The use of aminopenicillins in animals within the EU, emergence of resistance in bacteria of animal and human origin and its possible impact on animal and human health.

Aminopenicillins have been widely used for decades for the treatment of various infections in animals and humans in European countries. Following this extensive use, acquired resistance has emerged among human and animal pathogens and commensal bacteria. Aminopenicillins are important first-line treatment options in both humans and animals, but are also among limited therapies for infections with enterococci and Listeria spp. in humans in some settings. Therefore, there is a need to assess the impact of the use of these antimicrobials in animals on public and animal health. The most important mechanisms of resistance to aminopenicillins are the ß-lactamase enzymes. Similar resistance genes have been detected in bacteria of human and animal origin, and molecular studies suggest that transmission of resistant bacteria or resistance genes occurs between animals and humans. Due to the complexity of epidemiology and the near ubiquity of many aminopenicillin resistance determinants, the direction of transfer is difficult to ascertain, except for major zoonotic pathogens. It is therefore challenging to estimate to what extent the use of aminopenicillins in animals could create negative health consequences to humans at the population level. Based on the extent of use of aminopenicillins in humans, it seems probable that the major resistance selection pressure in human pathogens in European countries is due to human consumption. It is evident that veterinary use of these antimicrobials increases the selection pressure towards resistance in animals and loss of efficacy will at minimum jeopardize animal health and welfare.

Synergic toxic effects of food contaminant mixtures in human cells.

Humans are exposed to multiple exogenous substances, notably through food consumption. Many of these compounds are suspected to impact human health, and their combination could exacerbate their harmful effects. We previously observed in human cells that, among the six most prevalent food contaminant complex mixtures identified in the French diet, synergistic interactions between component appeared in two mixtures compared with the response with the chemicals alone. In the present study, we demonstrated in human cells that these properties are driven only by two heavy metals in each mixture: tellurium (Te) with cadmium (Cd) and Cd with inorganic arsenic (As), respectively. It appeared that the predicted effects for these binary mixtures using the mathematical model of Chou and Talalay confirmed synergism between these heavy metals. Based on different cell biology experiments (cytotoxicity, genotoxicity, mutagenesis and DNA repair inhibition experiments), a detailed mechanistic analysis of these two mixtures suggests that concomitant induction of oxidative DNA damage and decrease of their repair capacity contribute to the synergistic toxic effect of these chemical mixtures. Overall, these results may have broad implications for the fields of environmental toxicology and chemical mixture risk assessment.

Assessment of veterinary drug residues in food: Considerations when dealing with sub-optimal data.

The use of veterinary drugs in food-producing animals may lead to residues in animal-derived foodstuffs, potentially posing a risk to human safety. While the process of veterinary drug residue risk assessment continues to evolve as new data emerges, a recurring challenge is when sub-optimal or incomplete data are provided with the expectation of supporting a robust risk assessment. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) is comprised of international experts who routinely deal with such data challenges when performing veterinary drug residue evaluations. Recent developments in veterinary drug residue risk assessment are described, including specific consequences of sub-optimal data during the risk assessment process. When feasible, practical solutions to such challenges are also highlighted. Case examples from recent JECFA veterinary drug evaluations are provided to clearly quantify and illustrate the concepts described. The information provided is intended to facilitate the generation of improved quality data, enabling more timely and robust veterinary drug residue risk assessments.

The use of aminoglycosides in animals within the EU: development of resistance in animals and possible impact on human and animal health: a review.

Aminoglycosides (AGs) are important antibacterial agents for the treatment of various infections in humans and animals. Following extensive use of AGs in humans, food-producing animals and companion animals, acquired resistance among human and animal pathogens and commensal bacteria has emerged. Acquired resistance occurs through several mechanisms, but enzymatic inactivation of AGs is the most common one. Resistance genes are often located on mobile genetic elements, facilitating their spread between different bacterial species and between animals and humans. AG resistance has been found in many different bacterial species, including those with zoonotic potential such as Salmonella spp., Campylobacter spp. and livestock-associated MRSA. The highest risk is anticipated from transfer of resistant enterococci or coliforms (Escherichia coli) since infections with these pathogens in humans would potentially be treated with AGs. There is evidence that the use of AGs in human and veterinary medicine is associated with the increased prevalence of resistance. The same resistance genes have been found in isolates from humans and animals. Evaluation of risk factors indicates that the probability of transmission of AG resistance from animals to humans through transfer of zoonotic or commensal foodborne bacteria and/or their mobile genetic elements can be regarded as high, although there are no quantitative data on the actual contribution of animals to AG resistance in human pathogens. Responsible use of AGs is of great importance in order to safeguard their clinical efficacy for human and veterinary medicine.

Public health risk of antimicrobial resistance transfer from companion animals.

Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.

