In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri

The Veterinary Journal 214 (2016) 96–101

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In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri A. Paterna, J. Tatay-Dualde, J. Amores, M. Prats-van der Ham, A. Sánchez *, C. de la Fe, A. Contreras, J.C. Corrales, Á. Gómez-Martín Department of Animal Health, Faculty of Veterinary Sciences, Regional Campus of International Excellence ‘Campus Mare Nostrum’, Universidad de Murcia, Campus de Espinardo s/n., 30100 Murcia, Spain

A R T I C L E

I N F O

Article history: Accepted 14 May 2016 Keywords: Epsilometric test gyrA mutation Minimum inhibitory concentration Minimum mycoplasmacidal concentration Mycoplasma mycoides subsp. capri

A B S T R A C T

The minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of 17 antimicrobials against 41 Spanish caprine isolates of Mycoplasma mycoides subsp. capri (Mmc) obtained from different specimens (milk, external auricular canal and semen) were determined using a liquid microdilution method. For half of the isolates, the MIC was also estimated for seven of the antimicrobials using an epsilometric test (ET), in order to compare both methods and assess the validity of ET. Mutations in genes gyrA, gyrB, parC and parE conferring fluoroquinolone resistance, which have been recently described in Mmc, were investigated using PCR. The anatomical origin of the isolate had no effect on its antimicrobial susceptibility. Moxifloxacin and doxycycline had the lowest MIC values. The rest of the fluoroquinolones studied (except norfloxacin), together with tylosin and clindamycin, also had low MIC values, although the MMC obtained for clindamycin was higher than for the other antimicrobials. For all the aminoglycosides, spiramycin and erythromycin, a notable level of resistance was observed. The ET was in close agreement with broth microdilution at low MICs, but not at intermediate or high MICs. The analysis of the genomic sequences revealed the presence of an amino acid substitution in codon 83 of the gene gyrA, which has not been described previously in Mmc. © 2016 Elsevier Ltd. All rights reserved.

Introduction In some regions where contagious agalactia (CA) has been studied, including France (Chazel et al., 2010) and the Canary Islands (De la Fe et al., 2005), it has been reported that caprine CA is more commonly caused by Mycoplasma mycoides subsp. capri (Mmc) than by M. agalactiae. Mmc is an important cause of caprine CA in the Iberian Peninsula (Amores et al., 2012; Gómez-Martín et al., 2012). Thus, the economic losses caused by Mmc in dairy goats may be underestimated (Chazel et al., 2010). Antimicrobial chemotherapy is often used in an effort to reduce these losses (Gómez-Martín et al., 2013), and mainly consists of fluoroquinolones and macrolides, but few studies have examined the antimicrobial susceptibility of Mmc. Antunes et al. (2007) reported that Mmc had in vitro sensitivity to fluoroquinolones, macrolides, tetracyclines and lincosamides. However, massive use of antimicrobials in more recent times has led to the appearance of bacterial resistance, e.g. for M. bovis (Ayling et al., 2014). Therefore, the antimicrobial susceptibility of Mmc should be assessed in individual isolates in order to select the most appropriate therapy for each case. Changes in antimicrobial sus-

* Corresponding author. Tel.: +34 8688 84811. E-mail address: [email protected] (A. Sánchez). http://dx.doi.org/10.1016/j.tvjl.2016.05.010 1090-0233/© 2016 Elsevier Ltd. All rights reserved.

ceptibility may be due to mutations, as has been demonstrated under laboratory conditions by Antunes et al. (2015) for quinolone resistance determining regions (QRDRs) of Mmc, so it would be interesting to know if these changes also occur in field strains. Given the differences in the pharmacokinetic properties of different antimicrobials, we hypothesised that the antimicrobial susceptibility of an isolate from an anatomic location to which the antimicrobial can diffuse (e.g. fluoroquinolones to the mammary gland) could differ from that of an isolate from a location at which the antimicrobial concentration achieved is lower. The specialised growth requirements of mycoplasmas do not allow the use of the standardised antimicrobial susceptibility tests used for other bacteria. The guidelines by Hannan (2000) include several methods, which employ liquid or solid media for the determination of the minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of veterinary Mycoplasma species. However, these methods are expensive and timeconsuming, so several studies (Francoz et al., 2005; Filioussis et al., 2014) have used a commercial epsilometric test (ET), which is faster, but has not been standardised for Mycoplasma spp. Another difficulty in determining the antimicrobial susceptibility of Mycoplasma isolates is that few breakpoint antimicrobial concentrations for veterinary mycoplasmas have been established to allow classification of an isolate as sensitive (Hannan, 2000).

