Antimicrobial susceptibility of Mycoplasma bovis isolates from veal calves and dairy cattle in the Netherlands

Veterinary Microbiology 189 (2016) 1–7

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Antimicrobial susceptibility of Mycoplasma bovis isolates from veal calves and dairy cattle in the Netherlands Annet Heuvelink* , Constance Reugebrink, Jet Mars GD Animal Health, Arnsbergstraat 7, 7418 EZ, Deventer, the Netherlands

A R T I C L E I N F O

Article history: Received 11 December 2015 Received in revised form 6 April 2016 Accepted 9 April 2016 Keywords: Bovine Mycoplasma bovis Antimicrobial Susceptibility

A B S T R A C T

Control of Mycoplasma bovis infections depends on good husbandry practices and antibiotic treatment. To allow more prudent use of antimicrobial drugs, there is a need for information on the susceptibility profile of this pathogen. The objective of the present study was to analyse the in vitro antimicrobial susceptibility of clinical M. bovis isolates in the Netherlands. The collection comprised 95 bovine isolates, originating from lungs (n = 56), mastitis milk (n = 27), and synovial fluid (n = 12), collected between 2008 and 2014. Minimal inhibitory concentrations (MICs) were assessed by broth microdilution, both by using in-house prepared MIC plates and by using commercially available MIC plates. For each antimicrobial agent, the range of MIC results, the MIC50, and MIC90 values were calculated. M. bovis strains recently isolated in the Netherlands appeared to be characterized by relatively high MIC values for antimicrobial agents that, until now, have been recommended by the Dutch Association of Veterinarians for treating pneumonia caused by Mycoplasma species. Fluoroquinolones appeared to be the most efficacious in inhibiting M. bovis growth, followed by tulathromycin and oxytetracycline. The highest MIC values were obtained for erythromycin, tilmicosin, and tylosin. Future studies should be done on determining M. bovis specific clinical breakpoints, standardization of methods to determine MIC values as well as molecular studies on detection of antimicrobial resistance mechanisms of M. bovis isolates to develop PCR assays for determining resistance. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction In cattle, Mycoplasma bovis is able to cause respiratory disease, mastitis, arthritis, otitis, and reproductive disorders (Bürki et al., 2015). It is frequently implicated in cases of bovine respiratory disease (BRD) in calves. However, the role of M. bovis in the multifactorial BRD complex is not as easily defined; M. bovis mostly occurs in co-infection with viruses and/or other bacteria. M. bovis has characteristics that enable it to colonize and persist on mucosal surfaces, to invade tissues, and to persist at sites of disease despite an aggressive immune response (Bürki et al., 2015). In addition to economic costs, there are important animal welfare consequences of M. bovis infections. There is no effective vaccine, and therefore control of M. bovis infections depends on good husbandry practices and antibiotic treatment. However, M. bovis responds poorly to antimicrobial agents. Because mycoplasmas lack a cell wall, b-lactams are not effective against these pathogens. Similarly, mycoplasmas do not synthesize folic acid and are therefore

* Corresponding author. E-mail address: [email protected] (A. Heuvelink). http://dx.doi.org/10.1016/j.vetmic.2016.04.012 0378-1135/ã 2016 Elsevier B.V. All rights reserved.

intrinsically resistant to sulfonamides. Mycoplasmas as a class are generally susceptible to drugs that interfere with protein (aminoglycosides, lincosamides, macrolides, tetracyclines, and florfenicol) or DNA (fluoroquinolones) synthesis (Giguère, 2013). However, resistance against these drugs has been reported (Ayling et al., 2014; Gautier-Bouchardon et al., 2014; Kong et al., 2016). To ensure the prudent use of antimicrobial agents, it is important to effectively target antimicrobial treatment. Unsuitable treatment may lead to selection of resistant strains. However, assessing the antimicrobial susceptibility of mycoplasmas is difficult. This is related to their slow growth, small size and complex growth media requirements. Additionally, nutritional requirements, metabolic activities and fitness vary among species. Standard antimicrobial susceptibility procedures such as the disk diffusion method are therefore not suitable for mycoplasmas. Publications on the susceptibility of M. bovis to antimicrobial agents were carried out using broth microdilution (Ter Laak et al., 1993; Ayling et al., 2000; Rosenbusch et al., 2005; Gerchman et al., 2009; Soehnlen et al., 2011; Ayling et al., 2014; Kawai et al., 2014; Sulyok et al., 2014; Kong et al., 2016), agar dilution (Hirose et al., 2003; Uemura et al., 2010; Siugzdaite et al., 2012; GautierBouchardon et al., 2014), the E-test (Francoz et al., 2005; Gerchman

