Abstract
The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.
Introduction
Mycoplasma synoviae infections in poultry occur worldwide and most prominently in commercial layer chickens, turkeys and broilers, although their incidence in breeding stock has decreased considerably due to eradication programmes. M. synoviae can induce infectious synovitis in chickens and turkeys, and subsequently can cause considerable economic loss to the poultry industry. This mycoplasma species has also been associated with respiratory disease and condemnations due to airsacculitis (Kleven et al., Citation1972; Mohammed et al., Citation1987; Stipkovits & Kempf, Citation1996; Kleven, Citation2003), although subclinical infections seem predominant.
Economic losses caused by M. synoviae with an affinity for the respiratory tract or for joints can be reduced by antibiotic treatment at the beginning of infection. However, the antibiotic susceptibility profiles of the mycoplasma strains involved should be determined if treatment efficacy is to be maximized. During the past decades a number of studies describing the in vitro antibiotic susceptibility of M. synoviae strains have been published (Jordan & Knight, Citation1984; Bradbury et al., Citation1994; Jordan & Horrocks, Citation1996; Wang et al., Citation2001; Cerda et al., Citation2002). Nevertheless, recent reports on the minimum inhibitory concentrations (MICs) of M. synoviae isolates from European countries are scarce. In a more recent study by Dufour-Gesbert et al. (Citation2006), M. synoviae strains with intermediate susceptibility for enrofloxacin were described; however, the study only involved M. synoviae isolates originating from the respiratory tract without pathological lesions.
The emergence of M. synoviae infectious synovitis in both chickens and turkeys in the Netherlands (Landman & Feberwee, Citation2001; Van Beek et al., Citation2002) with associated economic losses prompted the need for efficacious antibiotic treatments. In order to provide the optimum choice for antibiotic therapy, knowledge of the susceptibility profiles of available compounds was needed. The present study was carried out due to the scarcity of recent data on antibiotic susceptibility of M. synoviae isolates from European countries and the complete lack of data on amyloidogenic M. synoviae strains. Thus, the antibiotic susceptibility of Dutch M. synoviae field isolates originating from both joint lesions and the respiratory tract of commercial poultry was determined against difloxacin, enrofloxacin, doxyxycline, tylosin and tilmicosin.
Materials and Methods
M. synoviae strains, culture and identification
M. synoviae type strain WVU 1853e (ATCC 25204) was used for control purposes and 17 M. synoviae field isolates gathered during the period 2000 to 2004 were included. The isolates were obtained from the trachea (n=14) and joints (n=3) of commercial poultry using sterile cotton swabs, which were dipped beforehand in Mycoplasma Experience (ME) broth (Mycoplasma Experience, Reigate, UK). The swabs were plated on ME agar, which was incubated under aerobic conditions at 37°C and examined for colonies every 2 days up to 28 days. If colony growth occurred, one separate colony was plated out on a fresh ME agar and approximately 2×0.5 cm2 agar with positive clones was transferred to 10 ml ME broth and incubated at 37°C as described above. Subsequently, 1 ml ME broth was used for identification of M. synoviae by real-time polymerase chain reaction (PCR). The real-time M. synoviae PCR was performed following the M. gallisepticum PCR protocol described by Mekkes & Feberwee (Citation2005). The forward primer 5′-GAG AAG CAA AAT AGT GAT ATC A and the reverse primer 5′-CAG TCG TCT CCG AAG TTA ACA (Genbank accession number X52083) were used. These primers amplify a 211 base pair sequence from the 16S ribosomal RNA gene of M. synoviae. Finally, isolates were freeze-dried and stored at 2 to 8°C as seed culture pending analysis.
Preparation of M. synoviae inocula
Each freeze-dried culture was suspended in 1 ml distilled water and transferred to 25 ml ME broth, which was then incubated at 37°C. When a colour change from red to yellow occurred, the ME broth was divided into portions of 1 ml each and stored at − 70°C. Throughout the experiment, ME broth was used without bacterial inhibitors.
To determine the concentration of the M. synoviae cultures, frozen M. synoviae stock was thawed and 10-fold dilutions were prepared in ME broth. Tubes were incubated for 14 days at 37°C and were observed daily for colour change. The final M. synoviae concentration was determined as the highest dilution where a colour change was observed and expressed as colour changing units per ml (CCU/ml) according to the Spearman–Karber method for quantal data (Finney, Citation1952). Inocula with a concentration of 103 to 105 CCU/ml were used for the broth microdilution test.
Tested antibiotics and quality control of ME medium
The antibiotics tested were enrofloxacin (lot number 258623a; Bayer B.V., Mijdrecht, the Netherlands), difloxacin HCl (lot number 62-114-GS Profarmaco lot number 0103; Solvay Duphar B.V., Weesp, the Netherlands), doxycycline (lot number 022H0119; Sigma Aldrich, Zwijndrecht, the Netherlands), tylosin tartrate (lot number 085H1016; Sigma-Aldrich) and tilmicosin (lot number 072KE5; Elanco Animal Health, Nieuwegein, the Netherlands).
