Antimicrobial susceptibility among respiratory tract pathogens isolated from diseased cattle and pigs from different parts of Europe.

AIMS: To survey antibiotic susceptibility of bacteria causing cattle and pig respiratory infections in 10 European countries. METHODS AND RESULTS: Non-replicate nasopharyngeal/nasal or lung swabs were collected from animals with acute respiratory signs during 2015-2016. Pasteurella multocida, Mannheimia haemolytica, Histophilus somni from cattle (n = 281), and P. multocida, Actinobacillus pleuropneumoniae, Glaesserella parasuis, Bordetella bronchiseptica, and Streptococcus suis from pigs (n = 593) were isolated. MICs were assessed following CLSI standards and interpreted using veterinary breakpoints where available. Histophilus somni isolates were fully antibiotic susceptible. Bovine P. multocida and M. haemolytica were susceptible to all antibiotics, except tetracycline (11.6%-17.6% resistance). Low macrolide and spectinomycin resistance was observed for P. multocida and M. haemolytica (1.3%-8.8%). Similar susceptibility was observed in pigs, where breakpoints are available. Resistance in P. multocida, A. pleuropneumoniae, and S. suis to ceftiofur, enrofloxacin, and florfenicol was absent or <5%. Tetracycline resistance varied from 10.6% to 21.3%, but was 82.4% in S. suis. Overall multidrug-resistance was low. Antibiotic resistance in 2015-2016 remained similar as in 2009-2012. CONCLUSIONS: Low antibiotic resistance was observed among respiratory tract pathogens, except for tetracycline.

Dose-dependent impact of enrofloxacin on broiler chicken gut resistome is mitigated by synbiotic application.

Fluoroquinolone agents are considered critical for human medicine by the World Health Organization (WHO). However, they are often used for the treatment of avian colibacillosis in poultry production, creating considerable concern regarding the potential spread of fluoroquinolone resistance genes from commensals to pathogens. Therefore, there is a need to understand the impact of fluoroquinolone application on the reservoir of ARGs in poultry gut and devise means to circumvent potential resistome expansion. Building upon a recent dose optimization effort, we used shotgun metagenomics to investigate the time-course change in the cecal microbiome and resistome of broiler chickens receiving an optimized dosage [12.5 mg/kg body weight (bw)/day], with or without synbiotic supplementation (PoultryStar®, BIOMIN GmbH), and a high dosage of enrofloxacin (50 mg/kg bw/day). Compared to the high dose treatment, the low (optimized) dose of enrofloxacin caused the most significant perturbations in the cecal microbiota and resistome of the broiler chickens, demonstrated by a lower cecal microbiota diversity while substantially increasing the antibiotic resistance genes (ARGs) resistome diversity. Withdrawal of antibiotics resulted in a pronounced reduction in ARG diversity. Chickens receiving the synbiotic treatment had the lowest diversity and number of enriched ARGs, suggesting an alleviating impact on the burden of the gut resistome. Some Proteobacteria were significantly increased in the cecal metagenome of chickens receiving enrofloxacin and showed a positive association with increased ARG burden. Differential abundance (DA) analysis revealed a significant increase in the abundance of ARGs encoding resistance to macrolides-lincosamides-streptogramins (MLS), aminoglycosides, and tetracyclines over the period of enrofloxacin application, with the optimized dosage application resulting in a twofold higher number of affected ARG compared to high dosage application. Our results provide novel insights into the dose-dependent effects of clinically important enrofloxacin application in shaping the broiler gut resistome, which was mitigated by a synbiotic application. The contribution to ameliorating the adverse effects of antimicrobial agents, that is, lowering the spread of antimicrobial resistance genes, on the poultry and potentially other livestock gastrointestinal microbiomes and resistomes merits further study.

Pharmacokinetics of Cannabidiol Following Intranasal, Intrarectal, and Oral Administration in Healthy Dogs.

The therapeutic potential of cannabidiol (CBD), a non-psychtropic component of the Cannabis sativa plant, is substantiated more and more. We aimed to determine the pharmacokinetic behavior of CBD after a single dose via intranasal (IN) and intrarectal (IR) administration in six healthy Beagle dogs age 3-8 years old, and compare to the oral administration route (PO). Standardized dosages applied for IN, IR and PO were 20, 100, and 100 mg, respectively. Each dog underwent the same protocol but received CBD through a different administration route. CBD plasma concentrations were determined by ultra-high performance liquid chromatography-tandem mass spectrometry before and at fixed time points after administration. Non-compartmental analysis was performed on the plasma concentration-time profiles. Plasma CBD concentrations after IR administration were below the limit of quantification. The mean area under the curve (AUC) after IN and PO CBD administration was 61 and 1,376 ng/mL*h, respectively. The maximal plasma CBD concentration (Cmax) after IN and PO CBD administration was 28 and 217 ng/mL reached after 0.5 and 3.5 h (Tmax), respectively. Significant differences between IN and PO administration were found in the Tmax (p = 0.04). Higher AUC and Cmax were achieved with 100 mg PO compared to 20 mg IN, but no significant differences were found when AUC (p = 0.09) and Cmax (p = 0.44) were normalized to 1 mg dosages. IN administration of CBD resulted in faster absorption when compared to PO administration. However, PO remains the most favorable route for CBD delivery due to its more feasible administration. The IR administration route is not advised for clinical application.