Prevalence of mcr-1 in commensal Escherichia coli from French livestock, 2007 to 2014.

Colistin resistance was investigated in 1,696 isolates collected from 2007 to 2014 within the frame of the French livestock antimicrobial resistance surveillance programme. The mcr-1 gene was detected in all commensal Escherichia coli isolates with a minimum inhibitory concentration to colistin above the 2 mg/L cut-off value (n=23). In poultry, mcr-1 prevalence was 5.9% in turkeys and 1.8% in broilers in 2014. In pigs, investigated in 2013, this prevalence did not exceed 0.5%. These findings support that mcr-1 has spread in French livestock.

The Monitoring of Triphenylmethane Dyes in Aquaculture Products Through the European Union Network of Official Control Laboratories.

Aquaculture has been the fastest growing animal production industry for the past four decades, and almost half of the fish eaten in the world are now farmed fish. To prevent diseases in this more intensive aquaculture farming, use of therapeutic chemicals has become a basic choice. The monitoring of malachite green, a triphenylmethane dye and one of the oldest and widely used chemicals in fish production, has gained more interest since the mid 1990s when this substance was finally proven to be toxic enough to be prohibited in seafood products destined for human consumption. The enforcement of the European Union (EU) regulation of this banned substance along with some other triphenylmethane dye congeners and their metabolites in its domestic production and in seafood imports was undertaken through the National Residue Monitoring Plans implemented in nearly all of the 28 EU member states. The reliability of the overall European monitoring of this dye contamination in aquaculture products was assessed by using the results of proficiency testing (PT) studies provided by the EU Reference Laboratory (EU-RL) in charge of the network of the EU National Reference Laboratories (NRLs). The proficiency of each NRL providing analytical support services for regulating dye residues was carefully checked during three PT rounds. In the process, the analytical methods developed and validated for this purpose have gradually been improved and extended over the last two decades.

Pleuromutilins: use in food-producing animals in the European Union, development of resistance and impact on human and animal health.

Pleuromutilins (tiamulin and valnemulin) are antimicrobial agents that are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. In pigs, tiamulin and valnemulin are used to treat swine dysentery, spirochaete-associated diarrhoea, porcine proliferative enteropathy, enzootic pneumonia and other infections where Mycoplasma is involved. There are concerns about the reported increases in the MICs of tiamulin and valnemulin for porcine Brachyspira hyodysenteriae isolates from different European countries, as only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to these antimicrobials is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions would present a considerable threat to pig health, welfare and productivity. In humans, only one product containing pleuromutilins (retapamulin) is authorized currently for topical use; however, products for oral and intravenous administration to humans with serious multidrug-resistant skin infections and respiratory infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA), are being developed. The objective of this review is to summarize the current knowledge on the usage of pleuromutilins, resistance development and the potential impact of this resistance on animal and human health.

PBPK Model To Predict Marbofloxacin Distribution in Edible Tissues and Intestinal Exposure in Pigs.

Marbofloxacin (MAR) is a fluoroquinolone antibiotic used in food-producing animals in European Union, especially in pigs. In this study, MAR concentrations in plasma, comestible tissues, and intestinal segments were determined in pigs injected with MAR. Based on these data and the literature, a flow-limited PBPK model was developed to predict the tissue distribution of MAR and estimate the withdrawal period after label-use in Europe. A submodel describing the different segments of the intestinal lumen was also developed to assess the intestinal exposure of MAR for the commensal bacteria. During model calibration, only four parameters were estimated. Then, Monte Carlo simulations were performed to generate a virtual population of pigs. The simulation results were compared with the observations from an independent data set during the validation step. A global sensitivity analysis was also carried out to identify the most influential parameters. Overall, the PBPK model was able to adequately predict the MAR kinetics in plasma and edible tissues, as well as in small intestines. However, the simulated concentrations in the large intestine were mostly underestimated, highlighting the need for improvements in the field of PBPK modeling to assess the intestinal exposure of antimicrobials in food animals.

Selection of antibiotic resistance in pigs after exposure to feed cross-contaminated with oxytetracycline.

Due to possible cross-contamination of animal feedstuff with antibiotics, food-producing animals may be exposed to undesirable low concentrations of antimicrobials. These sub-therapeutic levels of antibiotics can lead to the selection of resistant bacteria in the animal gut. The goal of this study was to assess, through analysis of the faeces of treated and control pigs, the risk of resistant E. coli being selected after daily exposure for three weeks to feed contaminated with oxytetracycline at 1% of the therapeutic dose. Liquid Chromatography coupled to tandem Mass Spectrometry was used to determine the oxytetracycline concentrations in faecal samples. In the treated group, concentrations were in the range of 4481.9 - 8671.2 µg/kg. In the control group, these concentrations were either below the method's limit of quantification or up to 60.5 µg/kg. After a transient increase in resistance in both groups, microbiological analysis showed that the treated group had a significantly higher oxytetracycline resistance rate by the end of the study than the control group (p < 0.001). Furthermore, the treated animals were found to select co-resistances to nalidixic acid and ampicillin. Finally, at tolerated antibiotic contamination levels of feed, the treated group had a higher proportion of multidrug-resistant isolates at the end of the study than the control one (p < 0.05). The present study demonstrates that, at the tolerated contamination rates, both antimicrobial resistance and multidrug-resistant bacteria can be selected and evidenced in the gut microbiota.

