Proposal of Epidemiological Cutoff Values for Apramycin 15 m g and Florfenicol 30 m g Disks Applicable to Staphylococcus aureus

Apramycin and ﬂorfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and ﬂor-fenicol. The susceptibility to apramycin and ﬂorfenicol was evaluated by disk diffusion of ﬁve S. aureus collections, comprising 352 isolates of animal ( n = 265) and human ( n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ‡ 15 mm (ECOFF = 15 mm) for apramycin and ‡ 21 mm for ﬂorfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas ﬁve NWT isolates were identiﬁed for ﬂorfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and ﬂorfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.


Introduction
S taphylococcus aureus is an important pathogen in veterinary medicine, mainly associated with skin and soft-tissue infections in companion animals, mastitis in cattle and systemic infections in poultry. 1,2The past decades have witnessed an increasing prevalence of antimicrobialresistant S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, in various animal species. 3As such, the occurrence of MRSA in animals has been identified as a microbiological hazard for human health. 4pramycin is an aminocyclitol antimicrobial agent produced by Streptomyces spp.approved only for use in veter-inary medicine for clinical purposes in Member States of the European Union. 5It is administered in oral formulations in feed or drinking water for the treatment of enteric infections caused by, for example, Salmonella enterica or Escherichia coli in pigs, calves, lambs, poultry, and rabbits. 5,6Florfenicol is a fluorinated thiamphenicol derivative.Similarly to apramycin, its use is restricted to veterinary medicine and it is administered parenterally for the treatment of respiratory bacterial infections in pigs, cattle, and ovine as well as in oral formulations in feed and drinking water for the treatment of bacterial infections in pigs, poultry, and fish. 6,7Florfenicol is also available, in combination with an antifungal and a steroid, for the topical treatment of otitis externa in dogs caused by Staphylococcus pseudintermedius and occasionally S. aureus. 6Importantly, both antimicrobial agents belong to classes that are considered as veterinary critically important antimicrobials, 8 as well as critically or highly important antimicrobials in human medicine. 9lthough apramycin is not used for the treatment of infections caused by staphylococci in animals, the presence of a resistance determinant to this antibiotic, apmA, was identified in MRSA isolates from cattle and pigs belonging to the clonal complex CC398. 10Since then, apmA has been described in MRSA isolates collected from a broiler, 11 from a nasal swab of a human working on a broiler farm and the environment of his residence 11 and from the environmental dust of a pig farm. 12his gene was also identified in a Staphylococcus lentus (recently reclassified as Mammaliicoccus lentus) 13 isolate from a veal calf 14 and in a Staphylococcus sciuri (recently reclassified as Mammaliicoccus sciuri) 13 isolate from an environmental sample of a pig farm. 15The apmA gene, which is usually located on small plasmids or large multiresistance plasmids, 16 encodes an acetyltransferase and is associated with elevated minimum inhibitory concentrations (MICs) of apramycin (32 to ‡128 mg/L) and gentamicin (8 mg/L). 10,12,16he florfenicol resistance gene fexA was first identified in a bovine S. lentus isolate. 17To date, this determinant has been described in various S. aureus and other staphylococcal isolates (including S. pseudintermedius and Staphylococcus simulans) collected from pigs, 18 cattle, 14,19 poultry, 20 cats, 21 a dog, 22 a horse, 23 and a marmot 24 as well as from soil surrounding pig farms. 25The fexA gene has also been reported in MRSA isolates from nasal swabs of pig farmers. 26his gene is located in the Tn558 transposon or its variants, which can be found on small or large plasmids, 3,27 or even integrated in the staphylococcal chromosomal DNA. 23It encodes the efflux pump FexA, a 14 transmembrane segment transporter of the Major Facilitator Superfamily and is associated with florfenicol MIC values ranging from 16 to 64 mg/L. 17,23,28Other determinants can convey resistance to florfenicol, namely, the cfr, optrA, and the poxtA genes.The cfr gene encodes an rRNA methylase and was first identified in a bovine S. sciuri isolate, 29 but its presence is now reported in several staphylococci, including S. aureus, from various animal origins. 3It is usually located on plasmids but can also be found in the staphylococcal chromosome. 3The optrA gene codes for an ABC-F protein that has a ribosomal protection effect and has been described in S. sciuri isolates from cats, dogs, and pigs. 3Recently, it has also been reported in MRSA isolates collected from pigs and poultry. 27The poxtA gene codes also for an ABC-F protein, which, however, is only distantly related to OptrA. 30Besides resistance to florfenicol, all three genes also mediate resistance to oxazolidinones.The gene cfr confers additional resistance to lincosamides, pleuromutilins, and streptogramin A. 3 The poxtA gene also confers elevated MICs to tetracycline. 30espite the occurrence of S. aureus strains carrying apramycin and florfenicol resistance genes, there are no inhibition zone-based epidemiological cutoff (ECOFF) values established to identify such strains in a S. aureus population during antimicrobial resistance surveillance studies.Hence, this study focused on proposing ECOFFs of S. aureus for apramycin and florfenicol based on inhibition zones that may allow a rapid identification of isolates with resistance mechanisms toward these two antimicrobial agents.
These isolates were analyzed at five independent laboratories; two located in Portugal (cities of Lisbon [Lab1] and Oeiras [Lab2]) and three located in Germany (two in Berlin [Lab3, Lab4], another in Wunstorf [Lab5]).In total, the collection comprised 178 MRSA isolates and 174 methicillin-susceptible S. aureus isolates.
This study involved only bacterial strains that were already isolated and thus, no ethics approval was necessary.

