Skip to content

Advertisement

You're viewing the new version of our site. Please leave us feedback.

Learn more

BMC Veterinary Research

Open Access

Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary

  • Dénes Grózner1,
  • Zsuzsa Kreizinger1,
  • Kinga M. Sulyok1,
  • Zsuzsanna Rónai2,
  • Veronika Hrivnák1,
  • Ibolya Turcsányi2,
  • Szilárd Jánosi2 and
  • Miklós Gyuranecz1Email author
Contributed equally
BMC Veterinary ResearchBMC series – open, inclusive and trusted201612:170

https://doi.org/10.1186/s12917-016-0799-0

Received: 2 March 2016

Accepted: 10 August 2016

Published: 19 August 2016

Abstract

Background

Mycoplasma sp. 1220 can induce inflammation primarily in the genital and respiratory tracts of waterfowl, leading to serious economic losses. Adequate housing and appropriate antibiotic treatment are promoted in the control of the disease. The aim of the present study was to determine the in vitro susceptibility to thirteen different antibiotics and an antibiotic combination of thirty-eight M. sp. 1220 strains isolated from geese and a duck in several parts of Hungary, Central Europe between 2011 and 2015.

Results

High MIC50 values were observed in the cases of tilmicosin (>64 μg/ml), oxytetracycline (64 μg/ml), norfloxacin (>10 μg/ml) and difloxacin (10 μg/ml). The examined strains yielded the same MIC50 values with spectinomycin, tylosin and florfenicol (8 μg/ml), while enrofloxacin (MIC50 5 μg/ml), doxycycline (MIC50 5 μg/ml), lincomycin (MIC50 4 μg/ml) and lincomycin-spectinomycin (1:2) combination (MIC50 4 μg/ml) inhibited the growth of the bacteria with lower concentrations. Tylvalosin (MIC50 0.5 μg/ml) and two pleuromutilins (tiamulin MIC50 0.625 μg/ml; valnemulin MIC50 ≤ 0.039 μg/ml) were found to be the most effective drugs against M. sp. 1220. However, strains with elevated MIC values were detected for all applied antibiotics.

Conclusions

Valnemulin, tiamulin and tylvalosin were found to be the most effective antibiotics in the study. Increasing resistance was observed in the cases of several antibiotics. The results highlight the importance of testing Mycoplasma species for antibiotic susceptibility before therapy.

Keywords

Antibiotic resistanceDuckGooseMICMicrobroth dilution Mycoplasma sp. 1220

Background

Mycoplasma sp. 1220 was first described as a new Mycoplasma species by Stipkovits et al. in 1986 [1]. This Mycoplasma species causes cloaca and phallus inflammation and testicular atrophy in the ganders [1, 2]. In the infected geese salpingitis and vaginitis are the main symptoms [1, 3]. The egg production activates the pathogen and the flared up mycoplasmosis could induce lethal pathological changes in the embryos [1, 4]. Airsacculitis and peritonitis are also common, and general symptoms such as changes in thirst, decreased food consumption, body weight losses, weakness, nasal discharge, impaired breathing, conjunctivitis, diarrhoea and nervous signs were also described in the affected waterfowl flocks [2, 58]. Mycoplasma infection of the birds can aggravate diseases caused by other agents and could lead to serious economic losses [3, 6]. Since there is no commercially available vaccine against M. sp. 1220, adequate housing and appropriate antibiotic treatment are promoted in the control of the diseases caused by this agent. Prophylactic medication could prevent economic losses if appropriate antibiotics are administered in the early weeks of life and in expected stress periods. Medication of the layers is essential to reduce the vertical transmission of M. sp. 1220 [2].

Mycoplasmas are resistant to β-lactam antimicrobials because of the lack of cell-wall and the bacteria are also resistant to membrane synthesis inhibitors [2, 9]. Antibiotics such as quinolones, tetracyclines, macrolides and pleuromutilins which induce DNA fragmentation or inhibition at the level of protein synthesis are the drugs of choice for the therapy of mycoplasmosis. Among the macrolides, erythromycin showed high effectiveness against Mycoplasma strains which could ferment glucose (e.g. M. sp. 1220), while arginine-hydrolysing strains proved to be less susceptible to this compound [2, 10]. Mycoplasma infected waterfowl and poultry flocks are usually treated with macrolides, pleuromutilins or with the combination of lincomycin and spectinomycin [3, 1118].

The aim of this study was to determine the susceptibility of 38 Hungarian M. sp. 1220 isolates to thirteen antibiotics and a drug combination using the microbroth dilution method.

