/PD cutoff, but may also have consequences for antimicrobials drug safety in clinical patients.

The Selection of Antibiotic- and Bacteriophage-Resistant Pseudomonas aeruginosa Is Prevented by Their Combination.

Bacteria developing resistance compromise the efficacy of antibiotics or bacteriophages (phages). We tested the association of these two antibacterials to circumvent resistance. With the Hollow Fiber Infection Model (HFIM), we mimicked the concentration profile of ciprofloxacin in the lungs of patients treated orally for Pseudomonas aeruginosa infections and, independently, mimicked a single inhaled administration of phages (one or two phages). Each treatment selects for antibiotic- or phage-resistant clones in less than 30 h. In contrast, no bacteria were recovered from the HFIM at 72 h when ciprofloxacin was started 4 h post phage administration, even when increasing the initial bacterial concentration by 1,000-fold. The combination of phages with antibiotics used according to clinical regimens prevents the growth of resistant clones, providing opportunities to downscale the use of multiple antibiotics. IMPORTANCE In the treatment of bacterial infections, the use of antibiotics or bacteriophages (phages) is limited by the ability of bacteria to develop resistance. The resistance frequency depends on the exposure to antibacterials. Therefore, determination of concentration profiles of antibiotics is key to define optimal regimens during treatments. In the laboratory, the Hollow Fiber Infection Model (HFIM) mimics concentration profiles observed in patients. In this study, we used the HFIM to evaluate the killing efficacy of the combination of phages and ciprofloxacin. We demonstrated that dosing schedule of phages first and the antibiotic second prevent the selection of resistant bacteria. These results demonstrate that combination efficacy relies on a strong initial reduction of the bacterial population by phages followed by antibiotics before any resistant arise.

Prediction of Minocycline Activity in the Gut From a Pig Preclinical Model Using a Pharmacokinetic -Pharmacodynamic Approach.

The increase of multidrug-resistant (MDR) bacteria has renewed interest in old antibiotics, such as minocycline, that can be active against various MDR Gram-negative pathogens. The elimination of minocycline by both kidneys and liver makes it suitable for impaired renal function patients. However, the drawback is the possible elimination of a high amount of drug in the intestines, with potential impact on the digestive microbiota during treatment. This study aimed to predict the potential activity of minocycline against Enterobacterales in the gut after parenteral administration, by combining in vivo and in vitro studies. Total minocycline concentrations were determined by UPLC-UV in the plasma and intestinal content of piglets following intravenous administration. In parallel, the in vitro activity of minocycline was assessed against two Escherichia coli strains in sterilized intestinal contents, and compared to activity in a standard broth. We found that minocycline concentrations were 6-39 times higher in intestinal contents than plasma. Furthermore, minocycline was 5- to 245-fold less active in large intestine content than in a standard broth. Using this PK-PD approach, we propose a preclinical pig model describing the link between systemic and gut exposure to minocycline, and exploring its activity against intestinal Enterobacterales by taking into account the impact of intestinal contents.

Destruction of Staphylococcus aureus biofilms by combining an antibiotic with subtilisin A or calcium gluconate.

In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca2+ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log10 CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 µg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log10 CFU/mL for 20-25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20-25%) to be susceptible to the combinations.

Dynamic interactions between cephalexin and macrophages on different Staphylococcus aureus inoculum sizes: a tripartite in vitro model.

BACKGROUND: The bactericidal activity of an antimicrobial drug is generally assessed by in vitro bacterial time-kill experiments which do not include any components of the immune system, even though the innate immunity, the primary host defence, is probably able to kill a large proportion of pathogenic bacteria in immunocompetent patients. We developed an in vitro tripartite model to investigate the joint action of C57Bl/6 murine bone-marrow-derived macrophages and cephalexin on the killing of Staphylococcus aureus. RESULTS: By assessing the bactericidal effects on four bacterial inoculum sizes, we showed that macrophages can cooperate with cephalexin on inoculum sizes lower than 106 CFU/mL and conversely, protect S. aureus from cephalexin killing activity at the highest inoculum size. Cell analysis by flow cytometry revealed that macrophages were rapidly overwhelmed when exposed to large inoculums. Increasing the initial inoculum size from 105 to 107 CFU/mL increased macrophage death and decreased their ability to kill bacteria from six hours after exposure to bacteria. The addition of cephalexin at 16-fold MIC to 105 and 106 CFU/mL inoculums allowed the macrophages to survive and to maintain their bactericidal activity as if they were exposed to a small bacterial inoculum. However, with the highest inoculum size of 107 CFU/mL, the final bacterial counts in the supernatant were higher with macrophages plus cephalexin than with cephalexin alone. CONCLUSIONS: These results suggest that if the bacterial population at the infectious site is low, as potentially encountered in the early stage of infection or at the end of an antimicrobial treatment, the observed cooperation between macrophages and cephalexin could facilitate its control.

