Maximum levels of cross‐contamination for 24 antimicrobial active substances in non‐target feed. Part 7: Amphenicols: florfenicol and thiamphenicol
2021; Wiley; Volume: 19; Issue: 10 Linguagem: Inglês
10.2903/j.efsa.2021.6859
ISSN1831-4732
AutoresKonstantinos Koutsoumanis, Ana Allende, Avelino Álvarez‐Ordóñez, Declan Bolton, Sara Bover‐Cid, Marianne Chemaly, Robert Davies, Alessandra De Cesare, Lieve Herman, Friederike Hilbert, Roland Lindqvist, Maarten Nauta, Giuseppe Ru, Marion Simmons, Panagiotis Skandamis, Elisabetta Suffredini, Dan I. Andersson, Vasileios Bampidis, Johan Bengtsson‐Palme, Damien Bouchard, Aude Ferran, Maryline Kouba, Secundino López Puente, Marta López‐Alonso, Søren Saxmose Nielsen, Alena Pechová, Mariana Petkova, Sebastien Girault, Alessandro Broglia, Beatriz Guerra, Matteo Lorenzo Innocenti, E. Liébana, Gloria López‐Gálvez, Paola Manini, Pietro Stella, Luísa Peixe,
Tópico(s)Pesticide Residue Analysis and Safety
ResumoEFSA JournalVolume 19, Issue 10 e06859 Scientific OpinionOpen Access Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 7: Amphenicols: florfenicol and thiamphenicol EFSA Panel on Biological Hazards (BIOHAZ), Corresponding Author EFSA Panel on Biological Hazards (BIOHAZ) biohaz@efsa.europa.eu Correspondence:biohaz@efsa.europa.euSearch for more papers by this authorKonstantinos Koutsoumanis, Konstantinos KoutsoumanisSearch for more papers by this authorAna Allende, Ana AllendeSearch for more papers by this authorAvelino Alvarez-Ordóñez, Avelino Alvarez-OrdóñezSearch for more papers by this authorDeclan Bolton, Declan BoltonSearch for more papers by this authorSara Bover-Cid, Sara Bover-CidSearch for more papers by this authorMarianne Chemaly, Marianne ChemalySearch for more papers by this authorRobert Davies, Robert DaviesSearch for more papers by this authorAlessandra De Cesare, Alessandra De CesareSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorFriederike Hilbert, Friederike HilbertSearch for more papers by this authorRoland Lindqvist, Roland LindqvistSearch for more papers by this authorMaarten Nauta, Maarten NautaSearch for more papers by this authorGiuseppe Ru, Giuseppe RuSearch for more papers by this authorMarion Simmons, Marion SimmonsSearch for more papers by this authorPanagiotis Skandamis, Panagiotis SkandamisSearch for more papers by this authorElisabetta Suffredini, Elisabetta SuffrediniSearch for more papers by this authorDan I Andersson, Dan I AnderssonSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorJohan Bengtsson-Palme, Johan Bengtsson-PalmeSearch for more papers by this authorDamien Bouchard, Damien BouchardSearch for more papers by this authorAude Ferran, Aude FerranSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorSøren Saxmose Nielsen, Søren Saxmose NielsenSearch for more papers by this authorAlena Pechová, Alena PechováSearch for more papers by this authorMariana Petkova, Mariana PetkovaSearch for more papers by this authorSebastien Girault, Sebastien GiraultSearch for more papers by this authorAlessandro Broglia, Alessandro BrogliaSearch for more papers by this authorBeatriz Guerra, Beatriz GuerraSearch for more papers by this authorMatteo Lorenzo Innocenti, Matteo Lorenzo InnocentiSearch for more papers by this authorErnesto Liébana, Ernesto LiébanaSearch for more papers by this authorGloria López-Gálvez, Gloria López-GálvezSearch for more papers by this authorPaola Manini, Paola ManiniSearch for more papers by this authorPietro Stella, Pietro StellaSearch for more papers by this authorLuisa Peixe, Luisa PeixeSearch for more papers by this author EFSA Panel on Biological Hazards (BIOHAZ), Corresponding Author EFSA Panel on Biological Hazards (BIOHAZ) biohaz@efsa.europa.eu Correspondence:biohaz@efsa.europa.euSearch for more papers by this authorKonstantinos Koutsoumanis, Konstantinos KoutsoumanisSearch for more papers by this authorAna Allende, Ana AllendeSearch for more papers by this authorAvelino Alvarez-Ordóñez, Avelino Alvarez-OrdóñezSearch for more papers by this authorDeclan Bolton, Declan BoltonSearch for more papers by this authorSara Bover-Cid, Sara Bover-CidSearch for more papers by this authorMarianne Chemaly, Marianne ChemalySearch for more papers by this authorRobert Davies, Robert DaviesSearch for more papers by this authorAlessandra De Cesare, Alessandra De CesareSearch for more papers by this authorLieve Herman, Lieve HermanSearch for more papers by this authorFriederike Hilbert, Friederike HilbertSearch for more papers by this authorRoland Lindqvist, Roland LindqvistSearch for more papers by