Methods

A total of 38 M. sp. 1220 strains isolated from geese and a duck originating from different parts of Hungary were tested in the study (Table 1, Fig. 1). The samples were collected during routine diagnostic examinations or necropsies between 2011 and 2015, thus ethical approval was not required for the study. Phallus lymph, cloaca swabs, tracheal swabs, follicules and lung samples were washed in 2 ml of Mycoplasma broth medium (pH 7.8) (ThermoFisher Scientific Inc./Oxoid Inc./, Waltham, MA) supplemented with 0.5 % (w/v) sodium pyruvate, 0.5 % (w/v) glucose and 0.005 % (w/v) phenol red and incubated at 37 °C in a 5 % CO2 atmosphere. The cultures were inoculated onto solid Mycoplasma media (Thermo Fisher Scientific Inc./Oxoid Inc./) after colour change of the broth, and were incubated at 37 °C and 5 % CO2 until visible colonies appeared (1–2 days). Purification of mixed cultures was performed by one-time filter cloning, minimizing the in vitro mutations of the isolates. The QIAamp DNA Mini Kit (Qiagen Inc., Hilden, Germany) was used for DNA extraction according to the manufacturers’ instructions for Gram-negative bacteria. The purity of the cultures was confirmed by a universal Mycoplasma PCR system targeting the 16S/23S rRNA intergenic spacer region in Mycoplasmatales followed by sequencing on an ABI Prism 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA), sequence analysis and BLAST search [19]. The number of colour changing units (CCU) was calculated by microbroth dilution method, from the lowest dilution showing colour change after one week of incubation [9].
Table 1

Background data and MIC values of the isolated Mycoplasma sp. 1220 strains

     

MIC values (μg/ml)

     

Fluoroquinolones

Aminoglycoside

Lincosamide

 