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA).

A pilot study for the intrinsic labeling of egg proteins with 15N and 13C.

The aim of this study was to produce intrinsically and uniformly doubly (15)N-(13)C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly (15)N-(13)C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The (15)N and (13)C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the (15)N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the (13)C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The (15)N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ(13)C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the (13)C labeling, adequate (13)C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway.

Validation of a LC-MS/MS method for the quantitative analysis of four antibiotics in pig tissues and plasma to assess the risk of transfer of residues to edible matrices after exposure to cross-contaminated feed.

Cross-contamination between medicated and non-medicated feed can occur during production, processing, transport or storage of animal feed. This may lead to the presence of low concentrations of antibiotics in supposedly drug-free feed for food production animals, which potentially could also harm consumers due to residues. In addition, consumption of sub-therapeutic concentrations of antibiotics may increase the risk of emergence of resistant bacteria. In this study, LC-MS/MS methods were developed to quantify four antibiotics (sulfadimethoxine, oxytetracycline, trimethoprim and amoxicillin) in several pig matrices, i.e. plasma, muscle, liver, kidneys and faeces. All methods were validated using the accuracy profile, except for amoxicillin in faeces, for which extraction could not be optimised for low concentrations. These methods were then applied as part of an animal study during which several pigs received contaminated feed at a concentration corresponding to 2% of therapeutic dose, in order to evaluate the risk of the presence of residues in animal faeces and tissues. The results showed that sulfadimethoxine is well absorbed and accumulates in the muscle, kidneys and liver, where concentrations were higher than the maximum residue limits (MRLs) authorised in EU legislation. Conversely, oxytetracycline was mostly found in faeces as its oral absorption is very low. Trimethoprim concentrations were slightly higher than the tolerated MRL in the kidneys, but they were below this level in the other tissues. Finally, amoxicillin concentrations remained below the lower limit of quantification of the methods in all matrices.

Review on methicillin-resistant Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an important opportunistic pathogen of companion animals, especially dogs. Since 2006 there has been a significant emergence of methicillin-resistant S. pseudintermedius (MRSP) mainly due to clonal spread. This article reviews research on MRSP with a focus on occurrence, methods used for identification, risk factors for colonization and infection, zoonotic potential and control options. Potential areas for future research are also discussed.

Estimation of transmission parameters of a fluoroquinolone-resistant Escherichia coli strain between pigs in experimental conditions.

Antimicrobial resistance is of primary importance regarding public and animal health issues. Persistence and spread of resistant strains within a population contribute to the maintenance of a reservoir and lead to treatment failure. An experimental trial was carried out to study the horizontal transmission of a fluoroquinolone-resistant Escherichia coli strain from inoculated to naïve pigs. All naïve contact pigs had positive counts of fluoroquinolone-resistant E. coli after only two days of contact. Moreover, re-infections of inoculated pigs caused by newly contaminated animals were suspected. A maximum likelihood method, based on a susceptible-infectious-susceptible (SIS) model, was used to determine the transmission parameters. Two transmission levels were identified depending on the quantity of bacteria shed by infected individuals: (i) low-shedders with bacterial counts of resistant E. coli in the faeces between 5*10(3) and 10(6) CFU/g (ßL = 0.41 [0.27; 0.62]), (ii) high shedders with bacterial counts above 10(6) CFU/g (ßH = 0.98 [0.59; 1.62]). Hence, transmission between animals could be pivotal in explaining the persistence of resistant bacteria within pig herds.

Should the Increased Awareness of the One Health Approach Brought by the COVID-19 Pandemic Be Used to Further Tackle the Challenge of Antimicrobial Resistance?

Several experts have expressed their concerns regarding the potential increase in antimicrobial resistance (AMR) during the COVID-19 pandemic as a consequence of the increase in antimicrobial and biocide use in humans globally. However, the impact of the pandemic on antimicrobial use (AMU) and AMR in animals has yet to be discussed and evaluated. Indeed, veterinary practices have been hugely impacted by the pandemic and its restrictive measures around the world. In this perspective, we call for more research to estimate the impact of COVID-19 on AMU and AMR in both humans and animals, as well as on the environment, in coherence with the One Health approach. In addition, we argue that the current pandemic is an opportunity to accelerate the implementation of a One Health approach to tackle the AMR crisis at the global scale. Indeed, the momentum created by the increased general awareness of both the public and decision-makers for the development and maintenance of effective drugs to treat human infections, as well as for the importance of a One Health approach to prevent the emergence of infectious diseases, should be used as a lever to implement global collaborative and sustainable solutions to the complex challenges of AMR.

