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.

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.

Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei.

Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei. It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei, whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen.

Genome sequence of Staphylococcus aureus Newbould 305, a strain associated with mild bovine mastitis.

Staphylococcus aureus is a major etiological agent of mastitis in ruminants. We report here the genome sequence of bovine strain Newbould 305, isolated in the 1950s in a case of bovine mastitis and now used as a model strain able to reproducibly induce chronic mastitis in cows.

Environmental contamination in a high-income country (France) by antibiotics, antibiotic-resistant bacteria, and antibiotic resistance genes: Status and possible causes.

Antimicrobial resistance (AMR) is a major global public health concern, shared by a large number of human and animal health actors. Within the framework of a One Health approach, actions should be implemented in the environmental realm, as well as the human and animal realms. The Government of France commissioned a report to provide policy and decision makers with an evidential basis for recommending or taking future actions to mitigate AMR in the environment. We first examined the mechanisms that underlie the emergence and persistence of antimicrobial resistance in the environment. This report drew up an inventory of the contamination of aquatic and terrestrial environments by AMR and antibiotics, anticipating that the findings will be representative of some other high-income countries. Effluents of wastewater treatment plants were identified as the major source of contamination on French territory, with spreading of organic waste products as a more diffuse and incidental contamination of aquatic environments. A limitation of this review is the heterogeneity of available data in space and time, as well as the lack of data for certain sources. Comparing the French Measured Environmental Concentrations (MECs) with predicted no effect concentrations (PNECs), fluoroquinolones and trimethoprim were identified as representing high and medium risk of favoring the selection of resistant bacteria in treated wastewater and in the most contaminated rivers. All other antibiotic molecules analyzed (erythromycin, clarithromycin, azithromycin, tetracycline) were at low risk of resistance selection in those environments. However, the heterogeneity of the data available impairs their full exploitation. Consequently, we listed indicators to survey AMR and antibiotics in the environment and recommended the harmonization of sampling strategies and endpoints for analyses. Finally, the objectives and methods used for the present work could comprise a useful example for how national authorities of countries sharing common socio-geographic characteristics with France could seek to better understand and define the environmental dimension of AMR in their particular settings.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 1: Methodology, general data gaps and uncertainties.

The European Commission requested EFSA to assess, in collaboration with EMA, the specific concentrations of antimicrobials resulting from cross-contamination in non-target feed for food-producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health, as well as the levels of the antimicrobials which could have a growth promotion/increase yield effect. The assessment was performed for 24 antimicrobial active substances, as specified in the mandate. This scientific opinion describes the methodology used, and the main associated data gaps and uncertainties. To estimate the antimicrobial levels in the non-target feed that would not result in emergence of, and/or selection for, resistance, a model was developed. This 'Feed Antimicrobial Resistance Selection Concentration' (FARSC) model is based on the minimal selective concentration (MSC), or the predicted MSC (PMSC) if MSC for the most susceptible bacterial species is unavailable, the fraction of antimicrobial dose available for exposure to microorganisms in the large intestine or rumen (considering pharmacokinetic parameters), the daily faecal output or rumen volume and the daily feed intake. Currently, lack of data prevents the establishment of PMSC and/or FARSC for several antimicrobials and animal species. To address growth promotion, data from an extensive literature search were used. Specific assessments of the different substances grouped by antimicrobial classes are addressed in separate scientific opinions. General conclusions and recommendations were made.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 2: Aminoglycosides/aminocyclitols: apramycin, paromomycin, neomycin and spectinomycin.

The specific concentrations of apramycin, paromomycin, neomycin and spectinomycin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for these antimicrobials, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for apramycin and neomycin, whilst for paromomycin and spectinomycin, no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these four antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 3: Amprolium.

The specific concentrations of amprolium in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for amprolium, it was not possible to conclude the assessment. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of amprolium in feed that showed to have an effect on growth promotion/increased yield were reported. The lack of antibacterial activity at clinically relevant concentrations for amprolium suggests that further studies relating to bacterial resistance are not a priority.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 4: ß-Lactams: amoxicillin and penicillin V.