Tetracyclines

Macrolides

Pleuromutilines

Phenicol

Sample ID

Sample source

Place

Animal

Date

Enrofloxacin

Norfloxacin

Difloxacin

Spectinomycin

Lincomycin

Lincomycin-spectinomycin (1:2) combination

Oxytetracycline

Doxycycline

Tylosin

Tilmicosin

Tylvalosin

Tiamulin

Valnemulin

Florfenicol

MYCAV 65

Phallus lymph

Rém

goose

2014

5

>10

10

16

4

4

32

5

0.5

0.5

≤0.25

1.25

0.078

8

MYCAV 34

Phallus lymph

Szentes

goose

2011

5

>10

10

8

4

2

64

2.5

1

4

≤0.25

0.625

≤0.039

4

MYCAV 35

Phallus lymph

Rém

goose

2012

5

>10

10

>64

4

4

64

10

1

4

≤0.25

1.25

≤0.039

8

MYCAV 36

Cloaca

Hajdúböszörmény

goose

2012

5

>10

>10

64

4

4

64

>10

1

4

≤0.25

1.25

≤0.039

8

MYCAV 38

Cloaca

Kelebia

goose

2012

2.5

>10

10

8

2

4

4

0.156

≤0.25

≤0.25

≤0.25

0.625

≤0.039

4

MYCAV 44

Cloaca

Nagykamarás

goose

2012

5

>10

10

8

4

4

8

0.312

8

>64

0.5

1.25

≤0.039

8

MYCAV 47

Lung

Tázlár

duck

2012

>10

>10

>10

16

>64

16

>64

5

16

>64

1

2.5

0.312

8

MYCAV 49

Phallus lymph

Tiszavasvári

goose

2013

5

>10

10

16

4

4

64

5

8

>64

0.5

0.625

≤0.039

8

MYCAV 50

Phallus lymph

Cered

goose

2013

>10

>10

>10

16

4

4

>64

5

2

2

≤0.25

0.625

≤0.039

8

MYCAV 51

Phallus lymph

Derekegyház

goose

2013

5

>10

10

32

4

4

>64

10

8

>64

0.5

0.625

≤0.039

8

MYCAV 53

Phallus lymph

Szentes

goose

2013

5

>10

10

16

4

4

>64

10

8

>64

0.5

0.625

≤0.039

8

MYCAV 54

Follicule

Hódmezővásárhely

goose

2013

5

>10

10

8

4

4

>64

5

8

>64

0.5

0.625

≤0.039

8

MYCAV 55

Follicule

Kiskunmajsa

goose

2013

10

>10

10

8

4

4

8

0.312

≤0.25

≤0.25

≤0.25

0.625

≤0.039

4

MYCAV 56

Phallus lymph

Sükösd

goose

2013

1.25

>10

1.25

8

4

4

4

0.312

8

>64

0.5

0.625

≤0.039

4

MYCAV 59

Follicule

Rém

goose

2013

5

>10

10

8

4

4

32

2.5

0.5

≤0.25

≤0.25

1.25

0.078

2

MYCAV 61

Phallus lymph

Tatárszentgyörgy

goose

2013

5

>10

10

16

2

4

2

0.078

≤0.25

≤0.25

≤0.25

0.312

≤0.039

4

MYCAV 63

Trachea

Sükösd

goose

2013

1.25

10

1.25

8

2

2

4

0.312

4

64

≤0.25

0.156

≤0.039

4

MYCAV 66

Phallus lymph

Tiszaföldvár

goose

2014

5

>10

10

16

4

4

>64

>10

≤0.25

≤0.25

≤0.25

0.625

≤0.039

4

MYCAV 67

Phallus lymph

Szentes

goose

2014

5

>10

10

8

>64

16

>64

5

>64

>64

16

2.5

0.078

4

MYCAV 68

Phallus lymph

Érpatak

goose

2014

5

>10

10

8

>64

32

>64

10

>64

>64

16

5

≤0.039

8

MYCAV 69

Phallus lymph

Ludas

goose

2014

5

>10

10

4

4

4

>64

5

8

>64

1

0.625

≤0.039

4

MYCAV 70

Phallus lymph

Cered

goose

2014

>10

>10

>10

16

4

4

>64

>10

16

>64

1

0.625

≤0.039

8

MYCAV 71

Phallus lymph

Sükösd

goose

2014

1.25

>10

1.25

8

2

4

8

0.625

8

>64

0.5

0.625

≤0.039

4

MYCAV 72

Phallus lymph

Nagykamarás

goose

2014

5

>10

10

8

4

4

4

0.312

8

>64

0.5

0.625

≤0.039

4

MYCAV 75

Phallus lymph

Dömsöd

goose

2014

5

>10

10

16

4

4

>64

10

≤0.25

≤0.25

≤0.25

0.625

≤0.039

8

MYCAV 76

Phallus lymph

Tiszabábolna

goose

2014

5

>10

10

32

8

4

64

5

8

>64

0.5

1.25

≤0.039

8

MYCAV 91

Phallus lymph

Hajdúsámson

goose

2011

10

>10

>10

8

8

4

64

2.5

≤0.25

≤0.25

≤0.25

0.625

≤0.039

8

MYCAV 93

Phallus lymph

Bojt

goose

2014

2.5

>10

1.25

8

2

4

8

0.312

≤0.25

≤0.25

≤0.25

0.312

≤0.039

8

MYCAV 94

Cloaca

Tiszabábolna

goose

2012

2.5

>10

5

16

4

4

>64

>10

≤0.25

≤0.25

≤0.25

0.625

≤0.039

8

MYCAV 160

Phallus lymph

Érpatak

goose

2015

>10

>10

>10

16

4

4

>64

10

>64

>64

2

0.625

≤0.039

8

MYCAV 161

Phallus lymph

Szilaspogony

goose

2015

>10

>10

>10

8

4

4

>64

>10

16

>64

0.5

0.625

≤0.039

8

MYCAV 162

Phallus lymph

Encsencs

goose

2015

2.5

>10

10

8

4

4

>64

5

16

>64

0.5

0.625

≤0.039

4

MYCAV 176

Phallus lymph

Cered

goose

2015

10

>10

5

8

4

4

>64

5

64

>64

4

0.625

≤0.039

16

MYCAV 177

Phallus lymph

Cered

goose

2015

>10

>10

10

8

4

4

>64

10

>64

>64

4

0.625

≤0.039

32

MYCAV 178

Follicule

Cered

goose

2015

5

>10

10

8

2

4

>64

5

4

>64

0.5

0.312

≤0.039

4

MYCAV 179

Trachea

Apátfalva

goose

2015

10

>10

10

16

4

4

4

0.312

4

4

0.5

1.25

≤0.039

8

MYCAV 180

Phallus lymph

Kisbér

goose

2015

5

>10

10

>64

4

4

4

0.312

32

>64

1

1.25

≤0.039

8

MYCAV 202

Cloaca

Kelebia

goose

2015

5

>10

5

16

4

4

32

2.5

0.5

0.5

≤0.25

1.25

≤0.039

8

Fig. 1

Map of Hungary showing the geographical origin of the Mycoplasma sp. 1220 isolates. Size of the circles indicates the number (n) of the strains. (The blank map was downloaded from an open source [28])