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Table 1 Antimicrobials and range of concentrations used in this study.

Aminoglycosides

Antimicrobial

Presentationa

Streptomycin Kanamycin

Powder Powder Strip Powder Strip Powder Powder Powder Powder Strip Powder Powder Powder Strip Powder Powder Strip Powder Powder Powder Strip Powder Strip Powder

Gentamicin

Fluoroquinolones

Neomycin Streptomycin Ciprofloxacin Enrofloxacin Marbofloxacin Danofloxacin Moxifloxacin

Tetracyclines

Norfloxacin Doxycycline

Macrolides

Tylosin Erythromycin Spiramycin

Lincosamides

Clindamycin Lincomycin

a

Manufacturer

Catalog number

Concentration range (μg/mL)

Sigma-Aldrich Sigma-Aldrich Liofilchem Sigma-Aldrich Liofilchem Sigma-Aldrich Sigma-Aldrich Fluka Fluka Liofilchem Tokyo Chemical Industry Fluka Cayman Chemical Company Liofilchem Sigma-Aldrich Sigma-Aldrich Liofilchem Fluka Fluka Sigma-Aldrich Liofilchem Cayman Chemical Company Liofilchem Sigma-Aldrich

S-6501 K-1377 92034 G3632 92009 N-1876 S-6501 17850 17849 92013 M2240 33700 14830 92090 N-9890 D-9891 92156 93806 45673 S-9132 92046 15006 92072 L-6004

8–128 8–128 0.016–256 8–128 0.016–256 8–128 8–128 0.006–12.8 0.006–12.8 0.002–32 0.006–12.8 0.006–12.8 0.006–12.8 0.002–32 0.1–12.8 0.006–12.8 0.016–256 0.006–12.8 0.1–12.8 0.1–12.8 0.002–32 0.006–12.8 0.016–256 0.1–12.8

Antimicrobials in powder form were diluted in sterile distilled water for the microdilution susceptibility test.

In the present study, we first determined the MIC and MMC of 41 Spanish caprine isolates of Mmc and two reference strains for 17 antimicrobials, using the microdilution susceptibility test. Secondly, we investigated possible differences in antimicrobial susceptibility depending on the anatomical origin of the isolate (external auricular canal or milk). The MIC of 22 isolates was also determined by ET, to compare both methods and assess the validity of ET for Mmc. Finally, in isolates that had high MIC and/or MMC values, we searched for possible mutations conferring resistance to fluoroquinolones. Materials and methods Mycoplasma isolates We studied 41 caprine isolates of Mmc obtained from 25 commercial dairy goat herds in Spain. Twenty isolates were obtained from milk specimens (from the bulktank or half udder milk), 20 from the external auricular canal (EAC) and one from semen (Appendix: Supplementary Table S1). Reference strains PG3 (NCTC 10137) and Y-goat (NCTC 11706) were also included. Pure cultures of all of the isolates were obtained by triple-cloning (Nicholas and Baker, 1998) and identified by PCR and sequencing of fusA gene (Manso-Silván et al., 2007). The isolates were cultured in PH medium (Gómez-Martín et al., 2012) until the stationary phase of microbial growth was reached. The concentrations of the inocula were determined as described previously (Albers and Fletcher, 1982), and then diluted with saline solution to the desired concentration. Microdilution susceptibility test The microdilution method was performed as described by Hannan (2000). The antimicrobials studied, the manufacturers and concentration ranges are provided in Table 1. A stock solution of each antimicrobial was prepared by dissolving it in sterile distilled water, from which two-fold dilutions were prepared. In 96-well sterile microtitre plates, 150 μL of PH medium containing 0.007% phenol red (Sigma-Aldrich), 25.6 μL of each antimicrobial dilution and the diluted inoculum (final concentration 103–105 CFU/mL) were added to each well. A positive control (without antimicrobial) and a sterility control (without inoculum) were included for each antimicrobial. The plates were sealed and incubated at 37 °C in a humid atmosphere with 5% CO2, and were read when a change of colour appeared in the positive control due to the acidification of the medium. Thus, the MIC was defined as the minimum concentration of antimicrobial at which no growth was observed. To determine the MMC, after reading the MIC plates, a 10 μL specimen from each well was transferred into 190 μL of fresh PH medium containing phenol red in a new 96-well microtitre plate, to dilute the antimicrobial below its MIC so that its bac-