Antimicrobial susceptibility testing
Inhibition zone diameters were determined for apramycin (15 mg) and florfenicol (30 mg) disks by the Kirby-Bauer method according to EUCAST (Lab1, for human isolates) or CLSI standards. 33,34Antibiotic disks were acquired from MAST Group Ltd. (Liverpool, United Kingdom) or Thermo ScientificÔ OxoidÔ (Basingstoke, United Kingdom or Wesel, Germany).In brief, fresh overnight cultures were obtained for each isolate on Tryptic Soy Agar (Thermo Scientific Oxoid) or blood agar plates (Thermo Scientific Oxoid), from which isolated colonies were transferred to 0.85% (p/v) NaCl to obtain a cellular suspension with turbidity equivalent to 0.5 McFarland.The cellular suspension was swabbed onto Mueller-Hinton agar (Thermo Scientific Oxoid) plates and the antibiotic disks were placed on the inoculated media within 15 minutes.After 5 minutes, plates were inverted and placed in an incubator at 35°C -1°C.After incubation for 18-20 hours, inhibition zone diameters were measured in millimeters.In agreement with the CLSI and EUCAST recommendations, S. aureus ATCC Ò 25923Ô and S. aureus ATCC Ò 29213Ô were used as quality control strains in this study. 33,34

Determination of ECOFF values
The inhibition zone-based ECOFF values were estimated using the normalized resistance interpretation (NRI) method. 35,36This method uses the distributions of inhibition zone diameters to make a least-square regression analysis to determine the putative wild-type (WT) population, the mean inhibition zone diameter and the associated standard deviation (SD) for each species-antimicrobial agent combination.The ECOFF corresponds to the smallest inhibition zone diameter presented by the putative WT population and is calculated at 2.5 • the SD above the mean value and rounded up to the lowest absolute value. 35,36Thus, the ECOFF allows the distinction between putative WT populations (devoid of phenotypically detectable acquired resistance mechanisms) and nonwild-type (NWT) populations (with phenotypically detectable acquired resistance mechanisms). 37The ECOFF estimated by the NRI method will include 99.4% of the WT population. 36The NRI method was used with permission from the patent holder, Bioscand AB, TA ¨BY, Sweden (European Patent No. 1383913, U.S. Patent No. 7,465,559).The automatic and manual excel programs were made available through courtesy by P. Smith, W. Finnegan, and G. Kronvall at www.bioscand.se/nri/.
The ECOFF values generated in this study are based on five data sets from five independent laboratories located in two countries, which provided data for 142 (Lab 1), 33 (Lab 2), 108 (Lab 3), 32 (Lab 4), and 37 (Lab 5) S. aureus isolates.