this authorMaarten Nauta, Maarten NautaSearch for more papers by this authorGiuseppe Ru, Giuseppe RuSearch for more papers by this authorMarion Simmons, Marion SimmonsSearch for more papers by this authorPanagiotis Skandamis, Panagiotis SkandamisSearch for more papers by this authorElisabetta Suffredini, Elisabetta SuffrediniSearch for more papers by this authorDan I Andersson, Dan I AnderssonSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorJohan Bengtsson-Palme, Johan Bengtsson-PalmeSearch for more papers by this authorDamien Bouchard, Damien BouchardSearch for more papers by this authorAude Ferran, Aude FerranSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorSøren Saxmose Nielsen, Søren Saxmose NielsenSearch for more papers by this authorAlena Pechová, Alena PechováSearch for more papers by this authorMariana Petkova, Mariana PetkovaSearch for more papers by this authorSebastien Girault, Sebastien GiraultSearch for more papers by this authorAlessandro Broglia, Alessandro BrogliaSearch for more papers by this authorBeatriz Guerra, Beatriz GuerraSearch for more papers by this authorMatteo Lorenzo Innocenti, Matteo Lorenzo InnocentiSearch for more papers by this authorErnesto Liébana, Ernesto LiébanaSearch for more papers by this authorGloria López-Gálvez, Gloria López-GálvezSearch for more papers by this authorPaola Manini, Paola ManiniSearch for more papers by this authorPietro Stella, Pietro StellaSearch for more papers by this authorLuisa Peixe, Luisa PeixeSearch for more papers by this author First published: 26 October 2021 https://doi.org/10.2903/j.efsa.2021.6859 Requestor: European Commission Question number: EFSA-Q-2021-00507 Panel members: Ana Allende, Avelino Alvarez-Ordóñez, Declan Bolton, Sara Bover-Cid, Marianne Chemaly, Robert Davies, Alessandra De Cesare, Lieve Herman, Friederike Hilbert, Konstantinos Koutsoumanis, Roland Lindqvist, Maarten Nauta, Luisa Peixe, Giuseppe Ru, Marion Simmons, Panagiotis Skandamis and Elisabetta Suffredini. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgements: The BIOHAZ Panel, leading Panel in charge of the adoption of the scientific opinion and assessment of Term of Reference 1 (ToR1, antimicrobial resistance) wishes to thank the following for the support provided to this scientific output: EFSA Panel on Animal Health and Welfare (AHAW Panel), who supported ToR1 assessments development and endorsement of those sections under their remit (animal production, main use of antimicrobials); EFSA Panel for Additives and Products or Substances used in Animal Feed (FEEDAP), in charge of the assessment and endorsement of ToR2, and providing advice and data needed for ToR1 assessments; European Medicines Agency (EMA), who was represented by an external expert and EMA secretariat as members of the Working Group (WG); Valeria Bortolaia, who was member of the WG until 17 April 2020; EFSA staff members: Angelica Amaduzzi, Gina Cioacata, Pilar García-Vello, Michaela Hempen, Rita Navarrete, Daniel Plaza and Anita Radovnikovic; EMA staff members: Barbara Freischem, Zoltan Kunsagi, Nicholas Jarrett, Jordi Torren, and Julia Fábrega (currently EFSA staff). The BIOHAZ Panel wishes also to acknowledge the EMA Committee for Medicinal Products for Veterinary Use (CVMP) and their experts. Adopted: 15 September 2021 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract 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. 1 Introduction The European Commission requested the European Food Safety Authority (EFSA) to assess, in collaboration with the European Medicines Agency (EMA), (i) 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 (term of reference 1, ToR1), and (ii) the levels of the antimicrobials which have a growth promotion/increase yield effect (ToR2). The assessment was requested to be conducted for 24 antimicrobial active substances specified in the mandate.11 Aminoglycosides: apramycin, paromomycin, neomycin, spectinomycin; Amprolium; Beta-lactams: amoxicillin, penicillin V; Amphenicols: florfenicol, thiamphenicol; Lincosamides: lincomycin; Macrolides: tilmicosin, tylosin, tylvalosin; Pleuromutilins: tiamulin, valnemulin; Sulfonamides; Polymyxins: colistin; Quinolones: flumequine, oxolinic acid; Tetracyclines: tetracycline, chlortetracycline, oxytetracycline, doxycycline; Diaminopyrimidines: trimethoprim. For the different substances (grouped by class if applicable)11 Aminoglycosides: apramycin, paromomycin, neomycin, spectinomycin; Amprolium; Beta-lactams: amoxicillin, penicillin V; Amphenicols: florfenicol, thiamphenicol; Lincosamides: lincomycin; Macrolides: tilmicosin, tylosin, tylvalosin; Pleuromutilins: tiamulin, valnemulin; Sulfonamides; Polymyxins: colistin; Quinolones: flumequine, oxolinic acid; Tetracyclines: tetracycline, chlortetracycline, oxytetracycline, doxycycline; Diaminopyrimidines: trimethoprim. , separate scientific opinions included within the 'Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed' series (Scientific Opinions Part 2 - Part 13, EFSA BIOHAZ Panel 2021b-l – see the Virtual Issue; for practical reasons, they will be referred as 'scientific opinion Part X' throughout the current document) were drafted. They present the results of the assessments performed to answer the following questions: Assessment Question 1 (AQ1), which are the specific antimicrobial concentrations in non-target feed below which there would not be emergence of, and/or selection for, resistance in the large intestines/rumen, and AQ2: which are the specific antimicrobial concentrations in feed of food-producing animals that have an effect in terms of growth promotion/increased yield. The assessments were performed following the methodology described in Section 2 of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (EFSA BIOHAZ Panel, 2021a, see also the Virtual Issue). The present document reports the results of the assessment for the amphenicols: florfenicol and thiamphenicol. 1.1 Background and Terms of Reference as provided by the requestor The background and ToRs provided by the European Commission for the present document are reported in Section 1.1 of the Scientific Opinion "Part 1: Methodology, general data gaps and uncertainties" (see also the Virtual Issue). 1.2 Interpretation of the Terms of Reference The interpretation of the ToRs, to be followed for the assessment is in section 1.2 of the Scientific Opinion "Part 1: Methodology, general data gaps and uncertainties" (see also the Virtual Issue). 1.3 Additional information 1.3.1 Short description of the class/substance Amphenicols are derivatives of dichloroacetic acid, with two other components: an aromatic nucleus with an alkyl group in the para position and an aminopropanediol chain, thereby possessing a 2,2-dichloro-N-[-1-hydroxy-1-(phenyl) propan-2-yl] acetamide structure. The chemical structure of thiamphenicol differs from chloramphenicol in having a sulfo-group instead of a nitro-group. Florfenicol has a fluorine atom instead of the hydroxyl group located at C-3 within the structure of chloramphenicol and thiamphenicol. This may allow florfenicol to be less susceptible to deactivation by bacteria with plasmid-borne resistance that involves acetylation of the C-3 hydroxyl group (USP, 2003). The amphenicols inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit and affecting the activity of the peptidyltransferase enzyme (Fisch and Bryskier, 2005). Amphenicols are broad spectrum bacteriostatic antimicrobials, active against both Gram-negative and Gram-positive bacteria, including some anaerobes (EMA/CVMP/CHMP, 2019). The spectrum of activity for both florfenicol and thiamphenicol is similar (although thiamphenicol has greater activity against some anaerobes) and includes most important enteric, respiratory and dermal/sepsis-related bacterial pathogens of food animals (Shin et al., 2005), including fish (Samuelsen et al., 1998). Florfenicol is more widely used in feed and water because of lower minimum inhibitory concentration (MIC) values for most important pathogens (other than anaerobes and mycoplasmas), superior pharmacokinetic (PK) characteristics (Ravizzola et al., 1984; Switała et al., 2007) and reduced susceptibility to inactivation by chloramphenicol transacetylase enzymes (Fukui et al., 1987; Michel et al., 2003). Each substance will therefore be assessed separately. 1.3.2 Main use22 Antimicrobials are currently used in food-producing animal production for treatment, prevention and/or metaphylaxis of a large number of infections, and also for growth promotion in non-EU countries. In the EU, in future, use of antimicrobials for prophylaxis or for metaphylaxis is to be restricted as addressed by Regulation (EU) 2019/6 and use in medicated feed for prophylaxis is to be prohibited under Regulation (EU) 2019/4. Florfenicol is widely used as a feed additive or water treatment for enteric and respiratory infections in pigs and can also be used in calves or poultry (EMEA/CVMP, 1999). Florfenicol feed premix is also indicated for the treatment of furunculosis caused by susceptible strains of Aeromonas salmonicida in salmon, and it is one of the