Abstract
Several Mycoplasma species can form biofilm, facilitating their survival in the environment, and shielding them from therapeutic agents. The aim of this study was to examine the biofilm-forming ability and its potential effects on environmental survival and antibiotic resistance in Mycoplasma anserisalpingitidis, the clinically and economically most important waterfowl Mycoplasma species. The biofilm-forming ability of 32 M. anserisalpingitidis strains was examined by crystal violet assay. Biofilms and planktonic cultures of the selected strains were exposed to a temperature of 50 °C (20 and 30 min), to desiccation at room temperature (16 and 24 h), or to various concentrations of eight different antibiotics. Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains, with positive staining in more than half of them. Biofilms were found to be more resistant to heat and desiccation than planktonic cultures, while no correlation was shown between biofilm formation and antibiotic susceptibility. Our results indicate that M. anserisalpingitidis biofilms may contribute to the persistence of the organisms in the environment, which should be taken into account for proper management. Antibiotic susceptibility was not affected by biofilm formation; however, it is important to note that correlations were examined only in vitro.
Introduction
Mycoplasma anserisalpingitidis is the most important waterfowl-pathogenic Mycoplasma species, both clinically and economically (Volokhov et al., 2020). It was first isolated in 1983 from a gander with phallus inflammation in Hungary (Stipkovits et al., 1984; Varga et al., 1986), and since then, it has also been confirmed to be present in other European and Asian countries as well (Stipkovits et al., 1986; Sprygin et al., 2012; Gyuranecz et al., 2020; Grózner et al., 2021). Mycoplasma anserisalpingitidis can cause chronic infections in geese and ducks (Razin and Jacobs, 1992; Grózner et al., 2019). Clinically manifested mycoplasmosis occurs during excessive stress with cloaca and phallus inflammation, salpingitis and testicular atrophy being the most frequent signs (Stipkovits et al., 1986; Hinz et al., 1994; Stipkovits and Kempf, 1996; Dobos-Kovács et al., 2009).
At present, control of the disease comprises the improvement of housing conditions and antibiotic therapy. Unfortunately, rapid development of multi-drug resistance in M. anserisalpingitidis has been described (Grózner et al., 2016; Gyuranecz et al., 2020). Bacterial biofilm formation is likely to contribute to an increase in the incidence of clinically untreatable infections (Reid, 1999; Daubenspeck et al., 2020).
Biofilm formation occurs when bacteria switch from a planktonic state to a surface-attached state (Coffey and Anderson, 2014). Bacterial biofilms consist of cells attached to a substratum or to each other, encased within an extracellular matrix composed of an aggregation of polysaccharides, polypeptides, nucleic acids and lipids (McAuliffe et al., 2006; Colvin et al., 2011; Daubenspeck et al., 2020). The physical and chemical properties of the extracellular matrix constituents coupled with their particular interactions allow the matrix to shield the biofilm cells from adverse environmental conditions (e.g. heat, desiccation, chemicals, invasion by other bacteria). The matrix also provides the mechanical properties to protect the cells from external forces and to ensure that the biofilm community remains attached to a surface (Yan and Bassler, 2019).
Compared with planktonic cells, biofilms are commonly found to be 10 to 1,000 times more resistant to antibiotics (Costerton et al., 1999; Mah and O'Toole, 2001). Besides the impeded antibiotic penetration into biofilms, increased antibiotic tolerance likely arises from the altered physiology of the biofilm cells. Cells dwelling inside thick biofilms could be in stationary phase, as penetration of nutrients and oxygen are known to be limited due to consumption by peripherally-located cells. Time-dependent antibiotic killing of a bacterial population shows that actively growing cells are killed first whereas cells in stationary phase are killed at a much lower rate (Yan and Bassler, 2019).