The following antimicrobial agents were examined during the microbroth dilution tests: the fluoroquinolones: enrofloxacin (batch SZBA336XV), difloxacin (SZBD178XV) and norfloxacin (batch SZBD099XV); the aminoglycoside: spectinomycin (batch SZBB166XV); the lincosamide: lincomycin (batch SZBC340XV); the tetracyclines: doxycycline (batch SZBD007XV) and oxytetracycline (batch SZBC320XV); the macrolides: tilmicosin (batch SZBC345XV) and tylosin (batch SZBB160XV); the pleuromutilins: tiamulin (batch SZBC026XV) and valnemulin (batch SZBE127XV); and the phenicol: florfenicol (batch SZBC223XV); all products originated from VETRANAL, Sigma-Aldrich, Germany. The macrolide tylvalosin (Aivlosin, ECO Animal Health Ltd., UK; LOT M102A) was also included in the examinations. Lincomycin and spectinomycin were applied also in combination at a ratio of 1:2. The antibiotics were diluted and stored according to the recommendations of Hannan [9]. Stock solutions of 1 mg/ml fluoroquinolones were prepared in 0.1 M NaOH; stock solution of 1 mg/ml florfenicol was prepared in 96 % ethanol and in sterile distilled water; and the rest of the stock solutions of 1 mg/ml were prepared in sterile distilled water. Dilutions of the antibiotics were freshly prepared for each microtest from the aliquots stored at −70 °C. Twofold dilutions were prepared in the range 0.039–10 μg/ml for fluoroquinolones, doxycycline and pleuromutilins, 0.25–64 μg/ml for spectinomycin, lincomycin, lincomycin-spectinomycin (1:2) combination, oxytetracycline and macrolides and 0.125–32 μg/ml for florfenicol.

The microbroth dilution examinations on 104–105 CCU/ml of the strains were performed according to Hannan [9]. Mycoplasma broth medium was used in the tests as well, and each 96-well microtiter plates contained growth controls (broth medium without antibiotic), sterility controls (broth medium without antibiotic and Mycoplasma inoculum) and pH controls (broth medium adjusted to pH 6.8). One clinical isolate (strain MYCAV 65) was selected to be used as quality control of minimal inhibitory concentration (MIC) determination throughout the experiments. The duplicates of three clinical isolates and the duplicate of the selected strain (MYCAV 65) were tested on each 96-well microtiter plates.

The MIC values were determined from the lowest concentration of the antibiotics where no pH and colour change of the broth was detected after one week of incubation, meaning that the growth of the bacteria was completely inhibited in the broth. MIC50 and MIC90 values were defined as the lowest concentrations that inhibited the growth of 50 % or 90 % of the strains [9].

Results

The quality control strain (MYCAV 65) showed consistent results throughout the study. Strains with elevated MIC values were found in the cases of all tested antibiotics (Tables 1 and 2).
Table 2

Summary of MIC range, MIC50 and MIC90 values of the isolated Mycoplasma sp. 1220 strains

Antibiotic class

Antibiotic agent

Range

MIC50

MIC90

Fluoroquinolones

Enrofloxacin

1.25 to >10

5

>10

Norfloxacin

10 to >10

>10

>10

Difloxacin

1.25 to >10

10

>10

Aminoglycoside

Spectinomycin

4 to >64

8

32

Lincosamide

Lincomycin

2 to >64

4

8

 

