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Article

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates

by
Lisa Käbisch
1,2,3,
Anne-Kathrin Schink
4,
Doris Hoeltig
5,
Jutta Verspohl
6,
Miklós Gyuranecz
7,8,
Joachim Spergser
9,
Corinna Kehrenberg
3 and
Stefan Schwarz
1,2,*
1
Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
2
Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
3
Institute for Veterinary Food Science, Department of Veterinary Medicine, Justus Liebig University Giessen, 35392 Giessen, Germany
4
LDG Laboratory Diagnostics Germany GmbH, 27472 Cuxhaven, Germany
5
Division for Pigs, Farm Animal Clinic, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
6
Institute of Microbiology, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Germany
7
HUN-REN Veterinary Medical Research Institute, H-1143 Budapest, Hungary
8
MolliScience Kft., H-2051 Biatorbágy, Hungary
9
Institute of Microbiology, University of Veterinary Medicine, 1210 Vienna, Austria
*
Author to whom correspondence should be addressed.
Microorganisms 2023, 11(12), 2881; https://doi.org/10.3390/microorganisms11122881
Submission received: 4 November 2023 / Revised: 16 November 2023 / Accepted: 26 November 2023 / Published: 29 November 2023
(This article belongs to the Special Issue Detection, Diagnosis, and Host Interactions of Animal Mycoplasmas)
Versions Notes

Abstract

:
Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

1. Introduction

Mycoplasma (M.) hyorhinis is a porcine pathogen of ubiquitous origin [1,2]. As a facultative pathogen, the clinical picture of the infected porcine host may vary from severe systemic infections to polyserositis and arthritis in nursery piglets or chronic arthritis, conjunctivitis, and meningitis in older pigs [1,2,3,4,5,6,7]. Nevertheless, M. hyorhinis is often also isolated from pigs showing no clinical symptoms. Because of the great variety of clinical manifestations, the economic impact of an infection with M. hyorhinis is difficult to enumerate. To the best of our knowledge, there is no literature available estimating the financial losses specifically for a M. hyorhinis-infected herd. Furthermore, while animals with clinical signs will be individually treated, in severe cases, a farm-specific vaccination is implemented; animals that need to be euthanized, especially within the finishing phase, will represent the greatest losses for farmers [8].
Due to the lack of a cell wall, Mycoplasma spp. are intrinsically resistant to β-lactams and other cell-wall-targeting antimicrobial agents. The lack of specific enzymes within the folic acid metabolism pathway also renders antimicrobial agents targeting this specific pathway, such as sulfonamides and trimethoprim, ineffective [9,10,11,12,13]. Nonetheless, several classes of antimicrobial agents are available for the treatment of M. hyorhinis infections, and various studies have shown differing antimicrobial susceptibility profiles of M. hyorhinis field isolates in the past [10,13,14,15,16,17,18,19,20]. However, since different antimicrobial susceptibility testing (AST) protocols and methodologies have been used in previously published studies, the respective AST data are difficult to compare [21,22].
For all Mycoplasma spp. of animal origin, the most effective antimicrobial classes are fluoroquinolones, macrolides, pleuromutilins, and tetracyclines [13,14,23]. Reduced efficacies toward several antimicrobial agents have been observed during the last decades, involving both human as well as veterinary Mycoplasma spp. [5,10,14,15,16,17,20,24,25,26]. In Mycoplasma spp., resistance development is mainly conferred by chromosomal mutations, such as the well-described point mutation within the 23S rRNA, conferring resistance to 14-membered macrolides [15,18,19,23,25,27,28,29,30,31].
Because of the observations of reduced efficacy toward certain antimicrobial agents, in addition to increased monitoring of antimicrobial resistance among bacterial species of animal origin and efforts toward reducing the usage of antimicrobial agents in farm animals, as implemented by many governments, a standardized AST methodology for monitoring studies needs to be established [21]. For a standardized AST methodology, organizations, including the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), as well as more local authorities, such as the American Food and Drug Administration (FDA), the German Deutsches Institut für Normung (DIN), or the French Société Française de Microbiologie (SFM), provide AST standards or guidelines for a wide variety of nonfastidious and fastidious bacteria of human or animal origin. However, until now, there has been no consensus method for Mycoplasma spp. of animal origin.
The use of diverging AST methodologies generally leads to inconsistent data, rendering the obtained results incomparable and, when considering monitoring studies, unusable. For M. hyorhinis, the available literature indicates several methodologies, whereas most studies agree on using the broth microdilution method [10,14,15,16,20,25]. On the other hand, a great variety of broth media is used for AST, wherefore the minimal inhibitory concentrations (MICs) of the tested isolates differ and are consequently incomparable or lead to misinformation, especially concerning the in vivo activity of antimicrobial agents against M. hyorhinis infections [9,14,15,16,18,19,32,33,34]. In addition, monitoring of the emergence of a decreased susceptibility profile of M. hyorhinis is not possible, not only due to the diverging methodology but also because of the lack of established interpretive criteria [21,22]. Until now, no species-specific clinical breakpoints have been available that allow the classification of M. hyorhinis isolates as susceptible, intermediate, or resistant to the tested antimicrobial agents.
We have recently published a broth microdilution method suitable for standardization including quality control (QC) strains for AST of M. hyorhinis [35,36]. In this study, we further evaluated this methodology by applying it to a variety of M. hyorhinis field isolates.

