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1 In vitro Evaluation of Various Antimicrobials against Field Mycoplasma

2 gallisepticum and Mycoplasma synoviae Isolates in Egypt

3 Running title: Antimicrobial Susceptibilities of Avian Mycoplasmas

4 Marwa I Abd El-Hamida#, Naglaa FS Awadb, Usama H. Abo-Shamac#*, Yousreya

5 MHd, Mahmoud A Abdel-Rahmane, Helal F. Hetta f,g Adel M Abdelazizb

6 aDepartment of Microbiology, Faculty of Veterinary Medicine, Zagazig University,

7 Zagazig 44519, Egypt

8 bDepartment of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig

9 University, Zagazig 44519, Egypt

10 cDepartment of Microbiology and Immunology, Faculty of Veterinary Medicine, Sohag

11 University, Sohag 82524, Egypt

12 dDepartment of Mycoplasma Research, Animal Health Research Institute, Giza 12622,

13 Egypt

14 eDepartment of Bacteriology, Animal Health Research Institute, Mansoura branch, Egypt

15 fDepartment of Medical Microbiology & Immunology, Faculty of Medicine, Assiut

16 University, Assiut, Egypt

17 gDepartment of Internal Medicine, University of Cincinnati College of Medicine,

18 Cincinnati, OH, USA

19 * Correspondence to:

20 Usama H. Abo-Shama, PhD

21 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Sohag

22 University, Sohag 82524, Egypt.

.CC-BY 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/726000doi: bioRxiv preprint

https://doi.org/10.1101/726000

http://creativecommons.org/licenses/by/4.0/

2

23 Phone: +20 01067344157

24 E-mail: [email protected]

25 [email protected]

26 # These authors contributed equally to this work.

27 Abstract

28 Among many avian mycoplasmas, Mycoplasma gallisepticum (MG) and

29 Mycoplasma synoviae (MS) are recognized as the main etiological agents of respiratory

30 diseases and infectious synovitis in chickens and turkeys causing tremendous economic

31 losses worldwide. Therefore, proper treatment is promoted for the control of these

32 diseases. This study was the first in Egypt to evaluate the in vitro efficacy of various

33 antimicrobials against field MG and MS isolates recovered from chicken and turkey

34 flocks using both conventional microdilution and quantitative real-time PCR (qRT- PCR)

35 assays. Totally, 47 mycoplasma isolates were recovered from 160 collected tracheal

36 samples (29.4%). Of these, 44 MG (27.5%) and 3 MS (1.9%) were identified using

37 conventional and molecular assays. The in vitro susceptibilities of 4 representative

38 mycoplasma isolates (3 MG and one MS) to 8 antibiotics and 4 essential oils were

39 investigated. The tested isolates showed various susceptibilities to tested antimicrobials.

40 Toldin CRD, followed by clove, cumin and cinnamon oils were commonly effective

41 against both MG and MS clinical isolates with MIC values ranging from 0.49 to 15.63

42 µg/mL. Similarly, tylvalosin was the most active antibiotic against MG and MS isolates

43 with the lowest MIC values (0.015-0.03 µg/mL). DNA copies of both MG mgc2 and MS

44 vlhA genes were markedly decreased upon treatment with majority of tested

45 antimicrobials confirming their effectiveness as was also evaluated by conventional MIC


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46 results. In conclusion, Toldin CRD and tylvalosin were found to be the most effective

47 antimicrobials in this study, which will contribute in controlling avian mycoplasma

48 infections.

49 Keywords: Mycoplasma species; Chicken; Turkey; Tylvalosin; Toldin; Microbroth

50 dilution; qRT-PCR.

51 Author Summary

52 Avian mycoplasmosis is considered as one of the most prominent economic problems in

53 the commercial poultry industry worldwide. Antimicrobial therapy is the most effective

54 tool for treatment of mycoplasmas. Owing to the side effects of antibiotics and the

55 development of resistance to the currently used drugs, an increased emphasis on the use

56 of alternative antimicrobials is of utmost importance. Here, we evaluate the in vitro

57 inhibitory effects of some essential oils and various commercial antibiotics against

58 Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) field isolates using

59 micro-broth dilution method and qRT- PCR assays. We found that toldin CRD, followed

60 by clove, cumin and cinnamon oils were effective against both MG and MS clinical

61 isolates. Similarly, tylvalosin was the most active antibiotic against MG and MS isolates.