Results
The five data sets generated in this study were analyzed individually before aggregation (Supplementary Table S1).Each distribution was validated, abiding the minimum number required of WT isolates (at least n = 15) 37 and an SD below the acceptable SD upper limit of 3.38 mm, recommended by the NRI method.
The aggregated distributions of inhibition zone diameters of apramycin and florfenicol for the 352 S. aureus isolates included in this study are shown in Fig. 1.Both aggregated distributions were bimodal, with inhibition zone diameters ranging from 6 ( = growth until the disk) to 30 mm for apramycin and from 9 to 38 mm for florfenicol.The aggregated distributions of the putative WT populations and associated ECOFF values of both antimicrobial agents were calculated using the NRI method (Table 1).Both normalized distributions of the WT populations were validated as they included >100 observations in the putative WT distribution 37 and the SDs were below the acceptable SD upper limit of 3.38 mm (Table 1).
For apramycin, the NRI analysis characterized a WT population ‡15 mm with an associated SD of 1.55 mm (Table 1).Applying the estimated ECOFF = 15 mm, an NWT population was identified comprising five isolates (1.4%) collected from cattle (n = 2) and pigs (n = 3).All these isolates were MRSA, showed no inhibition zone, and carried the apmA gene for apramycin resistance.
For florfenicol, a WT population with inhibition zone diameters ‡21 mm was estimated with an associated SD of 2.30 mm (Table 1).The application of the calculated ECOFF = 21 mm identified an NWT population of 1.4%, corresponding to five MRSA isolates collected from cattle (n = 2), pigs (n = 2), and a dog (n = 1).These isolates presented inhibition zone diameters ranging between 9 and 11 mm and all carried the fexA gene mediating resistance to florfenicol in staphylococci. 11

Discussion
The aggregated distribution analyzed in this study revealed an NWT population (1.4%) toward apramycin.This low frequency of NWT isolates is not surprising since dissemination of apramycin resistance determinants is still rare in S. aureus and is mainly associated with food-producing animals and humans with professional contact to them. 3,11n fact, all the five NWT isolates for apramycin in this study were collected from food-producing animals, either cattle or pigs.Apramycin is not affected by most aminoglycosidemodifying enzymes and in staphylococci, a single acetyltransferase encoded by the apmA gene has been identified as mediating resistance to this antimicrobial agent. 3In this study, the five isolates carrying apmA showed no inhibition zone for apramycin.All isolates of human origin were categorized as WT for apramycin, in agreement with Truelson et al., who analyzed the distribution of apramycin MICs for a collection of >100 S. aureus (mainly of human origin), proposing an MIC-based cutoff value of 32 mg/L and also finding no NWT population among human S. aureus. 38or florfenicol, the application of the ECOFF value proposed in this study detected the presence of an NWT population (1.4%), comprising five isolates collected from cattle, pigs, and a dog, all harboring fexA.The NWT MRSA isolate of canine origin belongs to the clonal lineage ST398 and presents a multidrug resistance phenotype, showing additional resistance to fluoroquinolones and tetracyclines. 39The ECOFF estimated in this study may complement the MIC-based ECOFF established by EUCAST for the combination florfenicol-S.aureus, ECOFF EUCAST = 8 mg/L.
The finding of NWT populations for these antimicrobial agents confirms the presence of apramycin and florfenicol resistance determinants in S. aureus isolates from foodproducing and companion animals.In this study, we propose ECOFF values for two antimicrobial agents used exclusively in veterinary medicine, apramycin and florfenicol, based on the inhibition zone diameter distributions obtained for a collection of 352 S. aureus independent isolates of animal and human origin from different geographic regions.The application of the proposed ECOFF values to other collections by other laboratories will be valuable in antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents, in a One Health context.The ECOFF values were determined based on the aggregated distributions of inhibition zone diameters for 352 S. aureus isolates.ECOFF, epidemiological cutoff; NWT, nonwild-type; SD, standard deviation; WT, wild-type.

FIG. 1 .
FIG. 1. Aggregated distributions of inhibition zone diameters of apramycin (A) and florfenicol (B) for the 352 Staphylococcus aureus isolates and respective estimated ECOFF values by the NRI method.Black columns represent the inhibition zone distributions, whereas the gray line indicates the NRI-generated normalized distribution of the WT populations.The estimated ECOFF values are shown by dashed vertical lines, whereas the WT and NWT populations are indicated by arrows.ECOFF, epidemiological cutoff; NRI, normalized resistance interpretation; NWT, nonwild-type; WT, wild-type.

Table 1 .
Epidemiological Cutoff Values of Staphylococcus aureus for Apramycin and Florfenicol Estimated Using the Normalized Resistance Interpretation Method