tericidal activity could be determined. These plates were incubated and read as described above. Epsilometric test For the ET, we selected 20 isolates by convenience sampling (EAC, n = 10; milk, n = 10) and both reference strains. The susceptibility of these isolates to representative antimicrobials of all the groups included in the microdilution test was assessed (Table 1). Commercial antimicrobial strips impregnated with a defined concentration gradient of antimicrobial, across 15 two-fold dilutions, were used. The concentrations of the inocula were adjusted to 108 CFU/mL (Loria et al., 2003) by diluting culture in saline solution. Subsequently, 50 μL of inoculum was spread with an L-shaped bacterial cell spreader onto 90 mm PH agar plates without antimicrobial. The antimicrobial strip was placed on the plate as described by the manufacturer and the plates were incubated as described above. As no references were available for this specific species and method, the plates were read after 24 h and 48 h to determine the optimum time of incubation. To obtain the MIC, the area of growth inhibition that intersected with the strip was observed using a microscope (10×), with the plate open and facing up, in sterile conditions. Statistical analysis Statistical analysis was performed with EpiInfo (Dean et al., 2011). The mean MIC values were obtained, and an ANOVA was used to compare the values for the isolates from different origins and different methods for determining susceptibility. Searching for antimicrobial resistance mutations We checked for the presence of mutations conferring resistance to fluoroquinolones in our isolates using PCR and sequencing. The target genes were gyrA, gyrB, parC and parE, which were amplified using the oligonucleotides and conditions described by Antunes et al. (2015), in an i-Cycler (Biorad) thermal cycler. PCR products were subjected to electrophoresis in 1% agarose gels containing 0.005% RedSafe (iNtRON Biotechnology) and the stained DNA visualised using UV transillumination. The amplicon was sequenced at the molecular biology service of the University of Murcia. Sequences were aligned using MEGA6 (Tamura et al., 2013).

Results The mean MICs for the milk and EAC isolates, MIC ranges, MICs at which 50% of the isolates were inhibited (MIC50), MICs at which 90% of the isolates were inhibited (MIC90), and MIC values for the semen and reference strains are provided in Table 2.

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Table 2 Mean ± standard deviation (SD) of the minimum inhibitory concentration (MIC) depending on the origin of the isolate, MIC90, MIC50, and range of MIC (μg/mL) for Mycoplasma mycoides subsp. capri type strains and field isolates and different antimicrobials. Antimicrobial SPT KAN GTM NEO STR CIP ENR MRB DAN MOX NOR DOX TYL ERY SPI CLI LIN

EAC (n = 20) Mean ± SD

Milk (n = 20) Mean ± SD

Range n = 40

MIC90 n = 40

MIC50 n = 40

Semen isolate

PG3

Y-goat

20.80a ± 8.77 30.00a ± 16.80 30.00a ± 16.80 126.40a ± 14.70 27.6a ± 17.50 0.06a ± 0.02 0.08a ± 0.04 0.16a ± 0.05 0.09a ± 0.04 0.03a ± 0.00 0.43a ± 0.13 0.03a ± 0.01 0.09a ± 0.02 10.09a ± 4.15 3.39a ± 4.57 0.18a ± 0.07 0.70a ± 0.63