62 We also found that DNA copies of both MG mgc2 and MS vlhA genes were markedly

63 decreased upon treatment with majority of tested antimicrobials. Our study provides new

64 insights into the control of avian mycoplasma infections.

65 Introduction

66 Avian mycoplasmosis has been deemed as one of the most prominent economic

67 problems in the commercial poultry industry all over the world. More than 20 species of


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68 genus Mycoplasma are known to infect avian hosts with Mycoplasma gallisepticum (MG)

69 and Mycoplasma synoviae (MS) being the most clinically relevant mycoplasmas [1].

70 Mycoplasma species are transmitted horizontally by susceptible birds through

71 direct or indirect contact with contaminated surfaces [2] and vertically from dam to

72 offspring through eggs [3].

73 In geographic localities where mycoplasmosis is enzootic, programs for

74 controlling their infections are the most practical ways to decrease the resulted economic

75 losses. Antimicrobial therapy remains the most prompt and effective tool for treatment of

76 mycoplasmas in poultry farms. Basically, MG and MS have shown in vitro and in vivo

77 susceptibilities to several antimicrobials such as macrolides, tetracyclines and quinolones

78 [4,5], but they are resistant to penicillins or other antibiotic inhibitors of cell wall

79 synthesis because of their cell-wall deficiency.

80 Veterinarians usually ignore the side effects of synthetic antibiotics while

81 recommending the therapy for avian mycoplasmosis. Moreover, the development of

82 resistance to currently used drugs [1,6] incorporating with their high costs increased

83 emphasis on the use of new and alternative antimicrobials.

84 An approach for the development of new antimicrobials mainly from plant origin

85 offers a great contribution in the fight against mycoplasma infections. To our knowledge,

86 few researchers reported the efficacy of medicinal plants against different species of

87 mycoplasma [7,8].

88 Performance of phenotypic antimicrobial susceptibility tests for mycoplasmas is

89 time-consuming and requires special techniques. Therefore, quantitative real time PCR

90 (qRT-PCR) assay has been recently developed as a faster, easier, sensitive and more


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91 specific method compared with conventional ones used in clinical microbiology

92 laboratories [9].

93 Based on the above background, this study was conducted to evaluate the in vitro

94 inhibitory effects of some essential oils and various commercial antibiotics against MG

95 and MS field isolates using micro-broth dilution method and qRT- PCR assays.

96 Materials and methods

97 Sample collection and study area

98 The current study was carried out from January 2017 to December 2018. Three

99 commercial broiler chicken flocks located in Faiyum, Giza and Sharkia Governorates,

100 Egypt as well as one turkey flock in El Dakhleya Governorate, Egypt were examined.

101 The birds from Giza (50), Sharkia (50) and El Dakhleya (10) were suffering from

102 respiratory manifestations in the form of conjunctivitis, nasal and ocular discharges,

103 sneezing, coughing, rales, gasping, depression and weakness, while those from Faiyum

104 were suffering from both respiratory disease and synovitis. All of these flocks were not

105 vaccinated against any mycoplasma diseases. From each flock, tracheal swabs from all

106 diseased birds were aseptically collected and transferred in test tubes with Frey’s broth

107 medium as a transport medium to the laboratory until being subjected to MG and MS

108 isolation and identification.

109 Ethical statement

110 All animal protocols were approved by the Institutional Animal Care and Use Committee

111 (IACUC), Faculty of Veterinary Medicine, Zagazig University.

112 Isolation and identification of M. gallisepticum and M. synoviae


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113 After incubating the broth media for 3-5 days, the broth culture was streaked onto

114 Frey’s agar medium and incubated for 21 days. Inoculated broth and agar media were

115 incubated under microaerophilic humid conditions (10% CO2) at 37ºC. When the growth

116 of the colonies was obtained, digitonin test was performed. Identification of MG and MS

117 was made by a combination of conventional biochemical methods (glucose fermentation,

118 arginine deamination and film and spot formation tests) and serologically via growth

119 inhibition test using specific antisera [10]. Further molecular identification of both MG

120 and MS was carried out as was described previously by PCR amplifications of mgc2 and

121 vlhA genes, respectively [11,12]. Known positive MG and MS reference strains as well as

122 negative controls were included in every set of PCR reactions. The sizes of expected

123 amplification products were 236-302 bp for mgc2 and 1.1 kbp for vlhA gene.