2

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7

et al., 2009), and also flow cytometry (Soehnlen et al., 2011) to determine minimum inhibitory concentrations (MICs). However, there are currently no MIC testing control standards for veterinary mycoplasmas, making it difficult to compare studies carried out using different methods. Breakpoints have not yet been determined and so MIC results cannot be defined as susceptible,

intermediate, or resistant, making it difficult to evaluate the likely in vivo therapeutic efficacy from MIC data established in vitro. Being scarce, recent publications on the antimicrobial susceptibility of M. bovis show an increase in MIC values (Ayling et al., 2014; Gautier-Bouchardon et al., 2014). There are no recent data on susceptibility patterns of M. bovis isolated from clinical samples in

Fig. 1. Distribution (%) of MIC values (mg/ml) of antimicrobial agents tested with in-house prepared MIC plates. The MIC50 and MIC90 values are marked. Additionally, when available, CLSI breakpoints for other bovine respiratory pathogens are indicated on the x-axis; isolates with MIC values less than or equal to the concentration indicated in the dotted-line arrow pointing to the left are susceptible, isolates with MIC values greater than or equal to the concentration indicated in the full-line arrow pointing to the right are resistant, the remaining isolates being intermediate.

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7

the Netherlands. Therefore, the objective of the current study was to examine the antimicrobial susceptibility of M. bovis field isolates, with a focus on antimicrobial agents approved to treat BRD in the Netherlands. The collection comprised respiratory, milk, and arthritis isolates from samples submitted to GD Animal Health (Deventer, the Netherlands) for routine diagnostic examinations or from necropsies during 2008 and 2014. 2. Materials and methods 2.1. Isolates In this study, 95 M. bovis isolates were tested. The isolates were collected during 2008 till 2014, all from diagnostic samples taken either at necropsy or from living animals. Isolates originated from the lungs of animals with respiratory problems (n = 56), from mastitis milk (n = 27), and from synovial fluid from animals with swollen joints (n = 12). Their identity was confirmed by PCR (Sachse et al., 2010), before they were tested for antimicrobial susceptibility. 2.2. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed following a previously described broth microdilution test using the colorimetric reagent alamarBlue (Rosenbusch et al., 2005) and the guidelines for MIC testing for veterinary mycoplasmas described by Hannan (2000). The minimum inhibitory concentrations (MIC), that is the concentrations required to completely suppress the growth of M. bovis, were determined of enrofloxacin (Sigma-Aldrich, Saint Louis, MO, United States), erythromycin (Sigma-Aldrich), oxytetracycline (Sigma-Aldrich), tilmicosin (Dr Ehrenstorfer, Augsburg, Germany), tulathromycin (Santa Cruz Biotechnology, Dallas, TX, United States), and tylosin (Sigma-Aldrich). In brief, of these antimicrobial agents twofold serial dilutions were made in filtered (0.2 mm pore size (Sartorius, Goettingen, Germany)) Mycoplasma broth (PPLO (pleuropneumonia-like organisms) broth without crystal violet (Becton, Dickinson and Company, Franklin Lakes, NJ, United States)) supplemented with horse serum (20%), yeast extract (1%), and 5% alamarBlue (Thermo Fisher Scientific, Wiltham, MA, United States) and 100 ml was transferred to each well of a Falcon 96-well microplate (Thermo Fisher Scientific). M. bovis isolates that had been pre-cultured and stored at 70  C pending MIC testing were diluted to approximately 103–105 colour-changing units (CCU) per ml in filtered Mycoplasma broth supplemented with horse serum (20%), yeast extract (1%), and 5% alamarBlue and 100 ml aliquots of these M. bovis suspensions were added to the wells of a 96-well microplate with the antimicrobial agents. Final test dilutions were 0.03–64 mg/ml for enrofloxacin, 0.25–512 mg/ml for erythromycin, 0.125–256 mg/ml for oxytetracycline and tylosin, and 0.5–1024 mg/ml for tilmicosin and tulathromycin. These levels were selected based upon previous research (Rosenbusch et al., 2005; Soehnlen et al., 2011). Additionally, per isolate four negative controls (no culture and no antimicrobial agent), and a series of positive growth controls (with culture and no antimicrobial agent) were included per plate. The tylosin stock solution was prepared in sterile distilled water. Stock solutions of enrofloxacin, erythromycin, tilmicosin, and tulathromycin were prepared in a 50/50 mix of methanol/sterile distilled water, while for the stock solution of oxytetracycline 0.1 M HCl was necessary. Therefore, positive growth controls were included consisting of twofold dilutions of these solvents in antibiotic-free filtered Mycoplasma broth supplemented with horse serum (20%), yeast extract (1%), and 5% alamarBlue in the same dilutions as those used in the broth microdilution test to exclude potential antimicrobial effects of the solvents on the