21.60a ± 7.83 38.40a ± 30.00 37.60a ± 27.60 129.30a ± 0.98 45.60a ± 39.97 0.08b ± 0.05 0.07a ± 0.05 0.15a ± 0.08 0.08a ± 0.05 0.03a ± 0.02 0.73b ± 0.43 0.04a ± 0.02 0.07b ± 0.04 12.10a ± 2.88 4.29a ± 3.66 0.47a ± 1.40 1.33a ± 2.82

8–128 8–128 8–128 64- > 128 8–32 0.05–0.2 0.025–0.2 0.1–0.4 0.025–0.2 0.01–0.1 0.2–1.6 0.025–0.1 0.025–0.2 1.6- > 12.8 0.2- > 12.8 0.01–6.4 0.2- > 12.8

64 64 64 >128 32 0.1 0.1 0.2 0.1 0.05 0.8 0.05 0.1 >12.8 12.8 0.2 0.8

16 32 32 >128 16 0.05 0.05 0.1 0.05 0.025 0.4 0.025 0.1 >12.8 3.2 0.2 0.8

8 8 16 128 8 0.025 0.025 0.01 0.025 0.01 0.4 0.01 0.01 >12.8 >12.8 0.01 0.4

32 64 64 >128 64 0.1 0.1 0.2 0.1 0.025 0.8 0.025 0.1 >12.8 12.8 0.025 >12.8

16 32 32 >128 16 0.05 0.1 0.1 0.1 0.025 0.4 0.05 0.1 6.4 >12.8 0.2 0.8

SPT, spectinomycin; KAN, kanamycin; GTM, gentamicin; NEO, neomycin; STR, streptomycin; CIP, ciprofloxacin; ENR, enrofloxacin; MRB, marbofloxacin; DAN, danofloxacin; MOX, moxifloxacin; NOR, norfloxacin; DOX, doxycycline; TYL, tylosin; ERY, erythromycin; SPI, spiramycin; CLI, clindamycin; LIN, lincomycin; EAC, external auditory canal. a,b Means with different superscripts within a row differ significantly (P < 0.05).

No significant differences were found between the mean values of the MIC obtained for milk and EAC isolates, except for ciprofloxacin (higher mean MIC in milk isolates, P = 0.005) and tylosin (higher mean MIC in EAC isolates, P = 0.03). The MICs of aminoglycosides for the isolates of Mmc had wide ranges and the MIC50 and MIC90 values were high. Fluoroquinolones (except norfloxacin), doxycycline, tylosin and clindamycin were the most effective antimicrobial agents, with MICs ranging from 0.01– 0.2 μg/mL. Moreover, moxifloxacin and doxycycline had the lowest MIC90s of the antimicrobials investigated. More than 90% of the isolates were resistant to erythromycin (MIC > 12.8 μg/mL). The MIC50 of norfloxacin, spiramycin and lincomycin were intermediate (0.4, 3.2 and 0.8 μg/mL, respectively), and a wide range of MICs was seen across the isolates. The Mmc isolate from semen had a higher antimicrobial susceptibility than isolates from EAC or milk, with MICs less than or equal to the minimum of the range for the EAC and milk isolates, except for gentamicin, neomycin, norfloxacin and lincomycin.

However, the semen isolate was resistant to erythromycin and spiramycin. The results for the MMC testing are provided in Table 3. No significant differences were seen between the mean values of MMC for milk or EAC isolates, except for enrofloxacin (higher mean in milk isolates, P = 0.04). All the isolates had MMC values >128 μg/mL for neomycin, and for most of the other aminoglycosides studied. The lowest values and narrowest ranges were those for fluoroquinolones (except norfloxacin), doxycycline and tylosin. At least 90% of the isolates had MMC values ≥128/12.8 μg/mL for norfloxacin, erythromycin, spiramicin and lincomicin, while the MMC90 for clindamycin was intermediate. The results from ET readings after 24 h or 48 h incubation, and the comparison of this technique with the microdilution method are shown in Table 4. Using the ET, there were no significant differences between reading the plates after 24 h or 48 h incubation, except for clindamycin, the mean of which increased over the second 24 h period. Reading of the result under the microscope was more