124 Mycoplasma reference strains

125 Mycoplasma gallisepticum and Mycoplasma synoviae reference strains with

126 accession numbers of HQ591357 and KT943466 were used for all PCR assays.

127 Natural substances

128 The essential oils of clove (Syzygium aromaticum), cinnamon (Cinnamomum

129 zeylanicum) and cumin (Cuminum cyminum) were purchased from National Research

130 Centre, Egypt. Additionally, Toldin CRD (B.NO, 00101) which is a prophylactic and

131 therapeutic agent for chronic respiratory disease (CRD) complex in poultry was kindly

132 supplied by Dawa International for Pharmaceutical Industries, Egypt. It contains many

133 active natural compounds such as aliumcepa, ginger, cinnamon, oregano, circumin,

134 liqurice, anise oils and propolis.

135 Antibiotics


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136 The following 8 antibiotic agents of 5 different groups; macrolides,

137 fluoroquinolones, tetracyclines, lincosamide and pleuromutilins were used for the

138 susceptibility tests: tylvalosin (Pharmgate Animal Health Canada, Inc), tilmicosin

139 (ELANCO, Geneva), tylosin (ELANCO, USA), enrofloxacin (INVESA, Spain),

140 doxycycline (Oxoid, UK), chlortetracycline (Oxoid, UK), lincomycin (Oxoid, UK) and

141 tiamulin (Sandoz GmbH, Basel, Switzerland).

142 Antimicrobial susceptibility testing of mycoplasma isolates using microdilution and

143 qRT-PCR assays

144 The antimicrobial susceptibility rates of four representative mycoplasma field

145 isolates (one isolate from each flock) were determined by both conventional broth

146 microdilution [13] and qRT-PCR [14] assays.

147 Briefly, each tested antimicrobial substance was twofold serially diluted starting

148 from a concentration of 1024 µg /mL for each antibiotic and 125 µg/mL for each

149 essential oil. Mycoplasma isolates were also diluted to contain 103- 104 color changing

150 unit/0.2 mL. Consequently, 0.1 mL volume of each diluted substance was mixed with 0.1

151 mL of each diluted mycoplasma isolate in the 96-well microtiter plates. Growth controls

152 (tested field mycoplasma strains grown into broth medium without any tested

153 substances), sterility controls (broth medium without neither tested substances nor

154 mycoplasma inoculum) and pH control (broth medium adjusted to pH 6.8) were included

155 in each plate. The plates were sealed, incubated at 37° C and inspected at regular intervals

156 for any changes in the color indicator. Each experiment was performed in triplicate and

157 repeated twice to confirm the results. In accordance with the previously mentioned

158 guidelines [13], the initial MIC was read when the change of color in the broth was first


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159 observed in the growth control wells. The final MIC was read at the time when no further

160 color change in the wells containing antimicrobials was observed.

161 At 1st, 2nd, 3rd and 4th weeks after the incubation, 50 µL aliquots from each well

162 was harvested and subjected for determination of DNA loads by qRT-PCR using MG and

163 MS specific primers. The number of DNA copies at all intervals was determined using

164 standard curves prepared from tenfold serial dilution of genome copies of MG and MS

165 reference strains. After amplification, a final melting curve analysis was performed to

166 determine the presence or absence of non-specific amplification products. The inhibition

167 rates of all tested substances were calculated according to the following formula:

168 Inhibition rate (%) = [(average of DNA loads in control wells- DNA load in test well) /

169 average of DNA loads in control wells] x 100 [14]. The MIC of each tested substance

170 was defined as the lowest concentration causing 99% inhibition of mycoplasma field

171 isolates.

172 Interpretation of antimicrobial susceptibility results is hampered by lacking of the

173 official breakpoints. In these cases, interpreting the data of our study is based on values

174 previously used in other publications [8,15].

175 Results

176 Prevalence and characterization of avian M. gallisepticum and M. synoviae

177 Fried-egg-shaped pure colonies of cultured mycoplasmas were seen on Frey’s

178 agar medium. All the colonies were found sensitive to 1.5% digitonin, ensuring that they

179 were mycoplasmas.

