In vitro susceptibility of contagious ovine digital dermatitis associated Treponema spp. isolates to antimicrobial agents in the UK.

BACKGROUND: Contagious ovine digital dermatitis (CODD) is an important cause of infectious lameness in sheep in the UK and Ireland and has a severe impact on the welfare of affected individuals. The three treponemal phylogroups Treponema medium/Treponema vincentii-like, Treponema phagedenis-like and Treponema pedis spirochaetes have been associated with clinical CODD lesions and are considered to be a necessary cause of disease. There are scant data on the antimicrobial susceptibility of the treponemes cultured from CODD lesions.
OBJECTIVE: The aim of this study was to determine in vitro the miniumum inhibitory concentration/ minimum bactericidal concentration (MIC/MBC) of antimicrobials used in the sheep industry for isolates of the three CODD associated treponeme phylogroups T. medium/T. vincentii-like, T. phagedenis-like and T. pedis. ANIMALS: Twenty treponeme isolates; from 19 sheep with clinical CODD lesions.
METHODS: A microdilution method was used to determine in vitro the MIC/MBC of 10 antimicrobial agents for 20 treponeme isolates (five T. medium/T. vincentii-like, 10 T. phagedenis-like and five T. pedis). The antimicrobials tested were penicillin G, amoxicillin, oxytetracycline, tilmicosin, lincomycin, spectinomycin, tylosin, tildipirosin, tulathromycin and gamithromycin.
RESULTS: The treponeme isolates tested showed low MICs and MBCs to all 10 antimicrobials tested. They were most susceptible to gamithromycin and tildipirosin (MIC90: 0.0469 mg/L), and were least susceptible to lincomycin, spectinomycin and oxytetracycline (MIC90: 48 mg/L, 24 mg/L and 3 mg/L, respectively).
CONCLUSIONS: These data are comparable to in vitro antimicrobial susceptibility data for treponemes cultured from bovine digital dermatitis lesions. Dependent on local licensing, penicillin and tilmicosin appear to be the best candidates for future in vivo studies.

Introduction

Contagious ovine digital dermatitis (CODD) is a cause of infectious lameness in sheep in the UK and Ireland and has been shown to have a severe impact on the welfare of affected individuals.1 Recent surveys have shown that CODD may affect approximately 35% of flocks in the UK; while on‐farm prevalence is typically low, it may affect up to 50% of the flock at any one time.1

Information about the microbial flora of CODD lesions is limited, although the bovine digital dermatitis (BDD) associated treponemes Treponema medium/T. vincentii‐like, Treponema phagedenis‐like and Treponema pedis are currently considered to be a necessary cause of disease.1 The recent characterization of treponemes associated with CODD demonstrated the presence of at least one BDD phylotype present in all 58 lesions studied, whereas these were totally absent from all healthy sheep foot tissues.2

There has been a wide range of empirically chosen treatments employed in clinical cases such as parenteral oxytetracycline and topical tylosin,3 with only one randomized controlled trial conducted comparing parenteral amoxicillin and simultaneous topical chlortetracycline with topical chlortetracycline alone.4

As with CODD, the successful treatment of BDD has remained problematic with many farms adopting management and control strategies as opposed to affecting a cure.5 In order to inform the development of effective therapeutic strategies for clinical cases of CODD, a greater understanding is required of the susceptibility of the treponemes found in CODD lesions to antimicrobials currently available for use in sheep.

The aim of this study was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of a panel of antimicrobials for representatives from each of the three treponeme phylogroups cultured as pure isolates from clinical CODD lesions.

Materials and methods

Bacterial isolates

Twenty treponeme isolates from CODD lesions from 19 sheep from six farms in England, Wales and Northern Ireland were used (Table 1). Included were five isolates from the T. medium/T. vincentii‐like group, 10 isolates from the T. phagedenis‐like group and five isolates from the T. pedis group.