2. Materials and Methods

2.1. Bacterial Strains

The type strains M. hyorhinis DSM 25591 (ATCC 17981), Enterococcus faecalis DSM 2570 (ATCC 29212), and Staphylococcus aureus DSM 2569 (ATCC 29213) were all purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). A total of 37 M. hyorhinis field isolates originating from Austria, Hungary, and Germany were included in the study. They were all obtained from diagnostic samples taken from different body sites (joint or synovial fluid, serosa, lung, nasal cavity) submitted for microbiological examination between 2002 and 2021 (Table 1). After arrival at our laboratory, they were cultured in modified Friis broth [35], and their identity was confirmed by a Mycoplasma-specific nested PCR and subsequent sequencing of the PCR products [37].

2.2. Media

The isolates were cultured and tested in modified Friis broth, as previously described [35]. The modifications, with regard to the composition of the medium, as provided by the DSMZ, included (i) an increase in porcine serum to ensure the survival of the bacteria during the freeze–thaw cycle without the addition of cryopreservatives, (ii) the doubling of the phenol red solution to intensify the color of the broth and make color changes more visible, and (iii) the adaptation of the amount of yeast extract solution and deionized water to maintain the equilibrium of the provided nutrients [38].
Approximately 176 mL of the modified Friis broth, composed of 0.82 g of porcine Brain and Heart Infusion (BHI) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), 0.87 g of Difco Mycoplasma PPLO Broth w/o CV (Becton Dickinson (BD), Franklin Lakes, NJ, USA), 50 mL of filter sterilized Hank’s Balanced Salt Solution (as provided by [39]), and 78 mL of deionized water, was prepared. Before autoclaving at 121 °C, 1 bar for 15 min, the pH was adjusted to 7.4. After cooling, 40.6 mL of heat-inactivated porcine serum (Biowest SAS, Nuaillé, France), 4.49 mL of 25% autoclaved yeast extract solution (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 0.361 mL of a 1% filter sterilized phenol red solution (phenol red sodium salt, Carl Roth GmbH), and 0.285 mL of autoclaved deionized water were added. For solid media, 1% agar-agar (Carl Roth GmbH) was added before autoclaving.

2.3. Culture, Storage, and Quantification of M. hyorhinis

For each isolate, a minimum of two subcultures was produced. An aliquot of 500 μL of the primary culture was diluted into 5 mL of freshly prepared modified Friis broth. The cultures were incubated at 37 °C, 7.5% CO2, until a visible color change occurred, or for up to 14 days. The second subculture was quantified (see description below), aliquoted, and stored at −80 °C until further use.
The quantification of the broth culture was carried out according to CLSI recommendations and as previously described [35,40]. A 10-fold serial dilution over five steps was set up, and finally, 20 μL of each dilution step, as well as the original broth culture, were dropped onto modified Friis agar, air-dried, and then incubated at 37 °C, 5% CO2, for three to 14 days, until individual colonies could be detected. Agar plates that did not show colonies after a maximum of 14 days of incubation were discarded as negative, and cultivation had to be repeated. After incubation, 30 to 300 individual colonies were counted, and the number of CFU/mL of the initial broth culture was calculated.
For controlling the correct inoculum size for each AST approach, this method of quantification was applied. Since the expected inoculum density was less than the respective broth culture, only two dilution steps were carried out.