180 The recovery rates of avian Mycoplasma species from 4 flocks located in different

181 localities are illustrated in Table 1. A total of 47 mycoplasma isolates out of 160 collected


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182 tracheal samples (29.4%) were identified biochemically into 2 groups. The first group

183 was positive for glucose fermentation test and negative for both arginine deamination and

184 film and spot formation tests, while the second group fermented glucose, did not

185 hydrolyse arginine and was positive for the film and spot formation test. According to

186 serological identification results using growth inhibition test, 44 (27.5%) and 3 (1.9%)

187 were inhibited by specific antisera to MG and MS, respectively. Moreover, PCR

188 confirmed the nucleic acid of all biochemically and serologically identified isolates with

189 the production of the predicted amplicons to MG and MS at 236-302 bpand 1.1 kbp,

190 respectively. The total recovery rate of Mycoplasma species from the examined chicken

191 and turkey flocks were 29.3% (44/150) and 30% (3/10), respectively. Cumulatively,

192 phenotypic and genotypic identification results confirmed that 41 isolates (27.3%) of 150

193 tracheal swabs from chicken flocks and 3 isolates (30%) of 10 tracheal swabs from the

194 turkey flock in El Dakhleyawere MG. In addition, MS were recovered from the chicken

195 flocks with a percentage of 2% (3/150).

196 In vitro antimicrobial activities of essential oils and antibiotics

197 Conventional broth microdilution assay

198 In the first part of the study, 4 selected mycoplasma isolates (3 MG and one MS)

199 representative for 4 flocks located in different localities were investigated. The in vitro

200 activities of 4 different essential oils and 8 antibiotics of 5 groups against these isolates

201 were detected by the microbroth dilution technique as illustrated in Tables 2-4. Herein,

202 MIC initial values were those used to evaluate the susceptibility of examined

203 mycoplasma isolates to different antimicrobials. The in vitro MIC results of selected

204 natural products revealed that clove, cinnamon, cumin and Toldin CRD essential oils


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205 were variously capable for inhibiting the growth of both MG and MS clinical isolates.

206 Toldin CRD was found to be the most effective natural substance against all tested

207 isolates with MIC values of 0.49- 0.98 µg/mL. Moreover, both clove and cumin oils had

208 excellent antimycoplasmal activities with MIC ranges of 0.49-1.95µg/mL and 0.98-3.91

209 µg/mL, respectively. On the other hand, cinnamon oil showed various activities from

210 excellent to good against mycoplasma isolates with MIC values of 1.95-15.63 µg/mL

211 with only one MG isolate showing an MIC value of 15.63 µg/mL.

212 The broadest ranges of MIC values were detected for antibiotics of the macrolide

213 group with MICs ranging from 0.015-0.25 µg/mL. Among the examined 3 macrolides

214 (tylvalosin, tilmicosin and tylosin), tylvalosin was found to be the most active antibiotic

215 in the examination with the lowest MIC values (0.015-0.03 µg/mL) against MG isolates

216 and 0.015 µg/mL against the MS isolate. Moreover, tilmicosin and tylosin demonstrated

217 also lower MICs against both MG and MS isolates (up to 0.06 µg/mL each). From the

218 pleuromutilins, the MIC values of tiamulin were low (0.06-0.5µg/mL). Among

219 tetracyclines, the MIC values of both doxycycline and chlortetracycline showed a wide

220 range (0.125 to 0.5 µg/mL and 0.5 to 16 µg/mL, respectively).

221 It was obviously noted that MS isolate was sensitive to all tested antibiotics. On the

222 other hand, all MG isolates were sensitive to tylvalosin, tilmicosin, tylosin, doxycycline,

223 lincomycin and tiamulin antibiotics and only 33.3% of the isolates were sensitive to each

224 of enrofloxacin and chlortetracycline.

225 A Comparison of initial and final MIC values showed zero to three-fold

226 differences for all antibiotics. Only in the case of 6 antibiotics (tylvalosin, tilmicosin,

227 doxycycline, chlortetracycline, lincomycin and tiamulin), initial and final MICs exhibited


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228 up to 3 fold differences among MG isolates. Values for initial and final MICs of

229 tylvalosin, tilmicosin, tylosin and enrofloxacin and tylvalosin, tilmicosin, tylosin and

230 tiamulin are the same for each of MG3 and MS isolates, respectively. Final MICs for

231 enrofloxacin and lincomycin differed from those of the initial MICs among each of MG2

232 and MS isolates, including changes of MIC profiles from sensitive to resistant.