To prepare the stock solutions of enrofloxacin 0.1 M NaOH was used, while for the stock solution of tilmicosin 0.1 M HCl was necessary. Therefore, the influence of these solvents on the pH of the ME medium as well as in the same dilutions as those used later on in the broth micro-dilution test was assessed. The induced pH variation did not exceed 0.1, making corrections unnecessary.
Also, beforehand, a potential effect of the media and growth conditions used was studied by determining the MICs for two reference strains (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213) in ME broth. The accepted quality control ranges of MICs for the tested antibiotics are published by the National Committee for Clinical Laboratory Standards (Citation2002), now the Clinical and Laboratory Standards Institute (CLSI).
Preparation of microtitre plates and the broth microdilution test
Broth microdilution tests were performed in two different studies. First a pilot study, which included three joint isolates, was performed once. In a second study, susceptibility tests were performed in duplicate and included 14 isolates originating from the respiratory tract. In both studies, M. synoviae ATCC 25204 (WVU 1853) was also added as a control strain and to determine the reproducibility of the test. Susceptibility tests were performed following the guidelines described by Hannan (Citation2000). In short, the wells of each microtitre plate (eight rows of 12 wells) (Greiner; Omnilabo B.V., Breda, the Netherlands) were filled with 50 µl ME broth. For each isolate–antibiotic combination, two rows were used. In the first row a two-fold dilution of the antibiotic was prepared, whereas in the second row a two-fold dilution of the solvent was prepared. This second row was used as a growth control row prepared to exclude potential antimicrobial effects of the solvents on the growth of M. synoviae. Plates were sealed after preparation and stored at − 70oC pending the test, which was carried out within 4 weeks.
Immediately before inoculation, sufficient volumes of M. synoviae culture were thawed and diluted to a concentration of 104 CCU/ml. As the inoculum, 50 µl/well of each M. synoviae culture was used. Immediately after inoculation the concentration of viable mycoplasmas was determined as described above.
The final test concentrations of all antibiotics were 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 and 0.015 µg/ml (excluding the rows used for control for M. synoviae growth). Plates were sealed and incubated aerobically at 37°C. They were examined after 1, 2, 3, 4, 7, 8, 9, 10, 11 and 14 days of incubation. The initial MIC was defined as the lowest antibiotic concentration to show no change in colour when the colour of the mycoplasma growth control changed (Hannan, Citation2000). The final MIC was read when no further colour change was observed (Bradbury et al., Citation1994; Hannan, Citation2000).
As the interpretation of the MIC depends on the reproducibility of the test, this was assessed using M. synoviae WVU 1853. The MIC breakpoints of avian mycoplasmas for enrofloxacin, doxycycline and tylosin were based on those determined by Hannan (1997, 2000). As the MIC breakpoints for avian mycoplasma of difloxacin and tilmicosin have not been given in the literature, those derived from the NCCLS were used. They are based on the MIC for other pathogens in other animal species ().
Table 1. MIC breakpoints of the tested antibiotics
Results and Discussion
Reports on the MIC profiles of M. synoviae field isolates are scarce both in Europe and elsewhere. During the past decade only one recent study has focused on the assessment of MICs of European M. synoviae field isolates (Dufour-Gesbert et al., Citation2006).
The antibiotics tested in the present study were chosen based on their availability in the Netherlands for commercial meat turkeys because outbreaks of M. synoviae-associated joint pathology and concomitant economic losses were increasing in such birds from 2000 to 2004. In contrast to Dufour-Gesbert et al. (Citation2006) we also examined the susceptibility of M. synoviae for difloxacin and tilmicosin. Another difference was that we included M. synoviae isolates from joints in addition to isolates from the respiratory tract. A third difference was that our study included M. synoviae isolates from different types of poultry (i.e. layers, meat type breeders and meat-turkeys).
Results of the initial MICs and results read at days 7 and 14 of the M. synoviae WVU 1853 () showed a variation of one to two dilutions for all antibiotics except for tilmicosin at day 7 and for doxycycline at day 14, where a difference of more than two dilutions was found to be 0.06 to 0.5 and <0.015 to 0.25, respectively. The occurrence of MIC profiles differing in more than two dilutions using mycoplasma inocula ranging from 103 to 105 CCU/ml was described previously by Hannan (Citation2000) for Mycoplasma hyopneumoniae and tylosin. Differences in mycoplasma growth were put forward as a possible explanation. In our reproducibility study only in two cases did MIC profiles differ in more than two dilutions, so we concluded that the overall reproducibility of the microdilution test was good.