most commonly used antimicrobials in aquaculture (USP, 2003; Romero et al., 2012; Kumar et al., 2018). Injectable formulations are also available for treating individual animals or serious cases in large animals (Papich, 2016; EMA/CVMP/CHMP, 2019). Thiamphenicol can also be used for the treatment and control of respiratory and intestinal infections in cattle, pigs and poultry by oral or intramuscular administration and is especially effective against anaerobes but is not commonly used for terrestrial food animals within the EU (EMA/ESVAC, 2020). It can also be used topically and by intra-mammary administration in both lactating and dry cows and for intrauterine administration in cows. Thiamphenicol is not permitted for use in laying hens (EMA/CVMP/CHMP, 2019). 1.3.3 Main pharmacokinetic data 1.3.3.1 Florfenicol The bioavailability of florfenicol administered by the oral route to calves, 2–5 weeks of age, is 89%, at a dose of either 11 or 22 mg/kg (USP, 2003); however, the absorption was widely variable and oral absorption may be reduced when florfenicol is administered mixed with milk replacers. One study reported bioavailability that ranged from 44% to 86% among calves when florfenicol was administered 5 minutes after feeding (Varma et al., 1986). In pigs, the bioavailability is around 100% in fasted animals (Liu et al., 2003) and no significant difference of the bioavailability between fasted and fed animals was reported (Jiang et al., 2006; De Smet et al., 2018). In broilers, the oral bioavailability appears to be lower in fed than in fasted animals. In one study, the reported bioavailability was 55% in non-fasted broilers (Afifi and Abo el-Sooud, 1997) whereas reported bioavailability ranged from 87% to 96% in fasted broilers (Shen et al., 2003; Anadón et al., 2008). For other species, the bioavailability of florfenicol after oral administration was 82% in fasted turkeys (Switała et al., 2007), 83% in fasted horses (McKellar and Varma, 1996), 76% in rabbits (no clear information on the fed or fasted status) (Park et al., 2007) and 96.5–99% in Atlantic salmon at a water temperature around 10°C (Horsberg et al., 1996; USP, 2003). The bioavailability of florfenicol administered by the oral route is high but the effect of the feed on the bioavailability is uncertain for most species because of the lack of data in fed animals. Only one study suggests that feed could decrease the bioavailability in broilers. Data on elimination for calves of less than 8 weeks of age suggests that approximately 50% of a 22 mg/kg intravenous dose is excreted unchanged in the urine within 30 h. For adult cattle, approximately 64% of a 20 mg/kg intramuscular dose administered twice, 48 h apart, is excreted as the parent drug in the urine. In horses, 6% of an oral dose is excreted unchanged in the urine within 30 h (USP, 2003). Florfenicol and some metabolites, such as monochloroflorfenicol and florfenicol oxamic acid, are also eliminated via the faecal route (USP, 2003). Quantitative data, especially on the percentage of the parent drug over metabolites, found in large intestines and in faeces is lacking for most species. An enterohepatic recirculation and/or a gastrointestinal secretion from blood to gut lumen of florfenicol is suggested by one study reporting high florfenicol concentrations in caecum or colon after both IM or oral administration (De Smet et al., 2018). Significant binding of florfenicol to elements within intestinal contents is unlikely, in view of the lack of binding and substantial survival times and high bioactivity associated with contamination of soil (Subbiah et al., 2011). However, there is currently no experimental study supporting this hypothesis. 1.3.3.2 Thiamphenicol Amphenicols were shown in one study to be relatively unstable in stored feed (Pietro et al., 2014), which could affect the actual levels presented to animals if cases of long-term administration. Thiamphenicol is efficiently absorbed from the gastrointestinal tract and it is principally excreted in urine, with only small amounts being found in faeces (Dowling, 2013). The bioavailability of thiamphenicol after oral administration in milk is around 60% in veal calves and pre-ruminant sheep (Mengozzi et al., 2002) but only of 30% in adult sheep (Abdennebi et al., 1994). Only one short communication reported a low bioavailability of 28% in fasted pigs (Haritova et al., 2002). In birds, the bioavailability seems high, at least for fasted animals. The bioavailability is of 69% in fasted turkeys, (Switała et al., 2007), 80% in 3-week-old broilers (Ocampo et al., 2000) and higher than 70% in ducks (Tikhomirov et al., 2019). Thiamphenicol is not readily metabolised in cattle, poultry, sheep (FAO, 1999). In rats, more than 30% of the dose was excreted in the faeces within 75 h (JECFA, 2002). In humans, renal insufficiency prolongs the half-life of thiamphenicol, but hepatic insufficiency, e.g. due to cirrhosis, did not increase the half-life, suggesting that enterohepatic circulation is not likely to be a significant factor that might prolong the presence in the gut (Walter and Heilmeyer, 1975). No data was found on the possible binding of thiamphenicol to intestinal contents. 