Table 3 Mean ± standard deviation (SD) of the minimum mycoplasmacidal concentration (MMC) depending on the origin of the isolate, MMC90, MMC50, and range of MMC/ (μg/ mL) for Mycoplasma mycoides subsp. capri type strains and field isolates and different antimicrobials. Antimicrobial SPT KAN GTM NEO STR CIP ENR MRB DAN MOX NOR DOX TYL ERY SPI CLI LIN

EAC (n = 20) Mean ± SD

Milk (n = 20) Mean ± SD

Range n = 40

MMC90 n = 40

MMC50 n = 40

Semen isolate

PG3

Y-goat

104.10a ± 40.96 88.10a ± 38.78 80.10a ± 38.24 128.00a ± 00.00 68.10a ± 38.97 0.76a ± 0.88 0.44a ± 0.39 0.93a ± 0.91 0.72a ± 0.57 0.13a ± 0.11 6.66a ± 4.90 0.79a ± 0.48 0.72a ± 0.40 12.80a ± 0.00 11.21a ± 3.62 4.68a ± 2.24 12.20a ± 1.99

103.10a ± 37.37 97.90a ± 35.45 93.30a ± 41.85 128.00a ± 00.00 86.00a ± 42.05 0.83a ± 0.87 0.72b ± 0.52 0.91a ± 0.76 0.92a ± 1.36 0.25a ± 0.34 6.10a ± 4.93 0.90a ± 0.64 0.51a ± 0.38 12.80a ± 0.00 11.51a ± 3.69 4.60a ± 3.44 10.60a ± 3.64

16- > 128 32- > 128 32- > 128 >128->128 16- > 128 0.05–3.2 0.05–3.2 0.1–3.2 0.1–3.4 0.01–1.6 0.8- > 12.8 0.05–3.2 0.05–1.6 12.8- > 12.8 1.6- > 12.8 0.8–12.8 3.2- > 12.8

>128 128 >128 >128 >128 1.6 1.6 1.6 1.6 0.4 12.8 1.6 0.8 >12.8 >12.8 6.4 >12.8

128 64 64 >128 64 0.4 0.4 0.8 0.4 0.1 3.2 0.8 0.8 >12.8 >12.8 3.2 12.8

8 16 16 >128 16 0.05 0.05 0.1 0.05 0.01 0.4 0.01 0.1 >12.8 >12.8 0.2 0.8

128 >128 128 >128 128 1.6 0.8 1.6 1.6 0.1 0.8 0.4 0.4 >12.8 >12.8 3.2 >12.8

32 32 32 >128 64 0.4 0.4 0.2 0.2 0.05 1.6 0.4 0.2 >12.8 >12.8 3.2 6.4

SPT, spectinomycin; KAN, kanamycin; GTM, gentamicin; NEO, neomycin; STR, streptomycin; CIP, ciprofloxacin; ENR, enrofloxacin; MRB, marbofloxacin; DAN, danofloxacin; MOX, moxifloxacin; NOR, norfloxacin; DOX, doxycycline; TYL, tylosin; ERY, erythromycin; SPI, spiramycin; CLI, clindamycin; LIN, lincomycin; EAC, external auditory canal. a,b Means with different superscripts within a row differ significantly (P < 0.05).

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Table 4 Mean of the minimum inhibitory concentration (MIC), MIC50, MIC90 and range of MIC (μg/mL) for Mycoplasma mycoides subsp. capri isolates and different antimicrobials depending on the susceptibility test employed.