3

growth of M. bovis. After sealing the plates, they were incubated at 37  C for 48 h. Plates were read visually for a colour change; red indicating growth of the isolate and blue no growth. The lowest concentration of an antimicrobial agent completely suppressing growth, as expressed by a blue colour, was recorded as the MIC for that antimicrobial. For each antimicrobial agent, the range of MIC results, the MIC50 (the lowest concentration that inhibits 50% of the isolates), and MIC90 values (the lowest concentration that inhibits 90% of the isolates), were obtained. Additionally, the percentage of isolates categorised as susceptible, intermediate and resistant was tentatively assessed. Because there are no CLSI-approved MIC cutoff values for veterinary Mycoplasma species, CLSI veterinary breakpoints for other respiratory bovine pathogens were used (VET01S) (CLSI, 2015), when available. Additionally, all isolates were tested for antimicrobial susceptibility using the commercially available Sensititre bovine/porcine MIC plates (Thermo Fisher Scientific), with dried twofold dilutions of the following antimicrobial agents on round-bottom 96-well plates: ampicillin, ceftiofur, chlortetracycline, clindamycin, danofloxacin, enrofloxacin, florfenicol, gentamycin, neomycin, oxytetracycline, penicillin, spectinomycin, sulphadimethoxine, tiamulin, tilmicosin, trimethoprim-sulphamethoxazole, tulathromycin, and tylosin tartrate. The cell wall active antimicrobial agents ampicillin, penicillin and ceftiofur were not expected to show activity against mycoplasmas, and thus served as negative controls. Similarly, sulphadimethoxine and trimethoprim-sulphamethoxazole were regarded as negative controls. A standard dilution of culture was added to each well so that 0.5  103–105 CCU per ml were delivered in a final 50 ml volume of filtered Mycoplasma broth supplemented with horse serum (20%), yeast extract (1%), and 7.5% alamarBlue. Plates were sealed, incubated, and read as described above for the in-house prepared MIC plates. To confirm that the inocula were within the acceptable range of CCU per ml, immediately after inoculation, the viable M. bovis concentrations were determined by preparing 10-fold dilutions of the cultures in filtered Mycoplasma broth supplemented with horse serum (20%), yeast extract (1%), and 5% alamarBlue without antimicrobial agents in 96-well microplates. The plates were incubated for 48 h at 37  C and observed for a colour change (red, indicating growth). The number of CCU was calculated using the most probable number (MPN) dilution technique (Blodgett, 2010). Each MIC run included the M. bovis type culture strain ATCC 25523 to verify whether MIC values were obtained matching with previously published values. Additionally, the standard quality control strains for broth microdilution susceptibility tests Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were included in order to validate the results obtained; the strains were

Table 1 MIC50 (the lowest concentration that inhibits 50% of the isolates) and MIC90 values (mg/ml) for respiratory, mastitis and arthritis isolates of Mycoplasma bovis collected from 2008 till 2014, determined by using the in-house prepared MIC plates. Antimicrobial agent

Enrofloxacin Erythromycin Oxytetracycline Tilmicosin Tulathromycin Tylosin

Origin of isolates Lung (n = 56)

Milk (n = 27)

Synovial fluid (n = 12)

MIC50

MIC90

MIC50

MIC90

MIC50

MIC90

0.25 128 4 512 8a,b 64

0.25 256 4 1024 1024 >256

0.25 64 4 256 0.5a 32

1 128 8 512 4 >256

0.25 128 8 256 1b 32

0.5 256 8 512 2 >256

Comparing median antimicrobial activities (MIC50) for isolates of different origin (lung, milk, synovial fluid), only for those values marked with the same superscript statistically significant (p < 0.05) differences were observed.