Tissue distribution, metabolism, and elimination of Victoria Pure Blue BO in rainbow trout: Main metabolite as an appropriate residue marker.

Victoria Pure Blue BO is a dye that bears some therapeutic activity and that can be retrieved in effluent or may be used in aquaculture as a prohibited drug. In this study, the metabolism and tissue distribution during uptake and depuration of VPBO were investigated in order to propose a residue marker of illegal treatment in fish. The dye was administered to rainbow trout (oncorhynchus mykiss) for one day by water bath at a dose of 0.1 mg.L-1. The concentrations of VPBO in all tissues increased rapidly during the treatment period, reaching a Cmax of 567 ± 301 µg.L-1 in plasma and 1846 µg kg-1 ±517 for liver after 2 h. After placing the rainbow trout in a clean water bath for a 64 day-period of depuration, the concentrations in the tissues and plasma decreased to reach comparable levels for muscle and for skin after 33 days. The concentrations measured were still above the LOQ at 2.26 ± 0.48 µg kg-1 for muscle and 2.85 ± 1.99 µg kg-1 for skin at the end of the depuration period. The results indicated the existence of 14 phase I metabolites and one glucuronide conjugated metabolite. Non-compartmental analysis was applied to assess the pharmacokinetic parameters. The half-life in edible muscle of the main metabolite detected, deethyl-leuco-VPBO, was found to be 22.5 days compared to a half-life of 19.7 days for the parent VPBO. This study provides new information to predict a VPBO drug treatment of aquacultured species via a proposed new residue marker.

Genomic evolution of antimicrobial resistance in Escherichia coli.

The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

Risk Factors for Antimicrobial Resistance in Turkey Farms: A Cross-Sectional Study in Three European Countries.

Food-producing animals are an important reservoir and potential source of transmission of antimicrobial resistance (AMR) to humans. However, research on AMR in turkey farms is limited. This study aimed to identify risk factors for AMR in turkey farms in three European countries (Germany, France, and Spain). Between 2014 and 2016, faecal samples, antimicrobial usage (AMU), and biosecurity information were collected from 60 farms. The level of AMR in faecal samples was quantified in three ways: By measuring the abundance of AMR genes through (i) shotgun metagenomics sequencing (n = 60), (ii) quantitative real-time polymerase chain reaction (qPCR) targeting ermB, tetW, sul2, and aph3'-III; (n = 304), and (iii) by identifying the phenotypic prevalence of AMR in Escherichia coli isolates by minimum inhibitory concentrations (MIC) (n = 600). The association between AMU or biosecurity and AMR was explored. Significant positive associations were detected between AMU and both genotypic and phenotypic AMR for specific antimicrobial classes. Beta-lactam and colistin resistance (metagenomics sequencing); ampicillin and ciprofloxacin resistance (MIC) were associated with AMU. However, no robust AMU-AMR association was detected by analyzing qPCR targets. In addition, no evidence was found that lower biosecurity increases AMR abundance. Using multiple complementary AMR detection methods added insights into AMU-AMR associations at turkey farms.

Emergence of resistant Klebsiella pneumoniae in the intestinal tract during successful treatment of Klebsiella pneumoniae lung infection in rats.

Antibiotic treatment of lung infections may lead to the emergence of resistance in the gut flora. Appropriate dosing regimens could mitigate this adverse effect. In gnotobiotic rats harboring intestinal Escherichia coli and Enterococcus faecium populations, a lung infection by Klebsiella pneumoniae was instigated with two different sizes of inoculum to represent an early or a late initiation of antibiotic treatment. The rats were treated with marbofloxacin, an expanded-spectrum fluoroquinolone, by a single-shot administration or a fractionated regimen over 4 days. Intestinal bacterial populations were monitored during and after treatment. At the infection site, bacterial cure without any selection of resistance was observed. Whatever the dosage regimen, fluoroquinolone treatment had a transient negative impact on the E. coli gut population but not on that of E. faecium. The intestinal flora was colonized by the pathogenic lung bacteria, and there was the emergence of intestine-resistant K. pneumoniae, occurring more often in animals treated with a single marbofloxacin dose than with the fractionated dose. Bacterial cure without resistance selection at the infection site with fluoroquinolone treatment can be linked to colonization of the digestive tract by targeted pulmonary bacteria, followed by the emergence of resistance.