Several Mycoplasma species are able to form biofilm, including Mycoplasma agalactiae, Mycoplasma bovis (McAuliffe et al., 2006), Mycoplasma gallisepticum (Chen et al., 2012) and Mycoplasma genitalium (Daubenspeck et al., 2020). An explanation for the chronicity associated with mycoplasmal diseases is that the bacteria are thought to persist in the host by the formation of an adherent biofilm, shielding the organism from host immune components such as antibodies or phagocytes, and therapeutic agents as well (Hall-Stoodley et al., 2004; McAuliffe et al., 2006; Feng et al., 2020). Furthermore, McAuliffe et al. (2006) provided evidence that biofilm formation facilitates the survival of Mycoplasma species in the environment as well. In this study, the biofilm-forming ability of M. anserisalpingitidis was examined by the crystal violet assay (McAuliffe et al., 2006). Additionally, the effect of biofilm formation on the resistance of M. anserisalpingitidis strains against heat, desiccation and eight different antibiotic agents was also investigated.
Materials and methods
Growth conditions and analysis of biofilm-forming ability of M. anserisalpingitidis
In total, 32 M. anserisalpingitidis strains were used in this study, including the ATCC BAA-2147 type strain, two Polish and 29 Hungarian field isolates (Table 1). All strains were stored at −70 °C until use. The medium used for the propagation of the organism consisted of Mycoplasma broth medium (pH 7.8) (Thermo Fisher Scientific Inc./Oxoid Inc., Waltham, MA, USA) supplemented with 0.5% (w/v) sodium pyruvate, 0.5% (w/v) glucose, 0.005% (w/v) phenol red and 0.15% (w/v) L-arginine hydrochloride. The number of microorganisms in the cultures used for the tests was determined by broth microdilution method (Hannan, 2000) and standardised (106 colour changing units, CCU mL−1) in order to obtain comparable results. The bacterial cultures were inoculated at 1:10 ratio in the broth medium in duplicates (cultures B and P), and were incubated at 37 °C for 48 h. Cultures B were left intact to grow biofilm, while cultures P were disrupted by vortexing, suspension and scratching the wall and bottom of the tubes three times daily, in order to prevent biofilm growth and remain in the form of planktonic cells in the broth. Analysis of biofilm growth was performed by crystal violet staining as described previously with minor modifications (McAuliffe et al., 2006). Tubes were emptied by discarding the broth cultures carefully, washed twice in tap water to remove non-adherent cells and stained with 0.5% crystal violet solution for 30 min. Washing steps were repeated before being left to dry at room temperature.
Background information and biofilm-forming ability of the 32 tested Mycoplasma anserisalpingitidis strains
| ID | Host | Sample | Origin | Year of isolation | Biofilm formationa |
| ATCC BAA-2147 | goose | phallus lymph | Hungary | 1983 | − |
| MYCAV47 | duck | lung and air sacs | Tázlár, Hungary | 2012 | ++ |
| MYCAV50 | goose | phallus | Cered, Hungary | 2013 | ++ |
| MYCAV55 | goose | ovarian follicle | Kiskunmajsa, Hungary | 2013 | − |
| MYCAV61 | goose | phallus lymph | Tatárszentgyörgy, Hungary | 2013 | − |
| MYCAV63 | goose | trachea | Sükösd, Hungary | 2013 | − |
| MYCAV66 | goose | phallus lymph | Tiszaföldvár, Hungary | 2014 | − |
| MYCAV67 | goose | phallus lymph | Szentes, Hungary | 2014 | ++ |
| MYCAV68 | goose | phallus lymph | Érpatak, Hungary | 2014 | − |
| MYCAV70 | goose | phallus lymph | Cered, Hungary | 2014 | ++ |
