Residues of veterinary antibiotics in manures from pig and chicken farms in a context of antimicrobial use reduction by implementation of health and welfare plans.

The use of antibiotics in food-producing animals can induce the presence of residual substances in manure, which are then released into the environment and may contribute to soil and groundwater contamination. During the on-farm implementation of strategies to improve animal health and welfare in chicken and pig farms, the consequences of antibiotic use were evaluated in terms of the occurrence and levels of antibiotic residues in manure. A set of 35 broiler farms from Cyprus, Greece, the Netherlands and 40 pig farms from France and Italy provided a total of 350 manure samples. The primary objective was to develop a specific LC/MS/MS method capable of quantifying antibiotic residues in both types of manure. The method was able to detect fifteen antibiotics belonging to nine classes, with validated limits of quantification of 10-20 µg/kg, and accuracies ranging from 81% to 138%. With the exception of amoxicillin, which was never detected in any manure, all antibiotics used were detected in manure from treated animals with typical concentrations ranging from 10 to 99198 µg/kg for both chickens and pigs. The occurrence of residual antibiotics was higher in chicken than in pig manure, especially for fluoroquinolones and doxycycline which were detected in 89% and 100% of the chicken manure, respectively, and in 28% of the pig manure. The impact of the health plans on the antibiotic load manure was assessed by measuring for each farm the ratio of the sum of all antibiotic concentrations measured after and before the implementation of the plan. The results showed that, in addition to the frequency of treatments, the class of antibiotic used is an important factor to consider as it strongly influences the stability/instability of the compounds, i.e. their ability to persist in the manure of food-producing animals.

Doxycycline serum protein binding in pigs reveals a relatively high free fraction.

Doxycycline is an antibiotic widely used in pig farming. As with all antibiotics, only the free concentrations are considered to be bacteriologically active. Historically, the free fraction (fu) in pig plasma has been estimated at 7%, which, given the effective dosage regime used in pigs, leads to free plasma concentrations of doxycycline largely lower than the minimum inhibitory concentrations of the target pathogens. This apparent inconsistency led us to reassess plasma protein binding of doxycycline in pigs. Using an equilibrium dialysis method, the extent of doxycycline binding was measured individually in 26 pigs for total doxycycline concentration ranging from 10 to 1000 µmol/L. Analysis of the data using a non-linear mixed-effects model demonstrated linearity of plasma protein binding with a mean fu value of 31% and a relatively low inter-subject variability of approximately 10%. This new data showing that the free fraction is four times greater than what could have been anticipated from historical data is discussed in particular for the calculation of the PK/PD cut-offs, which are used to establish the clinical breakpoints for antimicrobial susceptibility testing.

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.

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.

Comparison of hydroalcoholic rubbing and conventional chlorhexidine scrubbing for aseptic skin preparation in dogs.

OBJECTIVE: To compare preparation time, ease of application, and elimination of skin contamination of 3 skin preparation methods for asepsis. STUDY DESIGN: Experimental study. ANIMALS: Healthy dogs (n = 6) with no clinical signs of skin disease. METHODS: Three sites on each dog were randomly allocated to 1 of 3 preparation protocols for asepsis: (1) 5 scrubbings with chlorhexidine gluconate and rinsing (CHXG), (2) washing with mild soap prior to 3 rubbings with hydroalcoholic solution (soap-HAR), or (3) 3 rubbings with hydroalcoholic solution (HAR). The duration of each method of skin preparation was recorded. A Count-Tact agar plate was placed in the center of each site before, immediately after, 1 hour after, and 3 hours after antiseptic application. Plates were cultured, and colony forming units (CFU) were counted. RESULTS: Skin preparation lasted an average of 375 seconds for CHXG, 240 seconds for soap-HAR, and 190 seconds for HAR (P = .00049). Nine CFU (median) were cultured from the skin prior to preparation, with no difference between sites on any animal or for any method. Colony forming units were not detected at any time on any site in any dog after antiseptic application. CONCLUSION: Rubbing with hydroalcoholic (HA) solution was as effective as CHXG and prevented bacterial growth for at least 3 hours under these experimental conditions. Rubbing with hydroalcoholic solution was also faster and easier to perform. CLINICAL SIGNIFICANCE: Because there is currently no known resistance to HA solution, preparation of the surgical site with HAR should be considered to prevent the emergence of bacterial resistance to chlorhexidine as well as potential cross-resistances to antibiotics. Transfer to clinical animals requires additional investigation.

Impact of Low and High Doses of Marbofloxacin on the Selection of Resistant Enterobacteriaceae in the Commensal Gut Flora of Young Cattle: Discussion of Data from 2 Study Populations.