Table 2. Reproducibility of the test based on the initial MIC and antibiotic susceptibility profiles at day 7 and day 14 (µg/ml) of M. synoviae WVU 1853
The initial and final MICs of all isolates are outlined in . The MIC results of the control E. coli and S. aureus ATCC strains were within the accepted quality control range of the NCCLS, indicating that the growth conditions had no effect on the potency of the antibiotics. After thawing the plates, no change in broth colour due to possible pH changes was observed. The change in colour noted in the control rows was therefore attributed to mycoplasma growth. The results showed that all strains were susceptible to tylosin, doxycycline and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin; for these isolates, initial MICs varying from 2 to 4 µg/ml were found. Dufour-Gesbert et al. (Citation2006) recently found MICs of enrofloxacin varying from 0.5 to 1 µg/ml for French M. synoviae field isolates and concluded that there was intermediate susceptibility to this antibiotic. Moreover, these strains also showed intermediate susceptibility to difloxacin.
Table 3. Initial and final MIC (µg/ml) values and corresponding antibiotic sensitivity profiles of all M. synoviae isolates tested
In general, the reproducibility of the duplicate MIC values of the respiratory strains (both initial and final MICs) was good, with 53/70 (76%) of initial MICs and 69/70 (99%) of final MICs showing no more than one dilution step difference. As the test reproducibility of enrofloxacin, difloxacin, tylosin, doxycycline and tilmicosin was acceptable—that is, within the range of one to two dilutions ()—explanation for this variation could be, as already mentioned, a difference in growth rate of the same strain in each test despite a standardized inoculum of 103 to 105 CCU/ml (Hannan, Citation2000). Although the occurrence of a mixture of strains with different susceptibility could theoretically have occurred, this is not considered likely because all isolates were cloned prior to testing.
Several times the final MICs for enrofloxacin and difloxacin differed from that of the initial MICs (), including changes of profile from sensitive to intermediate (difloxacin) or resistant (enrofloxacin and difloxacin). There was little difference between initial and final MICs of most isolates, which is consistent with a mycoplasmacidal antibiotic. However, in two cases (isolates 2000.05 and 2003.08) there was a large difference between the initial and final MIC for enrofloxacin changing from sensitive to resistant. The difference between the initial and final MICs could not be attributed to slow mycoplasma growth as the initial MIC was read at day 2 and day 3, indicating rapid growth of these isolates. An alternative explanation is that resistance is developed in situ as described earlier by Hannan et al. (Citation1989), who showed a similar effect for M. hyopneumoniae versus gentamicin.
The last representative study that reported full susceptibility to the newer quinolones (enrofloxacin and danofloxacin) for all M. synoviae isolates analysed dates from 1997 (Hannan et al, 1997). However, five years later Stanley et al. (Citation2001) reported the occurrence of reduced effectivity of enrofloxacin for the treatment of an experimentally induced M. synoviae infection with a field isolate. The results of our study and the paper by Dufour-Gesbert et al. (Citation2006) suggest an increase in the occurrence of quinolone-resistant M. synoviae field isolates.
Descriptions of mechanisms involved in quinolone resistance in veterinary medicine are scarce (Reinhardt et al., Citation2002; Le Carrou et al., Citation2006). Reinhardt et al. (Citation2002) showed the presence of alterations on the four target genes encoding DNA gyrase and topoisomerase IV in M. gallisepticum enrofloxacin mutants. The studies of Le Carrou et al. (Citation2006) showed a significant increase of the resistance level of M. synoviae isolates under experimental conditions after a therapeutic dose of enrofloxacin. The development of resistance was associated with a parC gene substitution. This was also found for quinolone-resistant mutants of M. gallisepticum (Reinhardt et al., Citation2002).
In the Netherlands, clinically relevant M. synoviae isolates were not detected for decades until recently, when amyloid-inducing joint isolates causing considerable economic losses were observed (Landman & Feberwee, Citation2001; Van Beek et al., Citation2002). This prompted a substantial increase in antibiotic treatments as such treatments may reduce the economic impact of the disease if given at the beginning of infection. The increased use of antibiotics may at least explain in part the occurrence of antibiotic-resistant M. synoviae isolates. It is well known that there is a close relationship between antibiotic misuse and bacterial resistance. The use of sub-inhibitory concentrations but also the long-term use of therapeutic doses can contribute to the induction of antibiotic resistance. In an experimental M. synoviae infection of hens, Le Carrou et al. (Citation2006) demonstrated a significant increase in the resistance to enrofloxacin of five re-isolated M. synoviae clones after a second therapeutic treatment with this drug. Although in vitro resistance of M. synoviae strains to tylosin and oxytetracycline has been reported (Hannan et al., Citation1997; Cerda et al., Citation2002; Gautier-Bouchardon et al. Citation2002), all strains tested in our study were still susceptible to tylosin and doxycycline.
Based on the MIC breakpoints for M. synoviae, therapeutic failures are more likely to occur with quinolones than with oxytetracycline or tylosin. The MIC values found in this study further emphasize that M. synoviae infections should not be treated with quinolones. The application of quinolones may not only contribute to the selection of M. synoviae-resistant strains but may also affect other avian pathogens including zoonotic bacteria such as Campylobacte spp. Periodic assessment of the MICs of M. synoviae field isolates, and other bacteria in general, may contribute to a more rational and efficient use of antibiotics in the treatment of M. synoviae-affected flocks.
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