Determination of the pharmacokinetic-pharmacodynamic cut-off values of marbofloxacin in horses to support the establishment of a clinical breakpoint for antimicrobial susceptibility testing.

BACKGROUND: Marbofloxacin (MBX), a fluoroquinolone (FQ), is considered as a critical antibiotic requiring antimicrobial susceptibility testing (AST) for prudent use. No clinical breakpoint (CBP) currently exists to interpret the results of such tests in horses. OBJECTIVES: To compute PK/PD cut-offs (PK/PDCO ) that is one of the three minimum inhibitory concentrations (MICs) considered establishing a CBP for antimicrobial susceptibility test interpretation. STUDY DESIGN: A meta-analysis conducted by combining five sets of previously published pharmacokinetic data, obtained in clinical and nonclinical settings. METHODS: Horses (n = 131) received MBX intravenously at doses of either 2 or 10 mg/kg BW. They were richly sampled (five or six samples per horse). A population model was built to generate a virtual population of 5000 MBX disposition curves by Monte Carlo simulations (MCS) over 24 hours. The selected PK/PD index was the ratio of Area Under the free plasma concentration-time Curve divided by the MIC (fAUC/MIC). The PK/PDCO , which is the highest MIC for which 90% of horses can achieve an a priori selected critical value for the numerical value of the PK/PD index, was established for Gram-positive and Gram-negative bacteria for a dose of 2 mg/kg. RESULTS: The PK/PDCO of MBX in horses was 0.125 mg/L for Gram-positive pathogens and 0.0625 mg/L for Gram-negative pathogens. MBX MICs determined by broth microdilution for 54 Escherichia coli and 189 Streptococcus equi isolates are reported. MAIN LIMITATION: No clinical data are taken into account in the determination of a PK/PDco . CONCLUSION: The computed PK/PDco predicts that MBX may be efficacious in horses to treat infections associated with Enterobacteriaceae but unlikely to those involving Staphylococcus aureus or Streptococcus equi.

The pharmacokinetic/pharmacodynamic paradigm for antimicrobial drugs in veterinary medicine: Recent advances and critical appraisal.

Pharmacokinetic/pharmacodynamic (PK/PD) modelling is the initial step in the semi-mechanistic approach for optimizing dosage regimens for systemically acting antimicrobial drugs (AMDs). Numerical values of PK/PD indices are used to predict dose and dosing interval on a rational basis followed by confirmation in clinical trials. The value of PK/PD indices lies in their universal applicability amongst animal species. Two PK/PD indices are routinely used in veterinary medicine, the ratio of the area under the curve of the free drug plasma concentration to the minimum inhibitory concentration (MIC) (fAUC/MIC) and the time that free plasma concentration exceeds the MIC over the dosing interval (fT > MIC). The basic concepts of PK/PD modelling of AMDs were established some 20 years ago. Earlier studies have been reviewed previously and are not reconsidered in this review. This review describes and provides a critical appraisal of more recent, advanced PK/PD approaches, with particular reference to their application in veterinary medicine. Also discussed are some hypotheses and new areas for future developments.First, a brief overview of PK/PD principles is presented as the basis for then reviewing more advanced mechanistic considerations on the precise nature of selected indices. Then, several new approaches to selecting PK/PD indices and establishing their numerical values are reviewed, including (a) the modelling of time-kill curves and (b) the use of population PK investigations. PK/PD indices can be used for dose determination, and they are required to establish clinical breakpoints for antimicrobial susceptibility testing. A particular consideration is given to the precise nature of MIC, because it is pivotal in establishing PK/PD indices, explaining that it is not a "pharmacodynamic parameter" in the usual sense of this term.