Lincomycin-spectinomycin (1:2) combination

2 to 32

4

4

Tetracyclines

Oxytetracycline

2 to >64

64

>64

Doxycycline

0.078 to >10

5

>10

Macrolides

Tylosin

≤0.25 to >64

8

>64

Tilmicosin

≤0.25 to >64

>64

>64

Tylvalosin

≤0.25 to 16

0.5

4

Pleuromutilins

Tiamulin

0.156 to 5

0.625

1.25

Valnemulin

≤0.039 to 0.312

≤0.039

0.078

Phenicol

Florfenicol

2 to 32

8

8

Among the fluoroquinolones, the MIC values of enrofloxacin and difloxacin showed a wide range (1.25 to >10 μg/ml), while all strains had very high MIC values for norfloxacin (≥10 μg/ml) (Fig. 2a, b and c). The MIC50 was 8 μg/ml for spectinomycin and most of the strains yielded the MIC50 or higher MIC values (Fig. 2d). The MICs for lincomycin clustered around the MIC50 value (4 μg/ml) as well, but high MIC values (>64 μg/ml) were yielded in the case of three isolates (Fig. 2e). The MIC50 and the MIC90 values (4 μg/ml) for lincomycin-spectinomycin (1:2) combination was the same as the MIC50 value for lincomycin. In the case of lincomycin-spectinomycin (1:2) combination the highest concentration needed for inhibition was 32 μg/ml (Fig. 2f). Broad ranges of the MIC values were observed for tetracyclines (2 to >64 μg/ml for oxytetracycline and 0.078 to >10 μg/ml for doxycycline) with high MIC50 and MIC90 values (Fig. 2g and h). The broadest ranges of MIC values were detected for tylosin and tilmicosin (≤0.25 to >64 μg/ml) with high MIC50 and MIC90 values in the case of tilmicosin (Fig. 2i and j). While the MIC values for tylosin showed diverse distribution, the strains’ susceptibility profiles formed three groups in the case of tilmicosin (≤0.25, 4 and >64 μg/ml) (Fig. 2j). Among the examined three macrolides (tylosin, tilmicosin and tylvalosin), tylvalosin showed the lowest MIC50 value (0.5 μg/ml) against the strains (Fig. 2k). From the pleuromutilins the MIC values of tiamulin were higher than those of valnemulin, and the latter compound was found to be the most active antibiotic in the examinations (Fig. 2l and m). In the case of florfenicol, the susceptibility profiles of most strains were similar to each other and showed the MIC50 and MIC90 value (8 μg/ml) or its two-fold lower dilution (4 μg/ml) with few exceptions (Fig. 2n).
Fig. 2

MIC distribution of test antibiotics against Mycoplasma sp. 1220 isolates

M. sp. 1220 strains isolated year by year from the same farms and from the same tissue types (e.g. strains MYCAV 34, 53 and 67 from Szentes, strains MYCAV 50, 70, 176 and 177 from Cered, or strains MYCAV 38 and 202 from Kelebia) showed elevated MIC values from year to year in the cases of certain antibiotics. Higher MIC values were detected in subsequent isolates for lincomycin, lincomycin-spectinomycin combination, tetracyclines (both oxytetracycline and doxycycline), macrolides (tylosin, tilmicosin and tylvalosin), tiamulin and for florfenicol as well.

Discussion

Information about the susceptibility of M. sp. 1220 strains to antimicrobials is scarce, as until to date the sole published reference concerning the antibiotic susceptibility profile of this species is a review of Stipkovits and Szathmary [3]. Stipkovits and Szathmary determined the values of enrofloxacin, tylosin, chlortetracycline, oxytetracycline, doxycycline, tiamulin and lincomycin in Mycoplasma species affecting waterfowl (M. anatis, M. cloacale, M. anseris and M. sp. 1220), although detailed data of their method is lacking [3]. Thus we are facing the absence of reports about the antibiotic susceptibility of M. sp. 1220 and also of other Mycoplasma species occurring in waterfowl. Therefore, the results of the current study are also compared to data of antibiotic susceptibility of the well-studied Mycoplasma species of poultry: M. synoviae and M. gallisepticum.

Elevated MIC values were reported previously in the case of the fluoroquinolones, especially of enrofloxacin in M. sp. 1220 (MIC50 2 μg/ml and MIC90 4 μg/ml) and other Mycoplasma species of poultry [3, 13, 20, 21]. In addition, the increasing occurrence of quinolone-resistant M. synoviae and M. gallisepticum field isolates were also observed [13, 22]. In the current study, the detected MIC50 values (5 μg/ml for enrofloxacin, 10 μg/ml for difloxacin and ≥10 μg/ml for norfloxacin) were even higher than the ones reported before [3, 13, 2022], confirming the observation of increasing quinolone-resistance in Mycoplasma species. In order to save these antibiotics for human disease treatment the directive was to reduce the use of these agents in livestock. Former efforts for the prevention of the appearance of quinolone-resistant species are proved to be unsuccessful considering the observed dramatic elevations in the MIC values of these antibiotics in avian Mycoplasma species [13, 21, 23].

Administration of the combination of lincomycin and spectinomycin could reduce the egg infertility rates and increase the hatching rates and the egg production in M. sp. 1220 infected geese [11]. The lincomycin-spectinomycin therapy was proved to be effective against other Mycoplasma species as well; however, application of spectinomycin in monotherapy is not recommended for its insufficient effectiveness and relatively high MIC values in in vitro experiments [12]. In vitro effectiveness of lincomycin at 2 μg/ml MIC50 values against M. sp. 1220, M. anseris and M. anatis species has been reported [3]. In the present study, all isolates showed elevated MIC values for spectinomycin, lincomycin and lincomycin-spectinomycin combination. The growth of a couple of strains was not inhibited even at the highest concentrations used (64 μg/ml) for spectinomycin and lincomycin individually. The combination of the two antibiotics improved their effectiveness, as lincomycin-spectinomycin combination could inhibit the growth of all examined strains within the concentration range used (0.25 to 64 μg/ml) and lower MIC90 value was observed also.