1.3.4 Main resistance mechanisms The most frequently encountered mechanism of bacterial resistance to amphenicols is enzymatic inactivation by acetylation by a variety of types of chloramphenicol acetyltransferases (CATs). CATs can inactivate thiamphenicol but the replacement of the hydroxyl group at C-3 by a fluor residue makes florfenicol resistant to inactivation by CAT enzymes. There are numerous other mechanisms that can inactivate amphenicols, such as O-phosphorylation and hydrolytic degradation. A chloramphenicol acetate esterase (estDL136 gene) identified during a soil metagenomic study, was the first described hydrolytic mechanism capable of inactivating florfenicol (Tao et al., 2012). However, there are also reports of other resistance mechanisms, such as efflux systems, mutations in the target site genes and permeability barriers conferring resistance to amphenicols. Specific transporters involved in the export of amphenicols by bacterial efflux, including those encoded by fexA, fexB, floR, pexA or cmlB1 genes, have no known function in normal cell metabolism, but some multidrug transporters play an important role in the excretion of any cytotoxic compound, including amphenicols (Kadlec et al., 2007). Non-enzymatic resistance mechanisms based on permeability barriers involving alteration in membrane proteins such as porins have been described in various bacteria. The mar locus which is present in many Enterobacteriaceae has also been reported to contribute to resistance in E. coli. Mutations in the major ribosomal protein gene cluster of E. coli and B. subtilis as well as in the 23S rRNA gene of E. coli are known to confer resistance to amphenicols (Schwarz et al., 2004). Several publications describe other amphenicol resistance genes in bacteria of animal and human origin, including the ribosomal protection protein genes optrA and poxtA, the gene encoding RE-CmeABC, a functionally enhanced multidrug efflux pump variant (3–5), and cfr, encoding a 23S rRNA methyltransferase. Some of these mechanisms (e.g. cfr, optrA and poxtA) can also confer resistance to antimicrobials of other classes (e.g. lincosamides, oxazolidinones, pleuromutilins and streptogramin A) (Long et al., 2006). Amphenicol resistance genes often occur on MDR plasmids, so usage may co-select for resistance to other clinically relevant antimicrobials, e.g. extended-spectrum cephalosporins or colistin (Du et al., 2020; Wang et al., 2020). 2 Data and methodologies The data sources and methodology used for this opinion are described in a dedicated document, the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue). 3 Assessment 3.1 Introduction As indicated in the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue), exposure to low concentrations of antimicrobials (including sub-minimum inhibitory concentrations, sub-MICs) may have different effects on bacterial antimicrobial resistance evolution, properties of bacteria and in animal growth promotion. Some examples including emergence of and selection for antimicrobial resistance, mutagenesis, virulence and/or horizontal gene transfer (HGT), etc., for the antimicrobials under assessment are shown below. 3.1.1 Resistance development/spread due to sub-MIC concentrations of amphenicols florfenicol, thiamphenicol: examples There are numerous publications that report amphenicol resistance in a wide range of bacteria, but very few that report on the response to exposure. Co-resistance to florfenicol and other antimicrobials has been identified in various Enterobacteriaceae, which can increase the spread of multidrug resistance (MDR) and associated virulence or heavy metal tolerance genes (Doublet et al., 2004; Berge et al., 2005; Braibant et al., 2005; Higuera-Llantén et al., 2018; HPRA, 2018). Resistance genes are also being reported from a wider range of bacteria in situations where there is heavy usage (Tang et al., 2020), including cfr in LA-MRSA CC398 (Ruiz-Ripa et al., 2021). 