233 Quantitative real-time PCR assay

234 In the second part of our work, we quantitatively assessed the mgc2 and vlh1

235 gene copies when MG and MS isolates were grown in the presence of all tested

236 antimicrobials at 1st, 2nd, 3rd and 4th weeks of incubation, respectively (Tables 2 and 4,

237 Figure 1).

238 Fig 1. This is the Fig 1 Title. Growth inhibition of MG1 (A), MG2 (B), MG3 (C) and

239 MS (D) grown into mycoplasma medium with various concentrations of tylvalosin at

240 1, 2, 3 and 4weeks of incubation.

241 The CT values of mycoplasma isolates in medium inoculated with tested

242 substances increased with increasing the efficacy of such antimicrobials, implying that

243 the mycoplasma loads decreased. A remarkable reduction in the DNA loads of both MG

244 mgc2 and MS vlh1 genes was observed following treatment with majority of the screened

245 substances (up to 0.0014 and 0.010, respectively). DNA copy numbers of both MG and

246 MS mgc2 and vlh1 gene copies were markedly decreased upon treatment with tylvalosin

247 (up to 0.0032). Therefore, a representative model of growth inhibition rates of MG and

248 MS isolates after exposure to various concentrations of tylvalosin at 1st, 2nd, 3th and 4th

249 weeks of incubation is shown in figure 1. Mycoplasma synoviae isolate had a rapid

250 growth pattern as MIC for tylvalosin was determined early at 2 weeks after incubation.


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251 The MIC value was 0.062 µg/mL at 2nd, 3rd and 4th weeks of incubation. All MG isolates

252 grew slowly as MICs couldn't be detected at 1st and 2nd weeks. For MG1 and MG3, the

253 MIC value was 0.031µg/mL at both 3rd and 4th weeks and for MG2, the MIC was 0.062

254 µg/mL at the 4th week of incubation. Based on this model, MIC values of other examined

255 antimicrobials were determined and shown in Tables 2 and 4.

256 Comparison between conventional broth microdilution and qRT-PCR assays for

257 MICs determination of tested antimicrobials

258 In Tables 2 and 4, a comparison between all MIC results obtained by conventional

259 broth microdilution and real time PCR methods was carried out. The MICs determined

260 by qRT-PCR assay were similar to the initial MICs by the conventional method for

261 lincomycin (median, 1.5 versus 1.5 µg/mL), enrofloxacin (median, 0.5 versus 0.5

262 µg/mL), tylosin (median,0.125 versus 0.06 µg/mL), doxycycline (median, 0.25 versus

263 0.19 µg/mL), tiamulin (median, 0.38 versus 0.19 µg/mL), clove oil (median, 0.74 versus

264 0.98 µg/mL) and toldin (median, 0.49 versus 0.74 µg/mL), but higher for

265 chlortetracycline (median, 4 versus 1.5 µg/mL), cinnamon (median, 3.91 versus 5.86

266 µg/mL) and cumin (median, 0.98 versus 1.47 µg/mL).

267 Discussion

268 Avian mycoplasmosis is one of the most serious infectious diseases caused by

269 Mycoplasma species, especially MG and MS. Appropriate treatment is recommended in

270 the control of these infections in order to minimize their economic losses. In vitro

271 antimicrobial sensitivity testing of avian Mycoplasma species has been carried out using

272 broth microdilution method. Meanwhile, we are facing the absence of researches about

273 the inhibitory effects of various antimicrobial substances on clinical MG and MS strains


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274 by the use of qRT-PCR as a sensitive method. Therefore, this is the first work that

275 evaluated the in vitro susceptibilities of MG and MS isolated from chicken and turkey

276 flocks in Egypt against various antibiotics and essential oils using both conventional

277 broth microdilution method and qRT-PCR.