| MYCAV75 | goose | phallus lymph | Dömsöd, Hungary | 2014 | − |
| MYCAV76 | goose | phallus lymph | Tiszabábolna, Hungary | 2014 | ++ |
| MYCAV91 | goose | phallus | Hajdúsámson, Hungary | 2011 | ++ |
| MYCAV94 | goose | cloaca | Tiszabábolna, Hungary | 2012 | − |
| MYCAV160 | goose | phallus lymph | Érpatak, Hungary | 2015 | ++ |
| MYCAV161 | goose | phallus lymph | Szilaspogony, Hungary | 2015 | ++ |
| MYCAV162 | goose | phallus lymph | Encsencs, Hungary | 2015 | ++ |
| MYCAV176 | goose | phallus | Cered, Hungary | 2015 | − |
| MYCAV177 | goose | phallus | Cered, Hungary | 2015 | + |
| MYCAV178 | goose | ovarian follicle | Cered, Hungary | 2015 | + |
| MYCAV179 | goose | trachea | Apátfalva, Hungary | 2015 | − |
| MYCAV180 | goose | phallus | Kisbér, Hungary | 2015 | − |
| MYCAV270 | goose | cloaca | Szentes, Hungary | 2016 | ++ |
| MYCAV271 | goose | phallus lymph | Szentes, Hungary | 2016 | + |
| MYCAV415 | goose | phallus lymph | Hungary | 2017 | ++ |
| MYCAV494 | goose | phallus lymph | Hajdúsámson, Hungary | 2018 | + |
| MYCAV668 | goose | cloaca | Starogard Gdański, Poland | 1985 | ++ |
| MYCAV671 | goose | semen | Gödöllő, Hungary | 2019 | + |
| MYCAV675 | goose | cloaca | Zielona Góra, Poland | 1986 | + |
| MYCAV688 | goose | cloaca | Rém, Hungary | 2019 | − |
| MYCAV929 | goose | cloaca | Cered, Hungary | 2020 | + |
| MYCAV967 | goose | cloaca | Érpatak, Hungary | 2021 | − |
a no staining (−), slight staining (+), or strong staining (++) in the crystal violet assay.
Investigation of the effect of biofilm formation on heat resistance
To investigate the effect of biofilm formation on environmental survival, three Hungarian M. anserisalpingitidis strains with pronounced biofilm-forming ability (high-level staining in the crystal violet assay) were selected randomly (MYCAV70, MYCAV270, MYCAV415). As control, a clinical isolate exhibiting no biofilm formation was used (MYCAV55). After 48-h incubation at 37 °C, M. anserisalpingitidis biofilms and planktonic cell cultures were exposed to a temperature of 50 °C for 20 min or 30 min, then incubated at 37 °C for 1 h. After destroying the structure of biofilm by vortexing, suspension and scratching the wall and bottom of the tubes, the broth cultures were mixed well, divided into 500-µl aliquots, and stored at −70 °C until the counting of viable bacteria was performed. The number of colour changing units of the cultures was determined at 37 °C on 96-well microtitre plates with broth microdilution method by titrating a 10-fold dilution series of the Mycoplasma suspension. The plates were checked daily and the final results were read after 14 days. The highest dilution that showed a colour change was considered to contain 10° CCU mL−1 (Hannan, 2000).
Investigation of the effect of biofilm formation on resistance to desiccation
After 48-h incubation at 37 °C, biofilms and planktonic cell cultures of the selected four M. anserisalpingitidis strains (MYCAV70, MYCAV270, MYCAV415, and MYCAV55 as control) were harvested by centrifugation at 9,000×g for 9 min and the supernatant was removed. The pellet was exposed to desiccation at room temperature for 16 h or 24 h, then suspended in fresh broth and incubated at 37 °C for 1 h. After destroying the structure of biofilm by vortexing, suspension and scratching the wall and bottom of the tubes, the broth cultures were mixed well, divided into 500-µl aliquots, and stored at −70 °C until the counting of viable bacteria was performed as described above.