3.1.1.1 Effects of sub-MIC concentrations on selection for resistance and mutagenesis The MIC analysis for Piscirickettsia salmonis, strains which were cultured in a sub-lethal concentration of florfenicol (0.064 μg/mL) showed a mean increase of one dilution (equivalent to 0.5 μg/mL) when compared with the initial MIC value (Yañez et al., 2014). Salmonella Enteritidis, Klebsiella pneumoniae, Staphylococcus aureus and Listeria monocytogenes were exposed to a sub-inhibitory concentration of florfenicol (1, 20 μg/mL) for 24 h and 48 h and changes in resistance to human therapeutic antimicrobials were determined. Increases in MIC values for ampicillin, tetracycline, nalidixic acid and meropenem against Salmonella and Klebsiella were in the range of 20–1,000 μg/mL, 5–62.5 μg/mL, 5–125 μg/mL and 0.05–0.1 μg/mL, respectively, whereas increases in MICs against Staphylococcus and Listeria were 2.5–10 μg/mL, 2.5 μg/mL, 62.5–500 μg/mL and 0.1–0.2 μg/mL, respectively. Exposure to sub-inhibitory levels of florfenicol was therefore associated with increases in resistance to ampicillin, tetracycline and nalidixic acid ranging from 1.25- to 40-fold compared to unexposed bacteria, with the exception of meropenem (Singh and Bhunia, 2019). The minimal selective concentration (MSC) for a strain of E. coli was determined by analysing and comparing the growth rates of susceptible and resistant variants at different sub-MICs (0.031, 0.062, 0.125, 0.25, 0.5, 1.0 and 2.0 μg/mL) of florfenicol. The MSC of florfenicol for strain E. coli MG1655/pSD11 was determined to be 0.042 μg/mL, which was 1/100 of the MIC value for the isogenic susceptible E. coli strain (Zhang et al., 2019). After 12 passages at 1/8th of the MIC of a synergistic combination of florfenicol and thiamphenicol, there was no apparent development of resistance for either antimicrobial independently or in combination, with the only changes noted being a doubling in MIC for the combination exposure in the susceptible Actinobacillus pleuropneumoniae strain and for thiamphenicol exposure in the resistant A. pleuropneumoniae strain after the 9th passage (Rattanapanadda et al., 2019). 3.1.1.2 Effects of sub-MIC concentrations on horizontal gene transfer and virulence Exposure of MDR S. Typhimurium isolates to sub-inhibitory concentrations of florfenicol (16 μg/mL) for 30 min during early-log phase resulted in multiple genes associated with attachment and virulence genes located within the Salmonella pathogenicity islands being significantly up-regulated. Swimming and swarming motility were decreased due to antimicrobial exposure and florfenicol enhanced the cellular invasion of one isolate. Many of these genes may only be fully expressed in-vivo (Holman et al., 2018). However, in another study three clinical bovine S. Typhimurium DT104 isolates were exposed to sub-inhibitory concentrations of florfenicol (1 and 5 μg/mL) for 30 min. HEp-2 cellular invasion assays, as well as expression analyses of invasion-related genes, suggested that the invasiveness of the isolates was not noticeably enhanced after exposure to low levels of florfenicol (Brunelle, 2011). The effect of sub-inhibitory concentrations of florfenicol on adherence properties of susceptible and resistant Staphylococcus aureus strains was investigated in vitro. The susceptible S. aureus strain Newman and its resistant derivative carrying the resistance plasmid pSCFS1 from S. sciuri were incubated in the presence of 2 μg/mL florfenicol, and the resistant strain was also exposed to 64 μg/mL florfenicol. When grown in the presence of half the strain-specific MIC of florfenicol, both isolates showed significantly increased adherence to HEp-2 cells and to fibronectin-coated microtitre plates (Blickwede et al., 2004). 3.2 ToR1. Estimation of the antimicrobial levels in non-target feed that would not result in the selection of resistance: Feed Antimicrobial Resistance Selection Concentration (FARSC) As explained in the Methodology Section (2.2.1.3) of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue), the estimation of this value for these amphenicols under assessment for different animal species followed a two-step approach as described below. First, the minimal selective concentration (MSC) for a strain of E. coli (E. coli MG1655/pSD11), which was determined by analysing and comparing the growth rates of susceptible and resistant variants at different sub-MICs of florfenicol was used
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