Table 1

Treponemes tested for susceptibility to antimicrobial agents

Strain no.	Strain	Isolation date	UK Location	Nearest related organisma
1	G1F7C5	07/2013	Conwy	Treponema medium/Treponema vincentii
2	G1F9C27	07/2013	Conwy	Treponema medium/Treponema vincentii
3	G1OV11	08/2009	Gloucester	Treponema medium/Treponema vincentii
4	G2S2R	02/2009	Cheshire	Treponema medium/Treponema vincentii
5	ST27	07/2013	Conwy	Treponema medium/Treponema vincentii
6	3F2	02/2014	Anglesey	Treponema phagedenis
7	C2F	06/2009	Gloucestershire	Treponema phagedenis
8	G2S4F	02/2009	Cheshire	Treponema phagedenis
9	G2F3C12	07/2013	Conwy	Treponema phagedenis
10	G13F3	02/2014	Denbighshire	Treponema phagedenis
11	G2SL1	05/2014	Anglesey	Treponema phagedenis
12	G23F1	02/2014	Anglesey	Treponema phagedenis
13	S3R2	03/2009	Cheshire	Treponema phagedenis
14	G2ST24	07/2013	Conwy	Treponema phagedenis
15	C2R	06/2009	Gloucestershire	Treponema phagedenis
16	Ovine (G179)	2000	Northern Ireland	Treponema pedis
17	G3ST1	07/2014	Shrewsbury	Treponema pedis
18	G3S45	07/2014	Shrewsbury	Treponema pedis
19	G3T1	07/2014	Shrewsbury	Treponema pedis
20	G3T7	07/2014	Shrewsbury	Treponema pedis

As determined by 16S rRNA gene phylogenetic analysis.

In vitro antimicrobial susceptibility testing

The MIC/MBC for each antimicrobial was determined using a broth microdilution method as previously described.6 One small adjustment was made to the method such that prior to inoculation, bacterial counts were assessed by determining the optical density (OD) of the cultures using spectrometry with wavelength set at 540 nm. The T. medium/T. vincentii‐like cultures had an OD of 0.25; the T. phagedenis‐like cultures had an OD of 0.43 and the T. pedis cultures had an OD of 0.37. This corresponds to 8.75 × 107, 1.14 × 108 and 2.69 × 108 treponemal organisms/mL, respectively.6 At this time point, cultures were assessed by phase contrast microscopy to determine that the cultures were alive, of the correct morphology and lacking contaminants. The antimicrobials and their test ranges are listed in Table 2.

Table 2

Minimum inhibitory concentrations (MIC) of 10 antimicrobial agents tested against contagious ovine digital dermatitis associated treponemes

Strain no.a	Median MIC (mg/L)
	Penicillin	Amoxicillin	Oxytetracycline	Tilmicosin	Lincomycin	Spectinomycin	Tildipirosin	Tulathromycin	Gamithromycin	Tylosin
1	0.0750	0.5625	3	0.0703	24	12	0.0234	0.2930	0.0469	0.0469
2	0.0375	0.2813	3	0.0234	48	24	0.0234	0.2930	0.0234	0.0234
3	0.0375	0.5625	1.5	0.0117	48	12	0.0469	1.1719	0.0469	0.0234
4	0.0750	0.2813	1.5	0.0234	24	12	0.0469	0.2930	0.0234	0.0469
5	0.0750	0.5625	3	0.0117	24	24	0.0469	1.1719	0.0469	0.0469
6	0.0375	0.1406	0.75	0.0234	24	12	0.0469	0.5859	0.0469	0.0469
7	0.0188	0.1406	0.75	0.0469	12	12	0.0938	0.5859	0.0117	0.0469
8	0.0750	0.2813	0.375	0.0094	12	12	0.0469	0.2930	0.0117	0.0469
9	0.0375	0.1406	0.75	0.1875	24	12	0.0469	0.5859	0.0234	0.1875
10	0.0188	0.1406	0.75	0.0234	6	12	0.0117	0.5859	0.0469	0.0469
11	0.0188	0.2813	0.75	0.0234	3	6	0.0938	0.1465	0.0117	0.0469
12	0.0750	0.1181	0.375	0.0059	6	3	0.0469	0.2930	0.0029	0.0059
13	0.0375	0.1181	0.375	0.375	12	12	0.0234	0.5859	0.0938	0.1875
14	0.0750	0.1181	0.375	0.0938	48	12	0.0234	0.5859	0.0938	0.0234
15	0.0188	0.1406	1.5	0.1875	96	24	0.0469	0.2930	0.0234	0.0938
16	0.0750	0.2813	1.5	0.0234	24	24	0.0234	0.5859	0.0234	0.0938
17	0.0375	0.5625	6	0.0234	48	24	0.0234	0.5859	0.0469	0.0469
18	0.0750	0.5625	3	0.0234	48	12	0.0469	0.5859	0.0234	0.0469
19	0.0750	0.2813	1.5	0.0117	48	24	0.0469	0.5859	0.0234	0.0938
20	0.0750	0.5625	6	0.0117	96	24	0.0234	1.1719	0.0234	0.0938
MIC90 b	0.0750	0.5625	3	0.1875	48	24	0.0469	1.1719	0.0469	0.0938