2.4. Antimicrobial Susceptibility Testing of M. hyorhinis

For quality control purposes of each experiment, fresh overnight cultures of the quality control strains (QCs) E. faecalis DSM 2570 and S. aureus DSM 2569 were used as previously described [36]. The inoculum suspensions were prepared according to CLSI standards [41]. We prolonged the incubation periods to account for the slower growth rate of M. hyorhinis, and the modified Friis broth was used as the test medium instead of the cation-adjusted Mueller Hinton broth [36]. In addition, the type strain M. hyorhinis DSM 25591 was used to control for Mycoplasma growth [35].
The inoculum of all M. hyorhinis strains (type strain and field isolates) was calculated to be 1 × 105 CFU/mL, with an acceptable range between 5 × 104 CFU/mL and 5 × 105 CFU/mL, as described in the CLSI document for AST of human mycoplasmas [40]. The calculated volume of the slowly thawed frozen stock culture was transferred into the prewarmed (room temperature) modified Friis broth.
The prepared inoculum suspensions were preincubated for 2 h at 37 °C, 7.5% CO2, to restore fitness of the freshly thawed bacteria. This step of preincubation is equivalent to the fresh overnight culture of nonfastidious bacteria to generate a metabolically active culture.
From the preincubated inocula, 50 μL of the inoculum suspension was transferred into each well of the commercially available Sensititre microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) as described in the national resistance monitoring program GERM-Vet [42]. The microtiter plates were sealed with an adhesive foil and incubated at 37 °C, ambient air, until a color change in the growth controls, indicative of bacterial growth, was observed. If no color change occurred after a maximum incubation time of 14 days, the microtiter plates were discarded, and the AST was denoted as invalid and had to be repeated. The following antimicrobial agents were investigated: clindamycin (0.03–64 mg/L), doxycycline (0.06–128 mg/L), enrofloxacin (0.008–16 mg/L), erythromycin (0.015–32 mg/L), florfenicol (0.12–256 mg/L), gentamicin (0.12–256 mg/L), marbofloxacin (0.008–16 mg/L), tetracycline (0.12–256 mg/L), tiamulin (0.03–64 mg/L), tilmicosin (0.06–128 mg/L), tulathromycin (0.06–32 mg/L), and tylosin (0.06–128 mg/L).
The MIC values of the M. hyorhinis type strain and field isolates were recorded as described earlier [35]. Since growing M. hyorhinis does not cause turbidity in broth media, the indicator phenol red was used in order to evaluate the microtiter plates visually. Therefore, the MIC was defined as the first well, where the color change from red (no growth) to yellow (growth) was incomplete. Trailing (orange) was observed but defined as no growth. The microtiter plates were examined daily with the unaided eye by the same person. When the growth controls showed the expected color change and the MIC values were recorded, the microtiter plates were incubated for an additional 24 h, and the results were recorded again. The endpoint of the AST was determined as the point when the color change was complete, and no major changes (more than ± one dilution step compared to the previous recording) of the recorded MIC values were observed. When major changes were noted, and consistency in the repeated readouts could not be achieved, the test was termed invalid and was repeated.