In the context of requested decrease of antimicrobial use in veterinary medicine, our objective was to assess the impact of two doses of marbofloxacin administered on young bulls (YBs) and veal calves (VCs) treated for bovine respiratory disease, on the total population of Enterobacteriaceae in gut flora and on the emergence of resistant Enterobacteriaceae. In two independent experiments, 48 YBs from 6 commercial farms and 33 VCs previously colostrum deprived and exposed to cefquinome were randomly assigned to one of the three groups LOW, HIGH, and Control. In LOW and HIGH groups, animals received a single injection of, respectively, 2 and 10 mg/kg marbofloxacin. Feces were sampled before treatment, and at several times after treatment. Total and resistant Enterobacteriaceae enumerating were performed by plating dilutions of fecal samples on MacConkey agar plates that were supplemented or not with quinolone. In YBs, marbofloxacin treatment was associated with a transient decrease in total Enterobacteriaceae count between day (D)1 and D3 after treatment. Total Enterobacteriaceae count returned to baseline between D5 and D7 in all groups. None of the 48 YBs harbored marbofloxacin-resistant Enterobacteriaceae before treatment. After treatment, 1 out of 20 YBs from the Control group and 1 out of 14 YBs from the HIGH group exhibited marbofloxacin-resistant Enterobacteriaceae. In VCs, the rate of fluoroquinolone-resistant Enterobacteriaceae significantly increased after low and high doses of marbofloxacin treatment. However, the effect was similar for the two doses, which was probably related to the high level of resistant Enterobacteriaceae exhibited before treatment. Our results suggest that a single treatment with 2 or 10 mg/kg marbofloxacin exerts a moderate selective pressure on commensal Enterobacteriaceae in YBs and in VCs. A fivefold decrease of marbofloxacin regimen did not affect the selection of resistances among commensal bacteria.

Allometric scaling for predicting human clearance of bisphenol A.

The investigation of interspecies differences in bisphenol A (BPA) pharmacokinetics (PK) may be useful for translating findings from animal studies to humans, identifying major processes involved in BPA clearance mechanisms, and predicting BPA PK parameters in man. For the first time, a large range of species in terms of body weight, from 0.02 kg (mice) to 495 kg (horses) was used to predict BPA clearance in man by an allometric approach. BPA PK was evaluated after intravenous administration of BPA in horses, sheep, pigs, dogs, rats and mice. A non-compartmental analysis was used to estimate plasma clearance and steady state volume of distribution and predict BPA PK parameters in humans from allometric scaling. In all the species investigated, BPA plasma clearance was high and of the same order of magnitude as their respective hepatic blood flow. By an allometric scaling, the human clearance was estimated to be 1.79 L/min (equivalent to 25.6 mL/kg.min) with a 95% prediction interval of 0.36 to 8.83 L/min. Our results support the hypothesis that there are highly efficient and hepatic mechanisms of BPA clearance in man.

Low or high doses of cefquinome targeting low or high bacterial inocula cure Klebsiella pneumoniae lung infections but differentially impact the levels of antibiotic resistance in fecal flora.

The combination of efficacious treatment against bacterial infections and mitigation of antibiotic resistance amplification in gut microbiota is a major challenge for antimicrobial therapy in food-producing animals. In rats, we evaluated the impact of cefquinome, a fourth-generation cephalosporin, on both Klebsiella pneumoniae lung infection and intestinal flora harboring CTX-M-producing Enterobacteriaceae. Germfree rats received a fecal flora specimen from specific-pathogen-free pigs, to which a CTX-M-producing Escherichia coli strain had been added. K. pneumoniae cells were inoculated in the lungs of these gnotobiotic rats by using either a low (10(5) CFU) or a high (10(9) CFU) inoculum. Without treatment, all animals infected with the low or high K. pneumoniae inoculum developed pneumonia and died before 120 h postchallenge. In the treated groups, the low-inoculum rats received a 4-day treatment of 5 mg/kg of body weight cefquinome beginning at 24 h postchallenge (prepatent phase of the disease), and the high-inoculum rats received a 4-day treatment of 50 mg/kg cefquinome beginning when the animals expressed clinical signs of infection (patent phase of the disease). The dose of 50 mg/kg targeting the high K. pneumoniae inoculum cured all the treated rats and resulted in a massive amplification of CTX-M-producing Enterobacteriaceae. A dose of 5 mg/kg targeting the low K. pneumoniae inoculum cured all the rats and averted an outbreak of clinical disease, all without any amplification of CTX-M-producing Enterobacteriaceae. These findings might have implications for the development of new antimicrobial treatment strategies that ensure a cure for bacterial infections while avoiding the amplification of resistance genes of human concern in the gut microbiota of food-producing animals.

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.

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.

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.

Development of HPV16 mouse and dog models for more accurate prediction of human vaccine efficacy.

BACKGROUND: Animal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study. RESULTS: We first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre-lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis. CONCLUSIONS: In this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.

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.

Repeated Exposure of Escherichia coli to High Ciprofloxacin Concentrations Selects gyrB Mutants That Show Fluoroquinolone-Specific Hyperpersistence.