Isolates 1–5 are Treponema medium/Treponema vincentii‐like BDD spirochaetes with antibiotic test ranges (μg/L) of: penicillin G 0.75–0.0059; amoxicillin 2.25–0.0176; oxytetracycline 12–0.0938; tilmicosin 0.375–0.0029; lincomycin 192–1.5; spectinomycin 48–0.375 (Sigma‐Aldrich; Dorset, UK); tildipirosin 0.75–0.0059 (Zuprevo, MSD Animal Health; Milton Keynes, UK); tulathromycin 9.375–0.0732 (Draxxin, Zoetis UK Limited; London, UK); gamithromycin 0.188–0.0015 (Merial LLC; Duluth, Georgia, USA); and tylosin 0.375–0.0029 (Sigma‐Aldrich).

Isolates 6–15 are Treponema phagedenis‐like CODD spirochaetes and isolates 16–20 are Treponema pedis CODD spirochaetes with test ranges the same as those for Treponema medium/Treponema vincentii‐like BDD spirochaetes.

Cumulative susceptibility results across all treponemes tested are expressed as MIC90, the concentration at which 90% of CODD‐associated treponemes were inhibited.

Determination of MICs

The MIC for each antimicrobial was taken as the lowest concentration of antimicrobial that prevented growth in the wells observed at the same time points.6 Cell growth was determined by comparison of the absorbance measurement immediately after inoculation with the absorbance measured at the late exponential/early stationary phase. All of the absorbance measurements were at 540 nm using a Multiskan microtitre plate reader (Thermo Scientific; Hampshire, UK). The MIC values were taken as the median of three repeat experiments, performed on different days.

Determination of MBCs

The MBC for each antimicrobial was determined as previously described.6

Determination of MIC90 and MBC90

The cumulative inhibitory/bactericidal concentration for each antimicrobial tested across all the treponeme isolates was expressed as the concentration at which 90% of CODD‐associated treponemes were inhibited from growing (MIC90) or killed (MBC90).

Statistical analysis

Differences in MICs between the three different treponeme phylogroups were assessed using the Kruskal–Wallis test. The Kruskal–Wallis test and the nonparametric equality‐of‐medians test were used to compare the MICs for penicillin, oxytetracycline, lincomycin and spectinomycin with previous data,6 and for amoxicillin and gamithromycin.7 All statistical analyses were conducted using Stata IC 13 (Stata Corp; College Station, TX, USA) and statistical significance was set at P < 0.05.

Study validation

The MIC microdilution method described in this study was validated by comparing the results produced from four antimicrobials (penicillin, oxytetracycline, lincomycin and erythromycin) incubated with two control microorganisms T. phagedenis biotype Reiter and T. phagedenis‐like T320A against results previously obtained using a macrodilution method8 and also results obtained using a similar microdilution method.6, 7 The data were also compared statistically using linear regression.

Results

Antimicrobial susceptibilities of CODD‐associated treponemes

The individual MIC/MBCs of the antimicrobial agents to each treponeme isolate are summarized in Tables 2 and 3; in this study all isolates showed low MIC/MBCs to all of the antimicrobials tested. Using Table 2, all treponeme groups were most susceptible to gamithromycin and tildipirosin, and least susceptible to lincomycin, spectinomycin and oxytetracycline. The MIC90 for the other five antimicrobials were all relatively low, being <1.0 μg/mL. No bimodal distributions were identified.

Table 3

Minimum bactericidal concentrations (MBC) of 10 antimicrobial agents tested against contagious ovine digital dermatitis associated treponemes