4

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7

tested using the same broth with alamarBlue as used for the M. bovis isolates. 2.3. Statistical analysis Statistical analysis to determine whether there was a significant (p < 0.05) difference between the median antimicrobial activity (MIC50) for isolates of different origin (lung, milk, synovial fluid) was carried out using the Wilcoxon rank-sum (the Mann–Whitney two-sample) test. 3. Results MIC results obtained with the in-house prepared MIC plates are shown in Fig. 1; when available, CLSI breakpoints for other bovine respiratory pathogens are indicated on the x-axis. No colour change had been observed for the negative controls (no culture and no antimicrobial agent). The positive growth controls (with culture and no antimicrobial agent) changed colour from blue to red, indicating no antimicrobial effects of the solvents used on the growth of M. bovis. Additionally, MIC values obtained for the control E. coli and S. aureus ATCC strains were within the respected accepted quality control ranges of the CLSI (2015), indicating the antimicrobials used were effective under the testing conditions and the dilutions were correctly prepared. MIC values of enrofloxacin (0.25 mg/ml), oxytetracycline (0.5 mg/ml), tilmicosin (0.5 mg/ml) and tylosin (0.25 mg/ml) obtained for the M. bovis type culture strain ATCC 25523 matched with the ranges encountered in previously published studies applying broth microdilution tests (Ter Laak et al., 1993; Rosenbusch et al., 2005; Kawai et al., 2014; Sulyok et al., 2014). MIC values of tulathromycin for ATCC 25523 were not available in literature. All isolates had high MIC values of erythromycin (16 mg/ml). Also MIC values of tilmicosin (64 mg/ml) were high, with the exception of four isolates from milk having a MIC value of 0.5 mg/ml. The distribution of MICs of tylosin showed clustering around 32 mg/ml (34% of the isolates) and >256 mg/ml (31% of the isolates), and a distinctive population of isolates with MICs of 0.125 mg/ml (6% of the isolates). The distribution of MICs of tulathromycin was u-shaped, ranging from 0.5 to >1024 mg/ml, with 50% of the isolates yielding MICs of 1 mg/ml. For oxytetracycline, 41% of the isolates yielded a MIC of 4 mg/ml and 42% of 8 mg/ml. Enrofloxacin was found to be the most active compound, showing the lowest MIC values; the MIC50 and MIC90 values were 0.25 mg/ml and 0.5 mg/ml, respectively. In Table 1 the MIC results are presented grouped per isolate origin. Most MIC values did not differ between groups. Only for tulathromycin a significant difference in activity was seen for isolates of different origin; the MIC50 for respiratory isolates was higher than for both mastitis isolates and arthritis isolates. MIC results obtained with the commercial “Sensititre” MIC plates are summarized in Table 2. Only the results of the active antimicrobial agents, being aminoglycosides, clindamycin, florfenicol, fluoroquinolones, macrolides, and tetracyclines are given. The negative controls (ampicillin, penicillin, ceftiofur, sulphadimethoxine and trimethoprim-sulphamethoxazole) did not demonstrate activity against the M. bovis field isolates. Results of tiamulin, a pleuromutilin antibiotic, are not included since it is not registered for treatment of cattle. The MIC values found ranged from 0.125 to >2 mg/ml for enrofloxacin, 0.125 to >1 mg/ml for danofloxacin, 1 to 8 mg/ml for gentamicin, 4 to >32 mg/ml for neomycin, 8 to >64mg/ml for spectinomycin, both for chlortetracycline and oxytetracycline from 0.5 to >8 mg/ml, 4 to >64mg/ml for tilmicosin, 1 to >64 mg/ml for tulathromycin, and 0.5 to >32 mg/ ml for tylosin. The fluoroquinolones were found to be the most active compounds in vitro; the MIC50 and MIC90 values of both