278 In the present study, phenotypic and genotypic identification of the recovered

279 isolates revealed high isolation rates of Mycoplasma species from the cultured tracheal

280 swabs of the examined chicken (29.3%) and turkey (30%) flocks. Our findings are close

281 to those previously observed in other recent researches in Egypt, where high overall

282 recovery rates of Mycoplasma species from chicken tracheal swabs (55.65%) [16] and

283 from diseased turkey flocks (24%) [17] were recorded. Another research conducted

284 inEastern Algeria reported also a high isolation rate of Mycoplasma species from tracheas

285 of dead chickens (27%) [18]. The high mycoplasma prevalence rates in these investigated

286 flocks may refer to the already acknowledged vertical transmission characteristics of

287 mycoplasmas in addition to their horizontal transmission to the healthy susceptible birds,

288 which amplifies the disease incidence and the subsequent economic losses. In terms of

289 MG, the isolation rates from the diseased chickens and turkeys were 27.33 and 30%,

290 respectively. In this regard, MG was previously isolated from 40 and 20% of chicken and

291 turkey flocks, respectively [19]. Moreover, in other researches performed earlier, MG

292 prevalence rates from tracheal swabs collected from broilers and turkeys were 38.71%

293 [16] and 68% [20], respectively. With regards to MS, a lower incidence rate was recorded

294 from investigated chicken flocks (2%), although it could not be isolated previously from

295 broilers in Iran [21]. On the other hand, several researchers reported higher prevalence


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296 rates of MS from broiler chickens in Iran (31.25%) [22], Jordan (21.7%) [23] and Tehran

297 province (17.89%) [24].

298 From our findings, cinnamon, cumin, clove oil and Toldin CRD had various

299 antimycoplasmal activities from good to excellent with MIC values up to 0.49 µg/mL.

300 The potent activities observed for plant extracts against mycoplasma organisms may be

301 connected to the anatomical structure of mycoplasma which lacks a rigid cell wall. This

302 feature enables the plant extracts to diffuse into the mycoplasma microorganisms, thereby

303 causing inhibition of growth or death of mycoplasmas.

304 The extracts were analyzed by the previous established criteria which determines

305 that substances with MIC values < 10 μg/mL are considered to have an excellent

306 antibacterial activity, values between 10 and 100 μg/ mL are considered good, values

307 between 100 and 500 μg/mL are considered moderate, values between 500 and 1000

308 μg/mL are considered weak and the substances are considered inactive when MIC values

309 are above 1000 μg/mL [8].

310 Toldin CRD had the best activity with an MIC of 0.49-0.98 µg/mL for all

311 examined isolates. To our best knowledge, the antibacterial effects of Toldin CRD have

312 not been described yet. It is possible that, the strong antimycoplasmal activity of Toldin

313 CRD may be explained by the presence of many active compounds which are powerful

314 bacteriolytic agents such as aliumcepa, ginger, cinnamon, oregano, circumin, liqurice and

315 anise oils and propolis. The antibacterial activities of these compounds were identified

316 previously against a large variety of pathogenic bacteria [8,25].

317 Strong antimycoplasmal capacities were also determined for both clove and

318 cinnamon oils against both MG and MS isolates. In connection with previous researches


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319 performed on other Mycoplasma species, euganol; a major ingredient of the clove oil and

320 cinnamon possessed bactericidal activities against M. hominis in Czech Republic [26].

321 One of the most remarkable results of the present study was the pronounced antibacterial

322 property of cumin against screened mycoplasma isolates with an MIC value of 0.98-3.91

323 µg/mL. There are several studies affirmed that cumin exhibited a significant

324 antimicrobial activity against pathogenic bacteria including Bacillus subtilis, E. coli, S.

325 aureus, P. aeruginosa, K. pneumonea, Strept. feacalis and S. Typhimurium [27].

326 However, this is the first research that evaluates the antibacterial activity of cumin against

327 bacteria without cell wall like mycoplasma.

328 As our data show (Table 4), conventional microdilution results exhibited a

329 remarkable deviation when initial and final readings were compared. Only in the case of

330 6 antibiotics (tylvalosin, tilmicosin, doxycycline, chlortetracycline, lincomycin and

331 tiamulin), initial and final MICs exhibited up to 3 fold differences among MG isolates,

332 although it did not alter the interpretation of the data. On the other hand, final MICs for

333 enrofloxacin and lincomycin differed from those of the initial MICs among each of MG2

334 and MS isolates. The observed MIC differences of these antibiotics lead to the re-

335 categorization of these isolates from susceptible to resistant during interpretation of the

336 results. These results are consistent with a previous report carried out in Germany, where

337 the final MICs for enrofloxacin differed from that of the initial MICs including changes

338 of the profile from sensitive to resistant among most of MS isolates [4]. The

339 discrepancies may be attributed to the inactivation of the used antibiotics with the

340 continued incubation time [28] or the presence of a mixed colony in the field isolates

341 with a slower growing minor population [13,29]. Therefore, the initial MIC values are


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342 advised to be taken into consideration in the interpretation of the susceptibility results due

343 to these values compare the growth of each mycoplasma isolate in the presence and

344 absence of tested substances rather than determining the endpoint at a fixed time [13].