Enrofloxacin Dose Optimization for the Treatment of Colibacillosis in Broiler Chickens Using a Drinking Behaviour Pharmacokinetic Model.

Enrofloxacin is frequently administered via drinking water for the treatment of colibacillosis in broiler chickens. However, the EMA/CVMP has urged to re-evaluate historically approved doses, especially for antimicrobials administered via drinking water. In response, the objectives of this study were two-fold. First, to evaluate the pharmacokinetics (PK) of enrofloxacin following IV, PO and drinking water administration. Second, to predict the efficacy of a range of doses in the drinking water for the treatment of APEC infections. For the first objective, PK parameters were estimated by fitting a one-compartmental model with a zero-order IV infusion and an oral absorption lag function to the simultaneously modelled IV and PO data. After fixing these parameter values, a drinking behaviour pharmacokinetic (DBPK) model was developed for the description and prediction of drinking water PK profiles by adding three model improvements (different diurnal and nocturnal drinking rates, inter-animal variability in water consumption and taking account of dose non-proportionality). The subsequent simulations and probability of target attainment (PTA) analysis predicted that a dose of 12.5 mg/kg/24 h is efficacious in treating colibacillosis with an MIC up to 0.125 µg/mL (ECOFF), whereas the currently registered dose (10 mg/kg/24 h) reaches a PTA of 66% at ECOFF.

The Development of a Juvenile Porcine Augmented Renal Clearance Model Through Continuous Infusion of Lipopolysaccharides: An Exploratory Study.

Augmented renal clearance (ARC) as observed in the critically ill (pediatric) population can have a major impact on the pharmacokinetics and posology of renally excreted drugs. Although sepsis has been described as a major trigger in the development of ARC in human critically ill patients, mechanistic insights on ARC are currently lacking. An appropriate ARC animal model could contribute to reveal these underlying mechanisms. In this exploratory study, a state of ARC was induced in 8-week-old piglets. Conscious piglets were continuously infused over 36 h with lipopolysaccharides (LPS) from Escherichia coli (O111:B4) to induce sepsis and subsequently trigger ARC. To study the dose-dependent effect of LPS on the renal function, three different doses (0.75, 2.0, 5.0 µg/kg/h) were administered (two ♂ piglets/dose, one sham piglet), in combination with fluid administration (0.9% NaCl) at 6 ml/kg/h. Single boluses of renal markers, i.e., creatinine [40 mg/kg body weight (BW)], iohexol (64.7 mg/kg BW), and para-aminohippuric acid (PAH, 10 mg/kg BW) were administered intravenously to evaluate the effect of LPS on the renal function. Clinical parameters were monitored periodically. Blood sampling was performed to determine the effect on hematology, neutrophil gelatinase-associated lipocalin, and prostaglandin E2 plasma levels. All piglets that were continuously infused with LPS displayed an elevated body temperature, heart rhythm, and respiratory rate ~1-3 h after start of the infusion. After infusion, considerably higher total body clearances of iohexol, creatinine, and PAH were observed, independent of the administration of LPS and/or its dose. Since also the sham piglet, receiving no LPS, demonstrated a comparable increase in renal function, the contribution of fluid administration to the development of ARC should be further evaluated.

Evaluation of Fluoroquinolone Resistance in Clinical Avian Pathogenic Escherichia coli Isolates from Flanders (Belgium).

Fluoroquinolones are frequently used antimicrobials for the treatment of avian pathogenic Escherichia coli (APEC) infections. However, rapid development and selection of resistance to this class of antimicrobial drugs is a significant problem. The aim of this study was to investigate the occurrence and mechanisms of antimicrobial resistance against enrofloxacin (ENRO) in APEC strains in Flanders, Belgium. One hundred and twenty-five APEC strains from broilers with clinical colibacillosis were collected in Flanders from November 2017 to June 2018. The minimum inhibitory concentration (MIC) of all strains and the mutant prevention concentration (MPC) of a sample of sensitive isolates were determined using a commercial gradient strip test and via the agar dilution method, respectively. Non-wild type (NWT) isolates were further characterized using polymerase chain reaction (PCR), gel electrophoresis and gene sequencing. Forty percent of the APEC strains were NWT according to the epidemiological cut-off (ECOFF) measure (MIC > 0.125 µg/mL). With respect to clinical breakpoints, 21% were clinically intermediate (0.5 ≤ MIC ≤ 1 µg/mL) and 10% were clinically resistant (MIC ≥ 2). The MPC values of the tested strains ranged from 0.064 to 1 µg/mL, resulting in MPC/MIC ratios varying from 4 to 32. The majority (92%) of the NWT strains carried one or two mutations in gyrA. Less than a quarter (22%) manifested amino acid substitutions in the topoisomerase IV parC subunit. Only three of the NWT strains carried a mutation in parE. Plasmid mediated quinolone resistance (PMQR) associated genes were detected in 18% of the NWT strains. In contrast to the relatively large number of NWT strains, only a small percentage of APEC isolates was considered clinically resistant. The most common MPC value for sensitive strains was 0.125 µg/mL. Some isolates showed higher values, producing wide mutant selection windows (MSW). Chromosomal mutations in DNA gyrase and topoisomerase IV were confirmed as the main source of decreased antimicrobial fluoroquinolone susceptibility, de-emphasizing the role of PMQR mechanisms.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85-95% and 85-100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940-0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from -0.979 to -0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