Previously, tetracyclines (chlortetracycline, doxycycline and oxytetracycline) showed 1–2 μg/ml MIC values against M. sp. 1220 strains. Growth of other Mycoplasma species isolated from waterfowl were inhibited at 2–4 μg/ml MIC50 values using the same antibiotics [3]. Previously Mycoplasma species infecting poultry were observed to be inhibited by elevated MIC values, although with exceptions, as M. synoviae strains showed high susceptibility to doxycycline in the Netherlands [1214]. In the current study, although the M. sp. 1220 strains showed broad ranges of MIC values for oxytetracycline and doxycycline, more than 50 % of the strains were inhibited by only higher antibiotic concentrations (64 and 5 μg/ml, respectively) and MIC90 values exceeded the concentration ranges used for both compounds. These results show a dramatic increase of the MIC values of tetracyclines against M. sp. 1220 strains and reveals the presence of probably highly resistant strains in Hungary.

Macrolides, especially tylvalosin have good in vitro effectiveness against most Mycoplasma species infecting poultry, showing lower MIC values in previous examinations than quinolones and tetracyclines [3, 1215]. However, M. gallisepticum could develop resistance rapidly to these compounds, especially to tilmicosin [24]. Earlier, the MIC50 values in M. sp. 1220, M. anatis, M. anseris and M. cloacale strains were defined to be 2 μg/ml for tylosin [3]. In the current study, the MIC50 value (8 μg/ml) of tylosin was higher than the previous observation [3], and the MIC90 value exceeded the concentration range used in the experiment. However, high variability was observed in the susceptibility of the strains to this compound. Similarly, wide range of the MIC values was detected for tilmicosin, highlighting the necessity of susceptibility testing before antibiotic treatments. As opposed to the diverse susceptibility profiles of the strains for tylosin, the MIC values of tilmicosin were categorized into three separate groups. The observed distribution of the MIC values is likely in association with the capability of Mycoplasma sp. 1220 to develop resistance more rapidly to tilmicosin (i.e. with one or two mutations) than to other macrolides. The same phenomenon was described in other Mycoplasma species as well [24]. Out of the three macrolides examined in the study, tylvalosin proved to be the most effective agent against M. sp. 1220 strains, showing lower MIC50 value (0.5 μg/ml) against the pathogen than the majority of the antibiotics tested.

Pleuromutilins showed good in vitro effectiveness against avian Mycoplasma species in previous studies and low tendency of the development of resistance to these agents has been reported [1618, 21]. Tiamulin was used successfully for the treatment of mycoplasmosis and its effectiveness was similar to spectinomycin therapy in the treated geese [11]. Stipkovits and Szathmary described low MIC values (MIC50: 0.06 μg/ml, MIC90: 0.25 μg/ml) of tiamulin in the case of M. sp. 1220, and similarly low MIC50 values (0.125–1 μg/ml) were observed against M. anseris, M. anatis and M. cloacale [3]. In the present study, pleuromutilins were found to be the most effective antibiotic agents and the examined compounds, especially valnemulin showed high in vitro effectiveness against all tested isolates of the pathogen. However, it is noteworthy, that strains with elevated MIC values were detected for tiamulin (MIC: 2.5–5 μg/ml) and even for valnemulin (MIC: 0.312 μg/ml). Although the low MIC values of valnemulin against M. sp. 1220 strains in vitro are promising for its clinical use, it should be noted that in a previous study only a single mutation in M. gallisepticum could cause elevation in the MIC values of valnemulin [17]. To date, the use of pleuromutilins in humans is limited, as only one commercially available product is authorized containing this active substance. However, bacterial strains resistant to pleuromutilins have already been described and these strains also show multidrug resistance, which warrants the prudent use of these antibiotic agents [25].

Phenicols showed good in vitro activity against Mycoplasma species of poultry, but information about their effectiveness in waterfowl is lacking [26, 27]. In the present study, most of the M. sp. 1220 isolates yielded the same MIC values (4 or 8 μg/ml) for florfenicol, and only two isolates (originating from the same region) showed elevated MIC values compared to the MIC50 (8 μg/ml), one of them reaching the highest antibiotic concentration (32 μg/ml) used.