345 According to our results, various susceptibility rates of MG and MS isolates were

346 observed against the eight antibiotics. Macrolides proved to have good in vitro

347 effectiveness against both MG and MS isolates with lower MIC values ranging from

348 0.015-0.25 µg/mL. Lower MIC values for macrolides were also reported in previous

349 examinations [5,17] supporting the use of these antibiotics for treatment of mycoplasmas

350 infecting poultry in the field.

351 Among macrolides group, tylvalosin was the most effective drug against both MG

352 and MS isolates with an MIC range of 0.015-0.030 µg/mL. This finding is in a complete

353 agreement with other research results in Iran [30], UK [5] and Europe [15]. This could be

354 attributed to the recent introduction of tylvalosin as a newer antibiotic against MG and

355 MS isolates in Egypt. Our field experience supports this finding.

356 In the present investigation, DNA copies of both MG mgc2 and MS vlhA genes

357 were markedly decreased upon treatment with majority of the tested antimicrobials with

358 relevant MIC values determined by qRT-PCR assay confirming the good effectiveness of

359 these compounds. The MICs determined using qRT-PCR were almost similar to those

360 obtained by conventional microdilution method. Only, a previous report in Japan was

361 carried out to determine antibiotic susceptibility testing of M. genitalium by TaqMan 5'

362 nuclease qRT-PCR assay [14] . Recently, assessing the inhibitory effects of antibiotics

363 using qRT-PCR assay has been also described on common clinically relevant bacterial

364 species such as S. aureus, E. coli and Haemophilusinfluenzae in France [9].


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365 Conclusion

366 The current study showed that Toldin CRD and tylvalosin possessed strong in

367 vitro antimycoplasmal activities against both MG and MS field isolates. The MIC values

368 determined using qRT-PCR were similar to those obtained by the conventional

369 microdilution method. However, molecular detection of antimicrobial susceptibility using

370 qRT-PCR could be more sensitive than phenotypic methods.

371 Conflict of Interest: The authors declare that they have no conflict of interest.

372 Funding: This research did not receive any specific grant from funding agencies in the

373 public, commercial or not-for-profit sectors.

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Table 1 Prevalence and characterization of avian M. gallisepticum and M. synoviae in 4 flocks from different localities

MG: Mycoplasma gallisepticum, MS: Mycoplasma synoviae

Biochemical characterization Serological and molecular identificationFlock

No. Host LocalityNo. of

tracheal swabs

No. of positive mycoplasma isolates (%) Glucose

fermentation Arginine

deamination Film and spot

formationNo. of MG isolates (%)

No. of MS isolates (%)

1 Faiyum 50 3 (6) + - + 0 3 (6)2 Giza 50 25 (50) + - - 25 (50) 03

ChickenSharkia 50 16 (32) + - - 16 (32) 0

4 Turkey El-Dakhleya 10 3 (30) + - - 3 (30) 0Total 160 47 (29.4) 44 (27.5) 3 (1.9)


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Table 2In vitro antimicrobial activities of four essential oils against M. gallisepticum and M. synoviae isolates as determined by conventional broth microdilution and qRT-PCR methods

MIC values of essential oils (μg/mL)Clove Cinnamon Cumin ToldinCRD

Conventional Conventional Conventional Conventional

Strain ID or MIC range

Host Locality

I F qRT-PCR I F qRT-PCR I F qRT-PCR I FqRT-PCR

MG1 Chicken Giza 0.49 0.49 0.49 3.91 7.81 3.91 1.95 0.98 0.98 0.98 0.49 0.98MG2 Chicken Sharkia 0.98 0.49 0.98 7.81 1.95 3.91 0.98 0.98 0.98 0.49 0.49 0.49MG3 Turkey El Dakhleya 0.98 0.49 0.49 15.63 0.98 7.81 3.91 1.95 3.91 0.98 0.98 0.49MS Chicken Faiyum 1.95 0.98 3.91 1.95 0.98 1.95 0.98 0.98 0.49 0.49 0.49 0.49

MIC range - - 0.49-1.95 0.49-0.98 0.49-3.91 1.95-15.63 0.98-7.81 1.95-7.81 0.98-3.91 0.98-1.95 0.49-3. 91 0.49-0.98 0.49-0.98 0.49-0.98MG: Mycoplasma gallisepticum, MS: Mycoplasma synoviae, MIC: minimum inhibitory concentration, I: initial (profile), F: final,qRT-PCR: quantitative real time PCR.