ET 24 h incubation

ET 48 h incubation

Microdilution

Mean ± SD MIC50 MIC90 Range Mean ± SD MIC50 MIC90 Range Mean ± SD MIC50 MIC90 Range

KAN

GTM

ENR

MOX

DOX

SPI

CLI

31.36 ±18.45 24 64 6–64 51.44a ±44.36 48 >256 6- > 256 29.09a ±16.10 32 64 16–64

±29.30 48 96 12–128 54.66a ±30.23 48 96 12- > 256 29.82b ±13.34 32 32 16–64

±0.09 0.125 0.25 0.064–0.25 0.16a ±0.09 0.125 0.25 0.064–0.5 0.07b ±0.04 0.05 0.1 0.025–0.2

±0.03 0.023 0.047 0.012–0.125 0.03a ±0.03 0.023 0.047 0.012–0.125 0.03a ±0.02 0.025 0.05 0.01–0.1

±0.05 0.047 0.094 0.016–0.25 0.07a ±0.06 0.047 0.125 0.016–0.25 0.04a ±0.04 0.025 0.05 0.025–0.2

±0.30 0.125 0.19 0.064–1.5 0.28a ±0.29 0.19 0.38 0.064–1.5 2.59b ±3.13 1.6 6.4 0.2–12.8

0.65a ±0.29 0.5 0.75 0.19–1.5 0.93b ±0.35 1 1.5 0.19–1.5 0.18c ±0.07 0.2 0.2 0.05–0.4

a

49.27a

0.16a

0.03a

0.05a

0.20a

KAN, kanamycin; GTM, gentamicin; ENR, enrofloxacin; MOX, moxifloxacin; DOX, doxycycline; SPI, spiramycin; CLI, clindamycin; SD, standard deviation; ET, epsilometric test. a, b, c Means with different superscripts within a column differ significantly (P < 0.05).

accurate after 48 h of incubation. Comparisons of the mean values of MICs obtained using the ET and microdilution methods demonstrated significant differences (P < 0.05) for gentamicin, enrofloxacin, spiramycin and clindamycin. Only for spiramycin the MIC obtained using the microdilution method was higher than that obtained using the ET. Genetic analyses were performed on the reference strains, six field isolates with elevated MIC and/or MMC values for norfloxacin, and on one isolate with low values for these parameters. The QRDR genotype of these isolates is shown in the Appendix: Supplementary Table S2. No mutations were observed in gyrB, parC or parE in the positions described by Antunes et al. (2015). However, in gyrA (Table 5) a mutation resulting in an amino acid substitution was detected in codon 83 (E. coli numbering), Ser83Ile, in the two isolates with a MIC of 0.8 μg/mL and the highest MMC (>12.8 μg/mL). Discussion The origin of the isolate (milk or EAC) did not appear to influence antimicrobial susceptibility. Paterna et al. (2013) observed higher MIC values for M. agalactiae isolates obtained from herds with clinical signs of CA. The semen isolate was in general more sus-

ceptible than the rest of isolates studied but, as it was the only isolate from this location, no conclusions can be drawn from this observation. Our study of antimicrobial susceptibility confirmed that fluoroquinolones and tetracyclines are options for the treatment of CA caused by Mmc in Spain, in agreement with the observations of Antunes et al. (2007). A reduced susceptibility to norfloxacin was also observed for Mmc by Antunes et al. (2007) and for the closely related bovine pathogen Mycoplasma mycoides subsp. mycoides small-colony type (Mmm SC) by Ayling et al. (2005). Therefore, norfloxacin should not be considered for treatment of CA caused by Mmc. Together with doxycycline, moxifloxacin not only had the lowest MIC90 in the study, but also had MMC90 values below their suggested breakpoints (Hannan et al., 1997; Gerchman et al., 2009). Moreover, in goats, moxifloxacin has favourable pharmacokinetic properties with extensive penetration of the milk (Cárceles et al., 2007). Similarly, the pharmacokinetic properties of doxycycline in goats include the generation of concentrations in milk (Jha et al., 1989) that exceed the maximum value of MIC seen in our study, suggesting that an appropriate inhibitory concentration would probably be reached in mammary tissue.

Table 5 Analysis of codon 83 of the gyrA gene of Mycoplasma mycoides subsp. capri isolates with elevated minimum inhibitory concentration (MIC) and/or minimum mycoplasmacidal concentration (MMC) values for norfloxacin.

Letters in bold indicate the position of the nucleotide and amino acid changes.