The elevated MIC values of several antibiotics detected in subsequent isolates from the same farms from year to year are likely in association with the inconsistent use of antibiotics, the rapid development of antibiotic resistance and highlight the importance of susceptibility testing before therapy and responsible use of antimicrobial drugs.

Conclusion

In the present examinations the antibiotic susceptibility profiles of thirty-eight M. sp. 1220 strains isolated in Hungary were determined. To the best of our knowledge, this is the first detailed study about the antibiotic susceptibility of M. sp. 1220, a pathogen which could cause significant economic losses in waterfowl flocks. Valnemulin, tiamulin and tylvalosin were found to be the most effective antibiotics in the present study. Most of the isolates showed elevated MIC values for more than one agent, but none of the strains yielded high MIC values for all the examined antibiotics. Nevertheless, our results confirmed that increasing resistance could be observed in the cases of several antibiotics. These findings highlight the consistent use of antibiotics and the need for determination of antibiotic susceptibility of Mycoplasma species before treatment.

Abbreviations

MIC: 

Minimal inhibitory concentrations

Declarations

Funding

This work was supported by the Lendület (Momentum) programme (LP2012-22) of the Hungarian Academy of Sciences. The funders had no role in study design, data collection, analysis and interpretation, decision to publish, or preparation of the manuscript.

Availability of data and materials

All data supporting the findings is contained within the manuscript.

Authors’ contributions

All authors read and approved the final manuscript. DG, ZK and KMS analysed the data and wrote the manuscript. DG and VH performed the examinations. ZR, IT and SJ collected the samples, isolated the strains and edited the manuscript. MG designed the study, analysed the data and edited the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The ethics committees of the Institute for Veterinary Medical Research ruled that no formal ethics approval or consent were required as the samples were collected during routine diagnostic examinations or necropsies.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences
(2)
Veterinary Diagnostic Directorate, National Food Chain Safety Office