3. Results and Discussion

The antimicrobial agents were chosen according to therapeutic interest and the recommendations given by the CLSI, considering routine testing and relevance for swine [43]. Porcine mycoplasmas are expected to be susceptible to aminoglycosides, fluoroquinolones, phenicols, pleuromutilins, and tetracyclines [1,44]. Macrolide susceptibility is known to be variable [13]. Clindamycin was chosen as a representative for the class of lincosamides [43].
The QC strains E. faecalis DSM 2570 and S. aureus DSM 2569, as well as the type strain M. hyorhinis DSM 25591, were applied as published elsewhere, and MIC results were compliant with the published data [35,36].
The acquired MIC values of the 37 field isolates are shown in Table 2. Individual incubation times ranged from 72 h to 10 days.
For the antimicrobial agents gentamicin (0.25–2 mg/L), enrofloxacin (0.25–2 mg/L), marbofloxacin (0.25–2 mg/L), florfenicol (≤0.12–4 mg/L), erythromycin (2—≥64 mg/L), tiamulin (≤0.03–0.5 mg/L), doxycycline (≤0.06–1 mg/L) and tetracycline (≤0.12–2 mg/L), unimodal (i.e., with one peak) distributions of the MIC values were observed. A bimodal distribution (i.e., with two peaks) was noticed for the antimicrobial agents clindamycin (0.06–0.5 mg/L and 8–64 mg/L) and tilmicosin (0.12–2 mg/L and ≥256 mg/L). Tulathromycin and tylosin showed multimodal distributions (i.e., with multiple peaks).
In unimodal MIC distributions, as seen for tiamulin, doxycycline, and tetracycline, the respective bacteria represent the wild-type subpopulation. The isolates within the wild-type subpopulation are defined as those with no phenotypically detectable mechanisms of acquired resistance or reduced susceptibility for the antimicrobial agent being evaluated [45]. As a consequence, these wild-type isolates commonly display rather low MIC values. In our study, we also detected unimodal MIC distributions of M. hyorhinis isolates that displayed higher MICs of gentamicin, enrofloxacin, and marbofloxacin. As long as no clinical breakpoints are available that are applicable to M. hyorhinis and the aforementioned antimicrobial agents, as well as no information is provided about resistance-mediating mutations or resistance genes in the respective isolates, it is not possible to say whether the “wild-type” definition applies to these isolates. In bimodal and multimodal MIC distributions, usually, the subpopulation with the highest MIC values represents the non-wild-type subpopulation. It comprises isolates with presumed or known mechanisms of acquired resistance or reduced susceptibility for the antimicrobial agent being evaluated [45]. In our study, the same 15 M. hyorhinis isolates displayed tilmicosin MICs of ≥256 mg/L, tulathromycin MICs of 8–≥64 mg/L, tylosin MICs of 8–≥256 mg/L, erythromycin MICs of ≥64 mg/L, and clindamycin MICs of 8–64 mg/L.
According to the literature, M. hyorhinis is highly susceptible to gentamicin, fluoroquinolones, lincosamides, macrolides (except for erythromycin), pleuromutilins, and tetracyclines [13,16,46]. For most of these classes of antimicrobial agents, our results confirm these observations. However, high MIC values for macrolides and lincosamides were observed. About 40% (15/37) of the tested clinical isolates showed elevated MIC values (≥8 mg/L), occasionally above the highest available test concentrations. Reduced susceptibility to lincosamides and macrolides has also been observed in earlier studies [15,17,19]. Due to differing methodologies, the results are not directly comparable with those of other studies. However, the observed distributions of the tested field isolates in this study are mostly in accordance with other recent studies [14].
The high MICs observed for macrolides and lincosamides among the M. hyorhinis have serious clinical implications. Although clinical breakpoints that classify these isolates as “resistant” are currently not available, macrolides and lincosamides should not be used for therapeutic applications in these cases. The whole genome sequence of the type strain M. hyorhinis DSM 25591 showed the presence of a G2057A (Escherichia coli numbering) transition, which is known to confer resistance to 14-membered macrolides [27]. Földi and coworkers recently identified an A2058G transition via a mismatch amplification mutation assay in M. hyorhinis field isolates [47]. Moreover, an A2059G transition has been described in Japanese M. hyorhinis isolates [19].
When comparing the distribution of MIC values, the MIC50 and the MIC90 values with regard to time intervals (2002–2012 and 2013–2021), only minor trends in either direction could be observed (Table 3 and Table 4). Due to the small number of analyzed field isolates, statistical analysis was forgone. For the antimicrobial agents doxycycline, tetracycline, tiamulin, and erythromycin, no change could be observed. The distribution of MIC values of the tested field isolates between the two time intervals was comparable, and the MIC50 and MIC90 values were the same for both time periods. The MIC values of marbofloxacin were the only values that slightly increased from 2002–2012 to 2013–2021. This observation was also confirmed by an increase in both the MIC50 and MIC90 values. For the other antimicrobial agents (enrofloxacin, florfenicol, clindamycin, tilmicosin, tulathromycin, and tylosin), the recorded MIC values decreased, which was also observed in a decrease in the MIC50 values by one to five dilution steps for all aforementioned antimicrobial agents. The most pronounced decrease was observed for tylosin. A decrease in the MIC50 values indicates a larger number of isolates with lower MIC values. For tilmicosin, an at least two-fold decrease in the MIC90 values was also observed. The occurrence of bimodal and multimodal distributions, though not surprising, emphasizes the importance of the recognition that there are isolates with decreased susceptibility circulating within pig farms.
The observations in this study underline the importance of using a harmonized methodology and corresponding monitoring studies to register changes in antimicrobial susceptibility early on. In order to perform comparable surveillance studies, AST needs to be conducted in a harmonized manner. The recently proposed broth microdilution method appears to be a suitable AST method for M. hyorhinis field isolates of varying origins [35].