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Aminoglycosides are not commonly used for the treatment of mycoplasmal infections (Waites et al., 2014) and we saw wide ranges and high MIC and MMC values, as did Antunes et al. (2007) Antunes et al. (2008) for Mmc and M. putrefaciens, and Ayling et al. (2005) for Mmm SC, supporting the hypothesis of Antunes et al. (2007) that there is intrinsic resistance to aminoglycosides in the spiroplasma phylogenic group of mycoplasmas. Clindamycin was only moderately effective against Mmc, requiring at least 0.2 μg/mL to inhibit 90% of isolates. However, these results would classify our isolates as sensitive, considering the breakpoint used by Francoz et al. (2005) for lincomycin. Tylosin had good in vitro efficacy in contrast to the rest of the macrolides we tested. Erythromycin and spiramycin had lower MIC values in the study by Antunes et al. (2007). Our results suggest the acquisition of resistance to these antimicrobials, as the isolates studied by these authors were obtained a few years before ours. Other species belonging to the hominis group (e.g. M. agalactiae) seem to be innately resistant to erythromycin due to a mutation in the 23S rRNA gene (Furneri et al., 2001). As Mmc belongs to the spiroplasma group, which is phylogenetically distant from the hominis group, another mechanism may be involved in this resistance. The values observed for MMC may be due to the fact that the sizes of the inocula used for the test were unknown (Ayling et al., 2005), but conversely, they also reflect the inhibitory or biocidal ability of the antimicrobial compounds. Thus, all the antimicrobials employed in this study would have been inhibitory. The time of incubation of the ET for Mmc only seemed to influence the results for some antimicrobials. In other veterinary mycoplasma species, longer times of incubation have been used to perform ET (Francoz et al., 2005; Filioussis et al., 2014), depending on their different growth rates. Few studies have compared the ET with the microdilution test for the determination of MICs in mycoplasmas. Dósa et al. (1999) suggested that the ET was valid for the determination of MICs for M. hominis and Ureaplasma urealyticum. However, our results indicate that the ET would not be a valid method to determine the susceptibility of Mmc to many antimicrobial compounds. The Ser83Ile change in gyrA has not been previously described in Mmc or other agents involved in CA. The two strains that had the mutation showed MICs for norfloxacin of 0.8 μg/mL, which could be considered an intermediate level of resistance. However, their MMC for this antimicrobial was >12.8 μg/mL, indicating that at the MIC, most organisms would be able to resume replication when the antimicrobial concentration dropped. This change has been identified in isolates of the avian pathogen M. gallisepticum resistant to enrofloxacin, mainly when it occurs together with the change Ser80Leu of parC, but also individually (Lysnyansky et al., 2012). In E. coli this single mutation has played a vital role in increasing the MIC of ciprofloxacin (Nawaz et al., 2015). In our study, crossresistance to other fluoroquinolones was not detected. Hence, further studies are required to determine if, at this level of MIC and despite the presence of the mutation, norfloxacin and other fluoroquinolones can be used for the treatment of CA caused by Mmc, and to determine the usefulness of determining the MMC to detect the presence of resistance to fluoroquinolones. Conclusions Enrofloxacin, ciprofloxacin, marbofloxacin, danofloxacin, moxifloxacin, doxycycline, tylosin and clindamycin were the most effective agents in vitro against Mmc. Aminoglycosides, spiramycin and erythromycin are unlikely to be useful for the treatment of CA caused by Mmc. Lincomycin is likely to have intermediate efficacy. The origin of the isolate (EAC or milk) did not influence its antimicrobial susceptibility. The ET MIC estimates were in close agreement with those obtained using broth microdilution tests for

antimicrobials for which the MIC was low, but the agreement was poorer when the MIC by microdilution was intermediate or high. The first case of a mutation in a quinolone resistance-determining region of the gene gyrA in Mmc field isolates with high values of MMC for norfloxacin was also detected.

Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Acknowledgements This work has been supported by the Spanish Ministry of Economy and Competitiveness (Spanish Government) co-financed by FEDER funds, project AGL2013-44771-R. Ana Paterna Moran holds a fellowship awarded by the University of Murcia (F.P.U.).

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.tvjl.2016.05.010.

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