References

  1. Stipkovits L, Varga Z, Czifra G, Dobos-Kovacs M. Occurrence of mycoplasmas in geese affected with inflammation of the cloaca and phallus. Avian Pathol. 1986;15:289–99.View ArticlePubMedGoogle Scholar
  2. Stipkovits L, Kempf I. Mycoplasmoses in poultry. Rev Sci Tech. 1996;15:1495–525.PubMedGoogle Scholar
  3. Stipkovits L, Szathmary S. Mycoplasma infection of ducks and geese. Poult Sci. 2012;91:2812–9.View ArticlePubMedGoogle Scholar
  4. Stipkovits L, Varga Z, Glavits R, Ratz F, Molnar E. Pathological and immunological studies on goose embryos and one-day-old goslings experimentally infected with a Mycoplasma strain of goose origin. Avian Pathol. 1987;16:453–68.View ArticlePubMedGoogle Scholar
  5. Dobos-Kovacs M, Varga Z, Czifra G, Stipkovits L. Salpingitis in geese associated with Mycoplasma sp. strain 1220. Avian Pathol. 2009;38:239–43.View ArticlePubMedGoogle Scholar
  6. Hinz K-H, Pfützner H, Behr K-P. Isolation of mycoplasmas from clinically healthy adult breeding geese in germany. J Vet Med B. 1994;41:145–7.View ArticleGoogle Scholar
  7. Jordan FT, Gilbert S, Knight DL, Yavari C a. Effects of baytril, tylosin and tiamulin on avian mycoplasmas. Avian Pathol. 1989;18:659–73.View ArticlePubMedGoogle Scholar
  8. Stipkovits L, Glavits R, Ivanics E, Szabo E. Additional data on Mycoplasma disease of goslings. Avian Pathol. 1993;22:171–6.View ArticlePubMedGoogle Scholar
  9. Hannan PCT. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Vet Res. 2000;31:373–95.View ArticlePubMedGoogle Scholar
  10. Stipkovits L, Varga Z, Dobos-Kovacs M, Santha M. Biochemical and serological examination of some Mycoplasma strains of goose origin. Acta Vet Hung. 1984;32:117–25.PubMedGoogle Scholar
  11. Czifra G, Varga Z, Dobos-Kovacs M, Stipkovits L. Medication of inflammation of the phallus in geese. Acta Vet Hung. 1986;34:211–23.PubMedGoogle Scholar
  12. Behbahan NGG, Asasi K, Afsharifar AR, Pourbakhsh SA. Susceptibilities of Mycoplasma gallispeticum and Mycoplasma synoviae Isolates to Antimicrobial Agents in vitro. Int J Poult Sci. 2008;7:1058–1064.View ArticleGoogle Scholar
  13. Landman WJ, Mevius DJ, Veldman KT, Feberwee A. In vitro antibiotic susceptibility of Dutch Mycoplasma synoviae field isolates originating from joint lesions and the respiratory tract of commercial poultry. Avian Pathol. 2008;37:415–20.View ArticlePubMedGoogle Scholar
  14. Wang C, Ewing M, Aarabi SY. In vitro susceptibility of avian mycoplasmas to enrofloxacin, sarafloxacin, tylosin, and oxytetracycline. Avian Dis. 2001;45:456–60.View ArticlePubMedGoogle Scholar
  15. Forrester CA, Bradbury JM, Dare CM, Domangue RJ, Windsor H, Tasker JB, Mockett APA. Mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease. Avian Pathol. 2011;40:581–6.View ArticlePubMedGoogle Scholar
  16. Drews J, Georgopoulos A, Laber G, Shütze E, Unger J. Antimicrobial Activities of 81.723 hfu, a New Pleuromutilin Derivative. Antimicrob Agent Chemother. 1975;7:507–516.View ArticleGoogle Scholar
  17. Li B-B, Shen J-Z, Cao X-Y, Wang Y, Dai L, Huang S-Y, Wu C-M. Mutations in 23S rRNA gene associated with decreased susceptibility to tiamulin and valnemulin in Mycoplasma gallisepticum. FEMS Microbiol Lett. 2010;308:144–9.PubMedGoogle Scholar
  18. Xiao X, Sun J, Chen Y, Zou M, Zhao D-H, Liu Y-H. Ex vivo pharmacokinetic and pharmacodynamic analysis of valnemulin against Mycoplasma gallisepticum S6 in Mycoplasma gallisepticum and Escherichia coli co-infected chickens. Vet J. 2015;204:54–9.View ArticlePubMedGoogle Scholar
  19. Lauerman LH, Chilina AR, Closser JA, Johansen D. Avian mycoplasma identification using polymerase chain reaction amplicon and restriction fragment length polymorphism analysis. Avian Dis. 1995;39:804–11.View ArticlePubMedGoogle Scholar
  20. Lysnyansky I, Gerchman I, Mikula I, Gobbo F, Catania S, Levisohn S. Molecular characterization of acquired enrofloxacin resistance in Mycoplasma synoviae field isolates. Antimicrob Agents Chemother. 2013;57:3072–7.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Gautier-Bouchardon AV, Reinhardt AK, Kobisch M, Kempf I. In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae. Vet Microbiol. 2002;88:47–58.View ArticlePubMedGoogle Scholar
  22. Gerchman I, Lysnyansky I, Perk S, Levisohn S. In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys. Vet Microbiol. 2008;131:266–76.View ArticlePubMedGoogle Scholar
  23. European Medicines Agency, EMA/186029/2010. [http://www.ema.europa.eu/docs/en_GB/document_library/Referrals_document/quinolones_35/WC500094630.pdf]. Accessed 11 Nov 2015.
  24. Wu CM, Wu H, Ning Y, Wang J, Du X, Shen J. Induction of macrolide resistance in Mycoplasma gallisepticum in vitro and its resistance-related mutations within domain V of 23S rRNA. FEMS Microbiol Lett. 2005;247:199–205.View ArticlePubMedGoogle Scholar
  25. van Duijkeren E, Greko C, Pringle M, Baptiste KE, Catry B, Jukes H, Moreno MA, Pomba MCMF, Pyorala S, Rantala M, Ru auskas M, Sanders P, Teale C, Threlfall EJ, Torren-Edo J, Torneke K. Pleuromutilins: use in food-producing animals in the European Union, development of resistance and impact on human and animal health. J Antimicrob Chemother. 2014;69:2022–31.View ArticleGoogle Scholar
  26. Gharaibeh S, Al-Rashdan M. Change in antimicrobial susceptibility of Mycoplasma gallisepticum field isolates. Vet Microbiol. 2011;150:379–83.View ArticlePubMedGoogle Scholar
  27. Lin MY. In vitro comparison of the activity of various antibiotics and drugs against New taiwan isolates and standard strains of avian mycoplasma. Avian Dis. 1987;31:705–12.View ArticlePubMedGoogle Scholar
  28. Hungary outline. [http://d-maps.com/carte.php?num_car=23243&lang=en]. Accessed 10 Aug 2015.

Copyright

© The Author(s). 2016

Advertisement