Recent studies have shown that not only resistance, but also tolerance/persistence levels can evolve rapidly in bacteria exposed to repeated antibiotic treatments. We used in vitro evolution to assess whether tolerant/hyperpersistent Escherichia coli ATCC25922 mutants could be selected under repeated exposure to a high ciprofloxacin concentration. Among two out of three independent evolution lines, we observed the emergence of gyrB mutants showing an hyperpersistence phenotype specific to fluoroquinolones, but no significant MIC increase. The identified mutation gives rise to a L422P substitution in GyrB, that is, outside of the canonical GyrB QRDR. Our results indicate that mutations in overlooked regions of quinolone target genes may impair the efficacy of treatments via an increase of persistence rather than resistance level, and support the idea that, in addition to resistance, phenotypes of tolerance/persistence of infectious bacterial strains should receive considerations in the choice of antibiotic therapies.

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.

Combining sequential extraction and 3D fluorescence to investigate the behavior of antibiotic and polycyclic aromatic hydrocarbons during solar drying of sewage sludge.

Solar drying and liming are commonly used for sludge treatment, but little is known about their efficiency on antibiotics and Polycyclic Aromatic Hydrocarbons (PAHs) removal. This study aimed to investigate the removal of antibiotics and PAHs during solar drying of Limed Sludge (LS) and Non-Limed Sludge (NLS). Thus, organic matter fractionation and 3D fluorescence were used to assess the accessibility and the complexity of organic matter. 2 experiments have been conducted using LS and NLS for 45 days of drying in a pilot scale tunnel. Physicochemical results indicated significant decrease of water content (90%) for both sludge samples within 15 days of drying. For both treatments, the removal of total organic carbon and total nitrogen was low and similar for both treatments. Through this study, it has been confirmed that liming and drying contributed to a strong modification of the organic matter quality with an increase of its accessibility. On the other hand, drying alone increased the less accessible compartments, while the presence of lime affected the interconnexion between the organic matter pools. 3D fluorescence confirmed the obtained results and indicated that LS leads to obtaining more simple molecules in the most accessible compartments, while NLS leads to obtaining more complex molecules in the less accessible compartments. In addition, solar radiations and leaching may contribute to the significant removal (p < 0.01) of roxithromycin, benzo(a)anthracene, chrysene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo(g, h, i) perylene in the presence of lime. Furthermore, the evolution of organic matter pools in terms of accessibility and complexity may drive the bioavailability of these pollutants, leading to their significant removal.

Laparoscopic-assisted colopexy and sterilization in male dogs: short-term results and physiologic consequences.

OBJECTIVE: To describe laparoscopic-assisted colopexy and sterilization, and evaluate outcome and effects in healthy male dogs. STUDY DESIGN: Experimental study. ANIMALS: Male Beagle dogs (n=7). METHODS: A laparoscopic-assisted, extracorporeally sutured colopexy, and sterilization by ligation and section of the testicular vessels and ductus deferens were evaluated 11 weeks after surgery. Ex vivo tensile tests were performed on the colopexy sites and loss of testicular function was assessed by monitoring serum testosterone, and by ultrasonographic and histologic examinations of the testes. Systemic inflammation and potential iatrogenic colonic functional disorders were investigated by monitoring serum C-reactive protein (CRP) in the perioperative period and from a sulfapyridine (SP) kinetic profile obtained before and 10 weeks after surgery. RESULTS: No intraoperative complications were recorded and clinical outcome was considered fair in all dogs. A mean tensile force of 42 N was required to disrupt colopexies. No relevant postoperative increase in CRP concentrations or changes in SP kinetics were observed. Testicular function was lost. CONCLUSIONS: Laparoscopic-assisted colopexy achieved adhesion of the colon to the abdominal wall and testicular endocrine function and spermatogenesis were eliminated by laparoscopic castration.

A Large Impact of Obesity on the Disposition of Ivermectin, Moxidectin and Eprinomectin in a Canine Model: Relevance for COVID-19 Patients.

Ivermectin (IVM) and moxidectin (MOX) are used extensively as parasiticides in veterinary medicine. Based on in vitro data, IVM has recently been proposed for the prevention and treatment of COVID-19 infection, a condition for which obesity is a major risk factor. In patients, IVM dosage is based on total body weight and there are no recommendations to adjust dosage in obese patients. The objective of this study was to establish, in a canine model, the influence of obesity on the clearance and steady-state volume of distribution of IVM, MOX, and a third analog, eprinomectin (EPR). An experimental model of obesity in dogs was based on a high calorie diet. IVM, MOX, and EPR were administered intravenously, in combination, to a single group of dogs in two circumstances, during a control period and when body weight had been increased by 50%. In obese dogs, clearance, expressed in absolute values (L/day), was not modified for MOX but was reduced for IVM and EPR, compared to the initial control state. However, when scaled by body weight (L/day/kg), plasma clearance was reduced by 55, 42, and 63%, for IVM, MOX and EPR, respectively. In contrast, the steady-state volume of distribution was markedly increased, in absolute values (L), by obesity. For IVM and MOX, this obese dog model suggests that the maintenance doses in the obese subject should be based on lean body weight rather than total weight. On the other hand, the loading dose, when required, should be based on the total body weight of the obese subject.