Investigation of the effect of biofilm formation on antibiotic susceptibility
At first, possible correlations between biofilm-forming ability and the initial or final minimal inhibitory concentration (MICi or MICf) values were analysed. MIC values against the 32 examined M. anserisalpingitidis strains were previously determined (Grózner et al., 2016, 2022). The MICi of each strain was defined as the lowest concentration of the antibiotic that completely inhibited the growth at the time of colour change in the growth control (broth culture without antibiotics), while MICf was evaluated at the end of the incubation period (14 days). Additionally, the effect of preventing biofilm formation on antibiotic susceptibility in five selected M. anserisalpingitidis strains with biofilm-forming ability and high MIC values of certain antibiotics was investigated (MYCAV47, MYCAV67, MYCAV70, MYCAV178, MYCAV271). After 48-h incubation at 37 °C, M. anserisalpingitidis biofilms and planktonic cell cultures were supplemented with fresh broth in order to normalise pH and nutrient content. The cultures were then exposed to various concentrations of the following antibiotics: enrofloxacin (Batch No.: BCBZ6597), oxytetracycline (Batch No.: BCBR8034V), doxycycline (Batch No.: BCBS3626V), tiamulin (Batch No.: BCBW6530), lincomycin (Batch No.: BCBW4661), tylosin (Batch No.: BCBX0715), tilmicosin (Batch No.: BCBT8086), and tylvalosin (Batch No.: TVN1906054). All products originated from VETRANAL (Sigma-Aldrich Inc., St. Louis, USA) except for tylvalosin (Aivlosin), which was purchased from ECO Animal Health Ltd. (London, UK). The tested concentrations were selected based on the results of our previous studies: 0.078–10 μg mL−1 for enrofloxacin, doxycycline and tiamulin, and 0.125–64 μg mL−1 for the other antibiotics (Grózner et al., 2016, 2022). Initial MIC values of each strain were determined by broth microdilution method (Hannan, 2000) with a small modification (using Eppendorf tubes instead of microtitre plates). Cultures were incubated at 37 °C and checked three times daily until acidic colour change or 14 days.
Results
Biofilm-forming ability of M. anserisalpingitidis
Crystal violet staining revealed great diversity in the ability of the 32 tested M. anserisalpingitidis strains to form biofilm (Table 1). Three categories were distinguished based on the observed strength of staining. Some strains (n = 12) showed strong staining below the air/liquid interface. Other strains (n = 7) showed only slight staining regarded as weaker biofilm formation, while numerous strains (n = 13) including the ATCC BAA-2147 type strain exhibited no staining (i.e. no biofilm formation) at all (Fig. 1).
Biofilm-forming ability of the 32 tested M. anserisalpingitidis strains (in triplicate) by crystal violet staining. Abbreviations: numbers: ‘MYCAV’ strain ID numbers; ref: type strain BAA-2147; neg: negative control (no bacteria)
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00029
Biofilm-forming ability of the 32 tested M. anserisalpingitidis strains (in triplicate) by crystal violet staining. Abbreviations: numbers: ‘MYCAV’ strain ID numbers; ref: type strain BAA-2147; neg: negative control (no bacteria)
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00029
Biofilm-forming ability of the 32 tested M. anserisalpingitidis strains (in triplicate) by crystal violet staining. Abbreviations: numbers: ‘MYCAV’ strain ID numbers; ref: type strain BAA-2147; neg: negative control (no bacteria)
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00029
Effect of biofilm formation on heat resistance
Biofilm-forming M. anserisalpingitidis cultures (cultures B) of the strains MYCAV70, MYCAV270 and MYCAV415 were found to be more resistant to heat (50 °C) than their cultures consisting of planktonic cells only (cultures P) (Table 2). At least ten times more viable cells of each strain were detected in cultures B compared to cultures P, except for MYCAV270 in which no difference was observed between cultures B and P after 20 min of heat exposure. In case of the strain MYCAV55, which was found to be unable to form biofilm, no difference could be observed between cultures B and P in the 20-min heat assay, while culture P contained more viable cells in the 30th minute of heating.
Viable count (CCU mL−1) of four tested Mycoplasma anserisalpingitidis strains after exposure to a temperature of 50 °C for 20 or 30 min, or to desiccation for 16 or 24 h
| Strain ID | Culture type | Heat 20 min | Heat 30 min | Dryness 16 h | Dryness 24 h |
| MYCAV70 | culture B | 103 | 102 | 103 | 102 |
| culture P | 102 | 101 | 101 | 101 | |
| MYCAV270 | culture B | 102 | 102 | 102 | 102 |
| culture P | 102 | 100 | 101 | 100 | |
| MYCAV415 | culture B | 103 | 102 | 102 | 101 |
| culture P | 101 | 100 | 0 | 0 | |
| MYCAV55 | culture B | 102 | 100 | 101 | 100 |
| culture P | 102 | 101 | 101 | 100 |
Culture B: biofilm-forming culture; culture P: planktonic cell form culture.