enrofloxacin and danofloxacin were 0.25 mg/ml and 0.5 mg/ml, respectively. MIC values obtained for the control E. coli and S. aureus ATCC strains were within the accepted quality control ranges of the CLSI (2015) and those obtained for M. bovis ATCC 25523 matched with values reported in previous studies using broth microdilution tests (Ter Laak et al., 1993; Rosenbusch et al., 2005; Kawai et al., 2014; Sulyok et al., 2014). 4. Discussion We tested the antimicrobial susceptibility of a collection of 95 M. bovis isolates from clinical samples in the Netherlands, following a previously described broth microdilution procedure using the colorimetric reagent alamarBlue both with in-house prepared MIC plates and with commercially available plates. The in-house prepared plates contained large ranges of dilutions, of only antimicrobial agents approved to treat BRD in the Netherlands, to get a more detailed picture of the distributions of MIC values of these drugs. The commercial plates contained more antimicrobial agents, in smaller dilution ranges, but still including MIC breakpoints established by the CLSI for other bovine respiratory pathogens. In addition to collect information on the susceptibility for other potential effective antimicrobial agents, these plates were tested because of their ease of use and therefore, their potential in a routine diagnostic laboratory, providing a method to determine the agents that are most likely to be effective treatment options. Comparing the MIC results obtained with the in-house prepared MIC plates and the commercial plates showed equivalent MIC50 and MIC90 values of enrofloxacin, oxytetracycline, tilmicosin, tulathromycin, and tylosin. Since the highest concentrations of the antimicrobial agents tested with the commercial plates were lower than the concentrations of the respected agents tested with the in-house prepared plates, the exact MIC values could not always be compared. Results of the present study are in agreement with recent reports from other countries, as can be drawn from Table 2; only studies that tested isolates collected from 2000 onwards were included. MIC values of oxytetracycline appeared to be lower than recently reported by others. Isolates grouped by origin (lung, milk, synovial fluid) showed similar antimicrobial susceptibility levels, except for tulathromycin. The higher MIC50 of tulathromycin for respiratory M. bovis isolates (p < 0.05) can probably be explained by the frequent use of tulathromycin in case of respiratory infections. Tulathromycin is not registered to treat mastitis nor arthritis. Soehnlen et al. (2011) also found no significant difference in susceptibility levels for enrofloxacin, florfenicol, oxytetracycline, and tetracycline between quarter milk isolates and lung isolates. They only found a statistically significant difference for spectinomycin, with higher MICs for the milk isolates. Ayling et al. (2014), although based on small numbers, found higher MIC values of clindamycin, lincomycin, spectinomycin, tulathromycin, and tylosin for isolates from mastitis cases than for respiratory isolates, and hypothesized this could suggest that older animals have been exposed to more antimicrobial agents. In the Netherlands it is not common practice to treat M. bovis mastitis with antibiotics, so in the Netherlands the situation might be different. Since breakpoints have not yet been determined for M. bovis, in several publications CLSI interpretative criteria for other BRD pathogens (Mannheimia haemolytica,Pasteurella multocida, and Histophilus somni) have been used (CLSI, 2015). The conditions for reaching therapeutic concentrations in situ are theoretically equivalent (Gautier-Bouchardon et al., 2014). Doing so, determined by using the in-house prepared MIC plates, 95% of the isolates yielded MIC values higher than the tilmicosin threshold, indicating

Table 2 MIC50 (the lowest concentration that inhibits 50% of the isolates) and MIC90 values (mg/ml) for respiratory, mastitis and arthritis isolates of Mycoplasma bovis collected from 2008 till 2014, determined in this study by using the inhouse prepared MIC plates (BMD-1) and commercially available Sensititre bovine/porcine MIC plates (Thermo Fisher Scientific) (BMD-2) and a summary of recent literature reports testing isolates from 2000 onwards (A to K). Studya