The specific concentrations of amoxicillin and penicillin V in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for amoxicillin, whilst for penicillin V no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 5: Lincosamides: lincomycin.

The specific concentrations of lincomycin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of lincomycin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for lincomycin.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 7: Amphenicols: florfenicol and thiamphenicol.

The specific concentrations of florfenicol and thiamphenicol in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for florfenicol was estimated. However, due to the lack of data, the calculation of the FARSC for thiamphenicol was not possible until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for florfenicol, whilst for thiamphenicol no suitable data for the assessment were available. Uncertainties and data gaps associated to the levels reported were addressed. For florfenicol, it was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC, whereas for thiamphenicol, the recommendation was to generate the data required to fill the gaps which prevented the FARSC calculation.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 6: Macrolides: tilmicosin, tylosin and tylvalosin.

The specific concentrations of tilmicosin, tylosin and tylvalosin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tilmicosin and tylosin, whilst for tylvalosin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these three antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 8: Pleuromutilins: tiamulin and valnemulin.

The specific concentrations of tiamulin and valnemulin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tiamulin, while for valnemulin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 9: Polymyxins: colistin.

The specific concentrations of colistin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of colistin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed.Part 10: Quinolones: flumequine and oxolinic acid.

The specific concentrations of flumequine and oxolinic acid in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 11: Sulfonamides.

The specific concentrations of sulfonamides in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were identified for three sulfonamides: sulfamethazine, sulfathiazole and sulfamerazine. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 13: Diaminopyrimidines: trimethoprim.

The specific concentrations of trimethoprim in non-target feed for food-producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for trimethoprim was estimated. Uncertainties and data gaps associated to the levels reported were addressed. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for trimethoprim.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 12: Tetracyclines: tetracycline, chlortetracycline, oxytetracycline, and doxycycline.

The specific concentrations of tetracycline, chlortetracycline, oxytetracycline and doxycycline in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for these four tetracyclines was estimated. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tetracycline, chlortetracycline, oxytetracycline, whilst for doxycycline no suitable data for the assessment were available. Uncertainties and data gaps associated with the levels reported were addressed. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for these antimicrobials.

Contribution of sortase SrtA2 to Lactobacillus casei BL23 inhibition of Staphylococcus aureus internalization into bovine mammary epithelial cells.

Probiotics have been considered as a promising strategy to prevent various diseases in both humans and animals. This approach has gained interest in recent years as a potential means to control bovine mastitis. In a previous study, we found that several L. casei strains, including BL23, were able to inhibit the internalization of S. aureus, a major etiologic agent of mastitis, into bovine mammary epithelial cells (bMEC). This antagonism required a direct contact between L. casei and bMEC or S. aureus, suggesting the inhibition relied on interactions between L. casei cell surface components and bMEC. In this study, we have investigated the impact of some candidates which likely influence bacteria host cell interactions. We have shown that L. casei BL23 fbpA retained its inhibitory potential, indicating that L. casei BL23 antagonism did not rely (solely) on competition between S. aureus and L. casei fibronectin-binding proteins for adhesion to bMEC. We have then investigated the impact of four sortase mutants, srtA1, srtA2, srtC1 and srtC2, and a double mutant (srtA1-srtA2) on L. casei BL23 inhibitory potential. Sortases are responsible for the anchoring on the bacterial cell wall of LPXTG-proteins, which reportedly play an important role in bacteria-host cell interaction. All the srt mutants tested presented a reduced inhibition capacity, the most pronounced effect being observed with the srtA2 mutant. A lower internalization capacity of L. casei srtA2 into bMEC was also observed. This was associated with several changes at the surface of L. casei BL23 srtA2 compared to the wild type (wt) strain, including altered abundance of some LPXTG- and moonlighting proteins, and modifications of cell wall structure. These results strongly support the role of sortase A2 in L. casei BL23 inhibition against S. aureus internalization. Deciphering the contribution of the cell surface components altered in srtA2 strain in the inhibition will require further investigation.