Strain no.a	Median MBC (mg/L)
	Penicillin	Amoxicillin	Oxytetracycline	Tilmicosin	Lincomycin	Spectinomycin	Tildipirosin	Tulathromycin	Gamithromycin	Tylosin
1	0.0750	0.5625	6	0.0938	48	12	0.0469	0.5859	0.0469	0.0938
2	0.0750	0.5625	3	0.0469	96	24	0.0469	0.2930	0.0469	0.0938
3	0.0750	1.1250	3	0.0234	48	24	0.0469	1.1719	0.0469	0.0938
4	0.0750	0.5625	3	0.0469	48	24	0.0469	0.5859	0.0234	0.0938
5	0.0750	0.5625	6	0.0234	24	24	0.0469	1.1719	0.0469	0.0938
6	0.0375	0.5625	6	0.0469	48	12	0.0938	0.5859	0.0469	0.3750
7	0.0375	0.5625	3	0.1875	24	24	0.0938	0.5859	0.0234	0.0938
8	0.0750	0.5625	3	0.1875	24	12	0.0469	1.1719	0.0234	0.3750
9	0.0750	0.5625	6	0.1875	96	24	0.0938	0.1172	0.0469	0.1875
10	0.0375	0.2813	6	0.0234	96	12	0.0234	0.5859	0.0469	0.0469
11	0.0189	0.2813	3	0.0234	48	6	0.3750	0.5859	0.0117	0.0469
12	0.0750	0.2813	1.5	0.0117	24	6	0.0469	0.5859	0.0117	0.0117
13	0.0375	0.1181	0.75	0.1875	24	12	0.0234	1.1719	0.0938	0.1875
14	0.0750	0.5625	0.375	0.0938	48	12	0.0234	0.5859	0.0938	0.0938
15	0.0188	0.2813	0.75	0.1875	24	12	0.0469	0.5859	0.0234	0.0938
16	0.0750	0.5625	3	0.0234	48	24	0.0469	0.5859	0.0469	0.0938
17	0.0750	0.5625	6	0.0234	96	24	0.0469	0.5859	0.0469	0.0938
18	0.0750	0.5625	6	0.0469	48	12	0.0469	1.1719	0.0234	0.0938
19	0.0750	0.5625	6	0.0469	48	24	0.0469	1.1719	0.0469	0.0938
20	0.0750	0.5625	6	0.0234	96	24	0.0234	1.1719	0.0469	0.0938
MBC90 b	0.0750	0.5625	6	0.1875	96	24	0.0938	1.1719	0.0469	0.1875

1–5, Treponema medium/Treponema vincentii‐like; 6–15, Treponema phagedenis‐like; 16–20, Treponema pedis.

Cumulative susceptibility results across all treponemes tested are expressed as MBC90, the concentration at which 90% of CODD‐associated treponemes were killed.

Variation in MIC across the different treponeme phylogroups

There was no significant difference in MIC values between the three different phylogroups for five of the seven macrolides (P = 0.2), with phylogroup differences for lincomycin and spectinomycin approaching significance (P = 0.05). Whilst there was no significant difference between phylogroups in the case of penicillin MIC (P = 0.1), in the case of amoxicillin and oxytetracycline, T. phagedenis‐like bacteria were more susceptible compared to T. medium/T. vincentii‐like and T. pedis (P = 0.002 and P = 0.001, respectively).

Comparisons with data from previous studies

The MICs for penicillin, oxytetracycline, lincomycin, spectinomycin, amoxicillin and gamithromycin for the 20 isolates investigated here, were not significantly different to those previously reported.6, 7

The comparison described matched the previous results in all cases except for one antimicrobial (oxytetracycline), which was different by one serial dilution when compared with the macrodilution method.8 Linear regression for these comparisons showed strong correlations (R = 0.99 P < 0.004) indicating the efficacy and reproducibility of this microdilution method.

Discussion

Study validation and comparisons with data from previous studies

The methodological validations described reinforce the comparable nature of these current data with previous studies. Comparisons of these current data for penicillin, oxytetracycline, lincomycin and spectinomycin with previous studies do not reveal any statistically significant differences.6, 7 Therefore, these current data make a valuable contribution to the available data on the in vitro antimicrobial susceptibility of these treponemes.

Antimicrobial use in sheep with CODD

All of the isolates in this study were susceptible (in vitro) to all the antimicrobials tested, with gamithromycin, tildipirosin, penicillin, tylosin and tilmicosin demonstrating the lowest MICs and MBCs. This susceptibility, however, may not necessarily be reflected in vivo. To date, there have been very few robust in vivo studies examining effective treatment. In two clinical studies4, 9 systemic amoxicillin together with topical chlortetracycline was found to have a clinical cure rate in clinical cases of CODD of approximately 80%. Anecdotally, systemic tilmicosin was also proposed to be an effective treatment for sheep with CODD10 and systemic oxytetracycline together with a tylosin footbath were considered to be an effective preventative method.3

Currently, no antimicrobial product has a license for CODD in the UK. The antimicrobials studied here were selected to include antimicrobials that already have a license for sheep (penicillin, amoxicillin, oxytetracycline and tilmicosin) together with those that in the authors’ experience are already used (off label) in the sheep industry. Therefore, given this context and these data as a whole, penicillin and tilmicosin would appear to be the most likely candidates for future in vivo studies.

This study provides the first detailed examination of the in vitro antimicrobial susceptibilities of all three associated phylogroups of treponemes cultured from CODD lesions to antimicrobials. As such, these data provide important in vitro information on antimicrobials currently used to treat this disease and should help inform researchers planning further in vivo studies when considering which products to include.