4. Conclusions

With increased accounting of antimicrobial substance application in farm animals, as well as ongoing monitoring of respective bacterial pathogens and their antimicrobial susceptibility profiles, the availability of a standardized methodology is of utmost importance. The recently published harmonized method was used to successfully test a variety of 37 M. hyorhinis field isolates. For most isolates, MIC values within the lower concentration ranges of the tested antimicrobial substances were observed. A subset of field isolates showed elevated MIC values for antimicrobial agents within the classes of macrolides and lincosamides. Although further studies are needed to establish clinical breakpoints, this is the first study confirming that the previously described broth microdilution AST method is not only suitable for the type strain M. hyorhinis DMS 25591 (ATCC 17981) but also for M. hyorhinis field isolates from various sources.

Author Contributions

Conceptualization, L.K., A.-K.S., C.K. and S.S.; methodology, L.K., A.-K.S. and S.S.; validation, L.K., A.-K.S. and S.S.; formal analysis, L.K. and A.-K.S.; investigation, L.K.; resources, D.H., J.V., M.G., J.S. and L.K.; data curation, L.K.; writing—original draft preparation, L.K.; writing—review and editing, A.-K.S., D.H., J.V., M.G., J.S., C.K. and S.S.; visualization, L.K.; supervision, A.-K.S. and S.S.; project administration, C.K. and S.S.; funding acquisition, C.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the German Federal Ministry of Food and Agriculture (BMEL) and the Federal Office for Agriculture and Food (BLE), grant number FKZ 2818HS015.

Data Availability Statement

All data are contained within the article.

Acknowledgments

The publication of this article was funded by the Open Access Publication Fund of the Freie Universität Berlin.