Present study

Present study

A

B

C

D

E

F

G

G

H

H

I

J

K

Methodb Country

BMD-1 the Netherlands Respiratory, milk, arthritis

BMD-2 the Netherlands Respiratory, milk, arthritis

BMD China

BMD Great Britain

BMD Hungary

Agar dilution France

BMD Japan

Agar dilution Japan

BMD United States

BMD United States

BMD United States

E-test Canada

Respiratory, milk, arthritis

Respiratoryg

Respiratory

Milk

Respiratory

Milk

Respiratory

BMDc, E-testd Israel, importedf Respiratory, milk, arthritis

BMD Europe

Respiratory

BMDc, E-testd Israel, domestice Respiratory, milk

Respiratoryh

Respiratory, milk, arthritis, ear

n = 95

n = 93

n = 32

n = 45

n = 35

n = 46

n = 30

n = 29

n = 151

n = 33

n = 17

n = 18

n = 63

Respiratory, milk, arthritis, ear, eye, unknown n = 223

2008–2014

2008–2014

2011–2013

2004–2009

2010–2013

2010–2012

not specified

2008–2009

2007–2008

2007–2008

2005–2007

2005–2007

2004–2006

2002–2003

2001–2003

MIC50

MIC90

MIC50

MIC50

MIC90

MIC50

MIC50

MIC90

MIC50

MIC50

MIC90

MIC50

MIC50

MIC50

MIC90

MIC50

MIC90

MIC50

MIC50

MIC90

MIC50

MIC90

4 32 8

8 >32 >64

4

8

4

6

3

6

8

>32

256

256

>64

>64

2

8

>256

8

2

>1024

>1024

>1024

2

4

2

>1021

0.25 0.25

0.5 0.5

0.125

0.5

0.5 0.25

32 8

0.156 0.156

0.312 0.312

0.5 0.5

0.5 0.5

0.25 0.5

1 2

0.2

0.2

0.16 0.16

0.63 0.32

0.32 0.32

1.25 1.25

0.25

0.5

0.19

0.25

0.25

>16

0.5

256

8

>32

0.19

>256

256 128

128

128

>32 >32 >128 >32

128

32

>32 >32 2 8

>256

>64 >64 >32

32 16

>256

>64 1 32

128

2

4

2

4

8

32

8 4

>8 4

8

32

Origin of isolates

Number of isolates Year of isolation

Fluoroquinolones Enrofloxacin 0.25 Danofloxacin

MIC90

0.5

Lincosamide Clindamycin Macrolides Erythromycin Tilmicosin Tulathromycin Tylosin

128 512 1 32

>512 1024 1024 >256

Amphenicol Florfenicol Tetracyclines Chlortetracycline Oxytetracycline

4

8

MIC90

MIC90

128

>128 128 >64

>128 128 >64

4

8

8

8

64

64

>32

>32

0.5 0.5

MIC90

0.5 1

>128

>128

512 >512

>512 >512

64

128

128

128

>3.2

MIC90

MIC90

>3.2 128

>128

2

128

8

128

1

8

4

32

64

128 128

256 128

MIC90

4

4

6.4

6.4

4

8

2

4

32 64

>32 >128

1

4

4 2

16 16

>64

a A, Kong et al., 2016; B, Ayling et al., 2014; C, Sulyok et al., 2014; D, Gautier-Bouchardon et al., 2014; E, Kawai et al., 2014; F, Uemura et al., 2010; G, Soehnlen et al., 2011; H, Gerchman et al., 2009; I, Godinho, 2008; J, Rosenbusch et al., 2005; K, Francoz et al., 2005. b BMD, broth microdilution. c Enrofloxacin, danofloxacin, tilmicosin, tulathromycin, tylosin, oxytetracycline by BMD. d Gentamicin, spectinomycin by the E-test. e From domestic Israeli cattle. f From imported calves in quarantine. g One isolate originating from a lymph node. h From non-treated bovine.

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7

Antimicrobial MIC50 agents Aminoglycosides Gentamicin Neomycin Spectinomycin

n = 58

5

6

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7

resistance (by using the commercial Plates 94%). For oxytetracycline, this percentage was 50% (versus 6% by using commercial plates), for tulathromycin 27% (30%), and for enrofloxacin 4% (3%). For erythromycin and tylosin, there are no CLSI-approved breakpoints available for bovine respiratory pathogens, but based on their MIC50 values it is very likely that both agents are not effective at therapeutic doses, corresponding with experiences from the field. However, it should be stressed that a MIC distribution reflects the potency of a particular agent against a specific pathogen, and cannot predict the efficacy of an antimicrobial therapy. Additional factors, such as pharmacokinetic and—dynamic drug parameters, the commencement of the therapy, dosages, the presence of other bacterial agents as well as host characteristics also play an important role and should be taken into consideration. Although CLSI breakpoints are valid only for the stated organisms and only when using standardized CLSI methods, neither of these being available for M. bovis, the high MIC values observed for macrolides in our study as well as in recent publications indicate that they are losing their efficacy on M. bovis. In the Netherlands, erythromycin, tilmicosin, and tylosin are macrolides of first choice for treatment of calves with BRD caused by Mycoplasma species. The high MIC values found for erythromycin support publications stating that M. bovis is intrinsically resistant to erythromycin (Giguère, 2013). Therefore, its use in treating BRD caused by M. bovis should no longer be recommended. MIC values of oxytetracycline, also an antimicrobial of first choice, appear to concentrate around the breakpoint for other respiratory bovine pathogens. Therefore, it is difficult to conclude on the likely therapeutic effectiveness of oxytetracycline. The second choice antimicrobial for treatment of M. bovis-related pneumonia according to the Dutch guideline, tulathromycin, showed more or less two distinct populations with 52% (64% using the commercial MIC plates) of the isolates being characterized by a MIC value of 1 mg/ml and 26% (29%) of 128 mg/ml. Based upon the CLSI resistance breakpoint for other BRD-causing pathogens of 64 mg/ml, 27% (30%) of the isolates would be regarded as resistant. However, Godinho et al. (2005) reported that tulathromycin was effective in the treatment of calves with BRD following experimental infection with a M. bovis strain with a MIC value of >64 mg/ml. For enrofloxacin, together with difloxacin a third choice of antimicrobial agent for treatment of M. bovisrelated pneumonia, only 4% (3%) of the isolates are resistant based upon the CLSI resistance breakpoint for other BRD-causing pathogens, indicating that it is likely to be the most effective antimicrobial therapy of M. bovis infection. Several authors observed a significant increasing trend in MIC values of antimicrobial agents considered to be active on M. bovis in a relatively short period of time. Gautier-Bouchardon et al. (2014) determined the susceptibility of 27 and 46 M. bovis isolates obtained in 1978–1979 and in 2010–2012, respectively, from 73 distinct BRD outbreaks in young cattle all over France by using an agar dilution method. They reported a decrease in susceptibility for eight of the 12 antimicrobial agents tested. The MIC50 values for tylosin, tilmicosin, tulathromycin and spectinomycin, had increased from 2 to >64, 2 to >128, 16 to >128 and 4 to >64 mg/ml. For enrofloxacin, danofloxacin, marbofloxacin and oxytetracycline the MIC50 values had increased from 0.25 to 0.5, 0.25 to 0.5, 0.5 to 1, 32 to >32 mg/ml, respectively. No differences were observed for gamithromycin, tildipirosin, florfenicol and valnemulin with MIC50 values of 128, 128, 8, <0.03 mg/ml, respectively. Ayling et al. (2014) who tested 45 epidemiologically unrelated M. bovis isolates from cattle with respiratory, mastitis, and arthritis in Great Britain (by broth microdilution) also showed that the MIC values for the majority of the antimicrobial agents in their testpanel were high, and with the exception of the lincosamides appeared to have increased during the period 2004–2009. For lincomycin and