The impact of therapeutic-dose induced intestinal enrofloxacin concentrations in healthy pigs on fecal Escherichia coli populations.

BACKGROUND: Knowledge of therapy-induced intestinal tract concentrations of antimicrobials allows for interpretation and prediction of antimicrobial resistance selection within the intestinal microbiota. This study describes the impact of three different doses of enrofloxacin (ENR) and two different administration routes on the intestinal concentration of ENR and on the fecal Escherichia coli populations in pigs. Enrofloxacin was administered on three consecutive days to four different treatment groups. The groups either received an oral bolus administration of ENR (conventional or half dose) or an intramuscular administration (conventional or double dose). RESULTS: Quantitative analysis of fecal samples showed high ENR concentrations in all groups, ranging from 5.114 ± 1.272 µg/g up to 39.54 ± 10.43 µg/g at the end of the treatment period. In addition, analysis of the luminal intestinal content revealed an increase of ENR concentration from the proximal to the distal intestinal tract segments, with no significant effect of administration route. Fecal samples were also screened for resistance in E. coli isolates against ENR. Wild-type (MIC≤0.125 µg/mL) and non-wild-type (0.125 < MIC≤2 µg/mL) E. coli isolates were found at time 0 h. At the end of treatment (3 days) only non-wild-type isolates (MIC≥32 µg/mL) were found. CONCLUSIONS: In conclusion, the observed intestinal ENR concentrations in all groups showed to be both theoretically (based on pharmacokinetic and pharmacodynamic principles) and effectively (in vivo measurement) capable of significantly reducing the intestinal E. coli wild-type population.

Development and use of microbial-based cleaning products (MBCPs): Current issues and knowledge gaps.

Cleaning products containing microbes as active ingredients are becoming increasingly prevalent as an alternative to chemical-based cleaning products. These microbial-based cleaning products (MBCPs) are being used in domestic and commercial settings (i.e., households and businesses) and institutional settings (e.g., hospitals, schools, etc.), in a variety of cleaning activities (hard surface cleaning, odour control, degreasing, septic tank treatments, etc.). They are typically described as "environmentally friendly" and "non-toxic". Publicly available information sources (scientific literature, patent databases, commercial websites) were searched for information on microbial species contained in MBCPs, their mode of action, cleaning applications in which they are used, and their potential impacts on human health and the environment. Although information was found providing a broad indication of microbial genera/species used, information on specific species/strains and quantities produced and sold is generally lacking. This makes it difficult to conduct a meaningful examination of any risks to human health and the environment from the production and use of MBCPs and to determine how effective current policies and regulatory frameworks are in addressing these issues. These and other challenges were addressed at an international workshop in Ottawa, Canada in June 2013 by a number of stakeholders, including industry, government, academic and non-governmental organizations.

Hard surface biocontrol in hospitals using microbial-based cleaning products.

BACKGROUND: Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcare-associated pathogens, thus indicating the need for new and sustainable strategies. AIM: This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting. METHODS: The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected. RESULTS: Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3-4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities. CONCLUSIONS: This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAI-related microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment.

An environmentally representative stagnant-water model was developed to monitor the growth dynamics of Legionella pneumophila. This model was evaluated for three distinct water treatments: untreated tap water, heat-treated tap water, and heat-treated tap water supplemented with Pseudomonas putida, a known biofilm-forming bacterium. Bringing heat-treated tap water after subsequent cooling into contact with a densely formed untreated biofilm was found to promote the number of L. pneumophila by 4 log units within the biofilm, while the use of untreated water only sustained the L. pneumophila levels. Subsequent colonization of the water phase by L. pneumophila was noticed in the heat-treated stagnant-water models, with concentrations as high as 1 x 10(10) mip gene copies L(-1) stagnant water. Denaturing gradient gel electrophoresis in combination with clustering analysis of the prokaryotic community in the water phase and in the biofilm phase suggests that the different water treatments induced different communities. Moreover, boosts of L. pneumophila arising from heat treatment of water were accompanied by shifts to a more diverse eukaryotic community. Stimulated growth of L. pneumophila after heating of the water may explain the rapid recolonization of L. pneumophila in water systems. These results highlight the need for additional or alternative measures to heat treatment of water in order to prevent or abate potential outbreaks of L. pneumophila.