Levers to Improve Antibiotic Treatment of Lambs via Drinking Water in Sheep Fattening Houses: The Example of the Sulfadimethoxine/Trimethoprim Combination.

To limit the spread of bacterial diseases in sheep fattening houses, antibiotics are often administered collectively. Collective treatments can be delivered by drinking water but data on the drug's solubility in water or on plasma exposure of the animals are lacking. We first assessed the solubility of products containing sulfadimethoxine (SDM), associated or not with trimethoprim (TMP), in different waters. We then compared in lambs the SDM and TMP pharmacokinetic profiles after individual intravenous (IV) and oral administrations of SDM-TMP in experimental settings (n = 8) and after a collective treatment by drinking water with SDM-TMP or SDM alone in a sheep fattening house (n = 100 for each treatment). The individual water consumption during the collective treatments was also monitored to characterize the ingestion variability. We showed that TMP had a short terminal half-life and very low oral bioavailability, demonstrating that it would be unable to potentiate SDM by oral route. Conversely, SDM had a long terminal half-life of 18 h and excellent oral bioavailability. However, delivery by drinking water resulted in a very high interindividual variability of SDM plasma concentrations, meaning that although disease spread could be controlled at the group level, some individuals would inevitably be under- or over-exposed to the antibiotic.

Why Were More Than 200 Subjects Required to Demonstrate the Bioequivalence of a New Formulation of Levothyroxine with an Old One?

At the request of French Regulatory Authorities, a new formulation of Levothyrox® was licensed in France in 2017, with the objective of avoiding the stability deficiencies of an existing licensed formulation. Before launching the new formulation, an average bioequivalence (ABE) trial was conducted, having enrolled 204 subjects and selected for interpretation a narrow a priori bioequivalence range of 0.90-1.11. Bioequivalence was concluded. In a previous publication, we questioned the ability of an ABE trial to guarantee the switchability within patients of the new and old levothyroxine formulations. It was suggested that the two formulations should be compared using the conceptual framework of individual bioequivalence. The present paper is a response to those claiming that, despite the fact that ABE analysis does not formally address the switchability of the two formulations, future patients will nevertheless be fully protected. The basis for this claim is that the ABE study was established in a large trial and analyzed using a stringent a priori acceptance interval of equivalence. These claims are questionable, because the use of a very large number of subjects nullifies the implicit precautionary intention of the European guideline when, for a Narrow Therapeutic Index drug, it recommends shortening the a priori acceptance interval from 0.80-1.25 to 0.90-1.11.

Pharmacokinetics of Ertapenem in Sheep (Ovis aries) with Experimentally Induced Urinary Tract Infection.

Sheep are commonly used as animal models for human biomedical research, but descriptions of their use for studying the pharmacokinetics of carbapenem antimicrobials, such as ertapenem, are unavailable. Ertapenem is a critical antimicrobial for human infections, and the description of the pharmacokinetics of this drug is of value for research using ovine as models for human diseases, such as urinary tract infections (UTI). There are currently no ovine models for comparative biomedical research of UTI. The objective of this study was to report the pharmacokinetics of ertapenem in sheep after single and multiple dosing. In addition, we explored the effects of an immunomodulatory drug (Zelnate) on the pharmacokinetics of ertapenem in sheep. Eight healthy ewes (weight, 64.4 ± 7.7 kg) were used in an ovine bacterial cystitis model of human cystitis with Pseudomonas aeruginosa. After disease confirmation, each ewe received 1 g of ertapenem intravenously once every 24 h for 5 administrations. Blood was collected intensively (14 samples) during 24 h after the first and last administration. After multiple-dose administration, the volume of distribution was 84.5 mL/kg, clearance was 116.3 mL/h/kg, T1/2(λz) was 1.1 h, and the extraction ratio was 0.02. No significant differences in pharmacokinetic parameters or time points were found between groups treated with the immunostimulant and controls or after the 1st or 5th administration of ertapenem. No accumulation was noted from previous administration. Our ovine pharmacokinetic findings can be used to evaluate therapeutic strategies for ertapenem use (varying drug dosing schedules and combinations with other antimicrobials or immune modulators) in the context of UTI.