clindamycin, the MIC50 values had decreased from 8 to 1 mg/ml and >32 to 0.25 mg/ml, respectively, whereas for chloramphenicol, florfenicol, oxytetracycline, danofloxacin, enrofloxacin and marbofloxacin, the MIC50 values had increased. The highest increase had been observed for oxytetracycline, from 1 to 32 mg/ml. While the MIC50 values increased only one dilution, the MIC90 values for danofloxacin, enrofloxacin and marbofloxacin showed an increase from 0.25, 0.25 and 1 mg/ml in 2004 to 8, >32 and 32 mg/ml, respectively, in 2009. 5. Conclusion This study has shown that M. bovis strains recently isolated in the Netherlands are characterized by relatively high MIC values for antimicrobial agents that, until now, have been recommended for treating pneumonia caused by Mycoplasma species, both by using in-house prepared MIC plates and commercial MIC plates. This indicates that the choice of effective antimicrobial agents is becoming limited. These results are in agreement with recent publications from other countries that report increasing trends in MIC values of antimicrobial agents considered to be active on M. bovis in a relatively short period of time. Based on CLSI interpretative criteria for other respiratory bovine pathogens, fluoroquinolones (third choice) appeared to be the most efficacious in inhibiting M. bovis growth, followed by tulathromycin (second choice) and oxytetracycline (first choice). The highest MIC values were obtained for the remaining first choice antimicrobial agents, erythromycin, tilmicosin, and tylosin. Therefore, their use might no longer be recommended for treating M. bovis infection. Although in vitro susceptibility tests do not necessarily correspond to in vivo results, and additional factors play a role, they provide a method of determining the agents that are most likely to be effective treatment options. In order to use antimicrobial agents prudently and specifically, targeted susceptibility testing should be performed prior to application of antimicrobial agents. Future studies should be done on determining M. bovis specific clinical breakpoints, standardization of methods to determine MIC values as well as molecular studies on detection of antimicrobial resistance mechanisms of M. bovis isolates to develop PCR assays for determining resistance. Conflict of interest None to declare. Acknowledgements The research was part of the 1Health4Food—Dutch BRD Diagnostics Consortium. In this project also participate Adriaan Antonis, Tjeerd Kimman, Fimme Jan van der Wal, Thomas Hagenaars (Central Veterinary Institute, Wageningen UR, The Netherlands) and Theo Lam (GD Animal Health, Deventer, the Netherlands). The consortium is financed by the Dutch Ministry of Economic Affairs, the former Product Board for Livestock, Meat and Eggs, MSD Animal Health, the VanDrie Group, and Denkavit. References Ayling, R.D., Baker, S.E., Peek, M.L., Simon, A.J., Nicholas, R.A., 2000. Comparison of in vitro activity of danofloxacin florfenicol, oxytetracycline, spectinomycin and tilmicosin against recent field isolates of Mycoplasma bovis. Vet. Rec. 146, 745– 747. Ayling, R.D., Rosales, R.S., Barden, G., Gosney, F.L., 2014. Changes in antimicrobial susceptibility of Mycoplasma bovis isolates from Great Britain. Vet. Rec. 175, 486. Blodgett, R., 2010. Bacteriological analytical manual on-line. Appendix B, Most Probable Number from Serial Dilutions, . http://www.fda.gov/Food/ FoodScienceResearch/LaboratoryMethods/ucm109656.