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1. Background information of M. hyorhinis type strain and field isolates used in this study.
Table 1. Background information of M. hyorhinis type strain and field isolates used in this study.
Isolate IDOrigin
CountryYearTissue
M. hyorhinis DSM 25591 type strainUnknown1955Nasal cavity
M. hyorhinis 906L02Austria2002Lung
M. hyorhinis 1089L03Austria2003Lung
M. hyorhinis 2158N03Austria2003Nasal cavity
M. hyorhinis 259L08Austria2008Lung
M. hyorhinis 2618L08Austria2008Lung
M. hyorhinis 82L09Austria2009Lung
M. hyorhinis 265L09Austria2009Lung
M. hyorhinis 1191L09Austria2009Lung
M. hyorhinis 386S09Austria2009Serosa
M. hyorhinis 1255L10Austria2010Lung
M. hyorhinis 1533S10Austria2010Serosa
M. hyorhinis 158L11Austria2011Lung
M. hyorhinis 207L11Austria2011Lung
M. hyorhinis 507S11Austria2011Serosa
M. hyorhinis 67L12Austria2012Lung
M. hyorhinis 12048421L13Austria2013Lung
M. hyorhinis 3174S13Austria2013Serosa
M. hyorhinis 3081L13Austria2013Lung
M. hyorhinis 1606S14Austria2014Serosa
M. hyorhinis 3631L14Austria2014Lung
M. hyorhinis 3661N14Austria2014Nasal cavity
M. hyorhinis 1191L15Austria2015Lung
M. hyorhinis 1438L15Austria2015Lung
M. hyorhinis 57L15Austria2015Lung
M. hyorhinis 3565L16Austria2016Lung
M. hyorhinis MycSu 75Hungary2017Synovial Fluid
M. hyorhinis MycSu 111Hungary2017Lung
M. hyorhinis 3044/1/19Germany2019Joint
M. hyorhinis 4812/1/19Germany2019Synovial Fluid
M. hyorhinis 135S19Austria2019Serosa
M. hyorhinis 4236G19Austria2019Joint
M. hyorhinis 3741/1/20Germany2020Serosa
M. hyorhinis 30S20Austria2020Serosa
M. hyorhinis 222S20Austria2020Serosa
M. hyorhinis 289S20Austria2020Serosa
M. hyorhinis 450S20Austria2020Serosa
M. hyorhinis T/0423263Germany2021Lung (BALF a)
a Bronchoalveolar lavage fluid.
Table 2. Distribution of the MIC values of 37 M. hyorhinis field isolates, including MIC50/90 values.
Table 2. Distribution of the MIC values of 37 M. hyorhinis field isolates, including MIC50/90 values.
Number of Isolates and MIC Values Obtained (mg/L) *
Antimicrobial Agent0.0080.0150.030.060.120.250.51248163264128256512MIC50MIC90
Gentamicin -67168------- 12
Enrofloxacin-----120142--- 0.51
Marbofloxacin-----23257--- 12
Florfenicol 11716111------ 0.50.5
Clindamycin -17104---11112 0.532
Erythromycin -------1239616 32≥64
Tilmicosin -125113------15 1≥256
Tulathromycin 9921-1-1-212 0.25≥64
Tylosin 579--1-3146-1 0.2564
Tiamulin 716851------- 0.060.25
Doxycycline 1381411------- 0.120.25
Tetracycline 2394-1------- 0.120.5
* Concentrations not included within the test panels are depicted as gray-shaded areas. When no color change was visible, the MIC value was set as equal to or lower than the lowest test concentration. If growth was visible in all tested concentrations, the result was set as equal or higher than the next serially higher MIC value (counts shown as white numbers within gray-shaded areas).
Table 3. Distribution of the MIC values of 15 M. hyorhinis field isolates obtained during the time period 2002–2012.
Table 3. Distribution of the MIC values of 15 M. hyorhinis field isolates obtained during the time period 2002–2012.
Number of Isolates and MIC Values Obtained (mg/L) *
Antimicrobial Agent0.0080.0150.030.060.120.250.51248163264128256512MIC50MIC90
Gentamicin -4272------- 12
Enrofloxacin-----1671--- 11
Marbofloxacin-----591---- 0.50.5
Florfenicol -591-------- 0.50.5
Clindamycin --431---1141 0.532
Erythromycin -------1--437 32≥64
Tilmicosin ---152------7 2≥256
Tulathromycin 25-1-----16 0.5≥64
Tylosin 132--1-2-22-1 864
Tiamulin 2643-------- 0.060.25
Doxycycline 545-1------- 0.120.25
Tetracycline 941-1------- 0.120.5
* Concentrations not included within the test panels are depicted as gray-shaded areas. When no color change was visible, the MIC value was set as equal to or lower than the lowest test concentration. If growth was visible in all tested concentrations, the result was set as equal or higher than the next serially higher MIC value (counts shown as white numbers within gray-shaded areas).
Table 4. Distribution of the MIC values of 22 M. hyorhinis field isolates obtained during the time period 2013–2021.
Table 4. Distribution of the MIC values of 22 M. hyorhinis field isolates obtained during the time period 2013–2021.
Number of Tests and MIC Values Obtained (mg/L) *
Antimicrobial Agent0.0080.0150.030.060.120.250.51248163264128256512MIC50MIC90
Gentamicin -2596------- 12
Enrofloxacin------1471--- 0.51
Marbofloxacin-----12172--- 11
Florfenicol 1127-11------ 0.250.5
Clindamycin -1373-----71 0.2532
Erythromycin --------23539 32≥64
Tilmicosin -1373-----71 0.564
Tulathromycin 742--1-1-16 0.12≥64
Tylosin 446----1124- 0.2564
Tiamulin 510421------- 0.060.25
Doxycycline 8491-------- 0.120.25
Tetracycline 1453--------- 0.120.5
* Concentrations not included within the test panels are depicted as gray-shaded areas. If growth was visible in all tested concentrations, the result was set as equal to or higher than the next serially higher MIC value (counts shown as white numbers within gray-shaded areas).
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Käbisch, L.; Schink, A.-K.; Hoeltig, D.; Verspohl, J.; Gyuranecz, M.; Spergser, J.; Kehrenberg, C.; Schwarz, S. Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates. Microorganisms 2023, 11, 2881. https://doi.org/10.3390/microorganisms11122881

AMA Style

Käbisch L, Schink A-K, Hoeltig D, Verspohl J, Gyuranecz M, Spergser J, Kehrenberg C, Schwarz S. Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates. Microorganisms. 2023; 11(12):2881. https://doi.org/10.3390/microorganisms11122881

Chicago/Turabian Style

Käbisch, Lisa, Anne-Kathrin Schink, Doris Hoeltig, Jutta Verspohl, Miklós Gyuranecz, Joachim Spergser, Corinna Kehrenberg, and Stefan Schwarz. 2023. "Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates" Microorganisms 11, no. 12: 2881. https://doi.org/10.3390/microorganisms11122881

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