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Table 3 Summary of initial and final MIC ranges (μg/mL) of the isolated M. gallisepticum and M. synoviae isolates with the suggested non-official breakpoints

S: susceptible, R: resistant

Antibiotic class Antibiotic agentNon-official breakpoints

[15]

Initial MIC range

Final MIC range

Tylvalosin S ≤ 0.5; R > 2 0.015-0.03 0.015-0.125Tilmicosin S ≤ 8; R ≥ 32 0.06-0.25 0.25-0.50Macrolides

Tylosin S ≤ 1; R ≥ 4 0.06 0.06-0.25Fluoroquinolones Enrofloxacin S ≤ 0.5; R ≥ 2 0.25-2 1-2

Doxycycline S ≤ 4; R ≥ 16 0.125-0.5 0.25-1Tetracyclines Chlortetracycline S ≤ 4; R ≥ 16 0.5-16 2-16Lincosamide Lincomycin S ≤ 2; R ≥ 8 0.25-2 2-8

Pleuromutilins Tiamulin S ≤ 8; R ≥ 16 0.06-0.5 0.5


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Table 4 Comparison between MICs of various antibiotics for M. gallisepticum and M. synoviae isolates as determined by conventional broth microdilution and qRT-PCR methods with their corresponding susceptibility patterns

MG: Mycoplasma gallisepticum, MS: Mycoplasma synoviae, MIC: Minimum inhibitory concentration, TVN: Tylvalosin, TIL: Tilmicosin, TYL: Tylosin, EFX: Enrofloxacin, DX: Doxycycline, CTC: Chlortetracycline, LCM: Lincomycin, TIA: Tiamulin, I: initial (profile), F: final,qRT-PCR: quantitative real time PCR, *: Final MIC profiles differing from the initial ones, S: susceptible, R: resistant

MIC values (μg/mL)TVN TIL TYL EFX DX CTC LCM TIA

Conventional Conventional Conventional Conventional Conventional Conventional Conventional ConventionalStrain

IDI F

qRT-PCR I F

qRT-PCR I F

qRT-PCR I F*

qRT-PCR I F

qRT-PCR I F

qRT-PCR I F*

qRT-PCR I F

qRT-PCR

MG1 0.015(S) 0.125 0.031 0.125

(S) 0.25 0.5 0.06(S) 0.25 0.125 0.25

(S) 1 0.5 0.125(S) 1 0.25 0.5

(S) 4 4 0.25(S) 2 0.25 0.125

(S) 0.5 0.25

MG2 0.03 (S) 0.125 0.062 0.06

(S) 0.5 0.5 0.06 (S) 0.25 0.25 0.5

(S)2

(R) 0.5 0.5(S) 1 0.5 16

(R) 16 16 2(S)

8(R) 1 0.06

(S) 0.5 0.5

MG3 0.015(S) 0.015 0.031 0.25

(S) 0.25 0.25 0.06(S) 0.06 0.125 2

(R) 2 1 0.125(S) 0.25 0.125 1

(S) 2 1 1(S) 2 2 0.25

(S) 0.5 0.125

MS 0.015(S) 0.015 0.062 0.25

(S) 0.25 0.5 0.06(S) 0.06 0.06 0.5

(S)2

(R) 0.5 0.25(S) 1 0.25 2

(S) 4 4 2(S)

8(R) 2 0.5

(S) 0.5 0.5


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466 FIGURE LEGEND

467 Fig. 1 Growth inhibition of MG1 (A), MG2 (B), MG3 (C) and MS (D) grown into

468 mycoplasma medium with various concentrations of tylvalosin at 1, 2, 3 and 4weeks of

469 incubation.

470

471

472

473

474

475


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