A. Heuvelink et al. / Veterinary Microbiology 189 (2016) 1–7 Bürki, S., Frey, J., Pilo, P., 2015. Virulence: persistence and dissemination of Mycoplasma bovis. Vet. Microbiol. 179, 15–22. CLSI, 2015. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. second informational supplement, CLSI Document VET01S. 3rd ed. Clinical and Laboratory Standards Institute, Wayne, PA, USA. Francoz, D., Fortin, M., Fecteau, G., Messier, S., 2005. Determination of Mycoplasma bovis susceptibilities against six antimicrobial agents using the E test method. Vet. Microbiol 105, 57–64. Gautier-Bouchardon, A.V., Ferré, S., Le Grand, D., Paoli, A., Gay, E., Poumarat, F., 2014. Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30 years in France. PLoS One 9, e87672. Gerchman, I., Levisohn, S., Mikula, I., Lysnyansky, I., 2009. In vitro antimicrobial susceptibility of Mycoplasma bovis isolated in Israel from local and imported cattle. Vet. Microbiol. 137, 268–275. Giguère, S., 2013. Antimicrobial therapy of selected bacterial infections. In: Giguère, S., Prescott, J.F., Dowling, P.M. (Eds.), Antimicrobial Therapy in Veterinary Medicine. Wiley-Blackwell, pp. 421–430. Godinho, K.S., Rae, A., Windsor, G.D., Tilt, N., Rowan, T.G., Sunderland, S.J., 2005. Efficacy of tulathromycin in the treatment of bovine respiratory disease associated with induced Mycoplasma bovis infections in young dairy calves. Vet. Ther. 6, 96–112. Godinho, K.S., 2008. Susceptibility testing of tulathromycin: interpretative breakpoints and susceptibility of field isolates. Vet. Microbiol. 129, 426–432. Hannan, P.C., 2000. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species: international research programme on comparative mycoplasmology. Vet. Res. 31, 373–395. Hirose, K., Kobayashi, H., Ito, N., Kawasaki, Y., Zako, M., Kotani, K., Ogawa, H., Sato, H., 2003. Isolation of Mycoplasmas from nasal swabs of calves affected with respiratory diseases and antimicrobial susceptibility of their isolates. J. Vet. Med. B Infect. Dis. Vet. Public Health 50, 347–351.

7

Kawai, K., Higuchi, H., Iwano, H., Iwakuma, A., Onda, K., Sato, R., Hayashi, T., Nagahata, H., Oshida, T., 2014. Antimicrobial susceptibilities of Mycoplasma isolated from bovine mastitis in Japan. Anim. Sci. J. 85, 96–99. Kong, L.C., Gao, D., Jia, B.Y., Wang, Z., Gao, Y.H., Pei, Z.H., Liu, S.M., Xin, J.Q., Ma, H.X., 2016. Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China. J. Vet. Med. Sci. 78, 293–296. Rosenbusch, R.F., Kinyon, J.M., Apley, M., Funk, N.D., Smith, S., Hoffman, L.J., 2005. In vitro antimicrobial inhibition profiles of Mycoplasma bovis isolates recovered from various regions of the United States from 2002 to 2003. J. Vet. Diagn. Invest. 17, 436–441. Sachse, K., Salam, H.S., Diller, R., Schubert, E., Hoffmann, B., Hotzel, H., 2010. Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bovis infection in cattle herds with mastitis and respiratory disease. Vet. J. 186, 299– 303. Siugzdaite, J., Gabinaitiene, A., Kerziene, S., 2012. Susceptibility of Mycoplasma bovis field isolates to antimicrobial agents. Vet. Med. (Praha) 57, 575–582. Soehnlen, M.K., Kunze, M.E., Karunathilake, K.E., Henwood, B.M., Kariyawasam, S., Wolfgang, D.R., Jayarao, B.M., 2011. In vitro antimicrobial inhibition of Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory using flow cytometry and a broth microdilution method. J. Vet. Diagn. Invest. 23, 547–551. Sulyok, K.M., Kreizinger, Z., Fekete, L., Hrivnák, V., Magyar, T., Jánosi, S., Schweitzer, N., Turcsányi, I., Makrai, L., Erdélyi, K., Gyuranecz, M., 2014. Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe. BMC Vet. Res. 10, 256. Ter Laak, E.A., Noordergraaf, J.H., Verschure, M.H., 1993. Susceptibilities of Mycoplasma bovis Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. Antimicrob. Agents Chemother. 37, 317–321. Uemura, R., Sueyoshi, M., Nagatomo, H., 2010. Antimicrobial susceptibilities of four species of Mycoplasma isolated in 2008 and 2009 from cattle in Japan. J. Vet. Med. Sci. 72, 1661–1663.