Effect of biofilm formation on resistance to desiccation
In agreement with the results of the heat assay, cultures B of the biofilm-forming MYCAV70, MYCAV270 and MYCAV415 strains were more resistant to drying than their cultures P (Table 2). At least a tenfold difference was detected in the number of viable cells between cultures B and P of each strain. In case of MYCAV415, no surviving planktonic cells could be detected after 16-h drying, whereas biofilm cells were still viable even after 24 h. No difference could be observed between the survival rates of cultures B and P of the MYCAV55 strain that was unable to form biofilm.
Effect of biofilm formation on antibiotic susceptibility
No correlation was observed between biofilm-forming ability and the MIC values determined previously (Grózner et al., 2016, 2022) in the 32 examined M. anserisalpingitidis strains, as high MIC values of each antibiotic were determined against strains lacking this ability as well (Table 3). Accordingly, the antibiotic susceptibility profiles of B and P cultures of the five tested M. anserisalpingitidis strains were highly similar (Table 4), and these strains were mostly inhibited by a concentration close to the previously determined MIC value (no more than one dilution step difference). A fourfold initial MIC difference was observed in three cases: enrofloxacin with MYCAV47 P and oxytetracycline with MYCAV70 B and MYCAV178 P. However, no greater than one dilution step difference was observed between cultures B and P of each strain, and this difference could not be consistently attributed to either biofilms or planktonic cultures.
Previously determined (Grózner et al., 2016, 2022) initial (MICi) and final (MICf) minimal inhibitory concentration values (µg mL−1) of the 32 tested Mycoplasma anserisalpingitidis strains
| Strain ID | Biofilma | enrofloxacin | oxytetracycline | doxycycline | tiamulin | lincomycin | tylosin | tilmicosin | tylvalosin | ||||||||
| MICi | MICf | MICi | MICf | MICi | MICf | MICi | MICf | MICi | MICf | MICi | MICf | MICi | MICf | MICi | MICf | ||
| MYCAV47 | ++ | 10 | >10 | n.a. | >64 | 0.312 | 5 | 0.625 | 2.5 | >64 | >64 | 4 | 16 | 64 | >64 | n.a. | 1 |
| MYCAV50 | ++ | n.a. | >10 | n.a. | >64 | 1.25 | 5 | 0.078 | 0.625 | n.a. | 4 | 0.5 | 2 | 0.5 | 2 | 0.25 | 0.25 |
| MYCAV67 | ++ | n.a. | 5 | >64 | >64 | 0.312 | 5 | 0.625 | 2.5 | >64 | >64 | >64 | >64 | >64 | >64 | 8 | 16 |
| MYCAV70 | ++ | n.a. | >10 | 32 | >64 | n.a. | >10 | n.a. | 0.625 | 2 | 4 | 4 | 16 | 64 | >64 | 0.25 | 1 |
| MYCAV76 | ++ | 2.5 | 5 | 16 | 64 | 1.25 | 5 | 0.312 | 1.25 | 8 | 8 | 1 | 8 | 16 | >64 | 0.25 | 0.5 |
| MYCAV91 | ++ | n.a. | 10 | n.a. | 64 | 0.625 | 2.5 | n.a. | 0.625 | n.a. | 8 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV160 | ++ | 5 | >10 | 64 | >64 | 0.625 | 10 | n.a. | 0.625 | 4 | 4 | n.a. | >64 | n.a. | >64 | 1 | 2 |
| MYCAV161 | ++ | 5 | >10 | 64 | >64 | 0.312 | >10 | 0.312 | 0.625 | 4 | 4 | 4 | 16 | >64 | >64 | n.a. | 0.5 |
| MYCAV162 | ++ | 1.25 | 2.5 | 32 | >64 | 0.039 | 5 | n.a. | 0.625 | 2 | 4 | 8 | 16 | 64 | >64 | n.a. | 0.5 |
| MYCAV270 | ++ | n.a. | n.a. | n.a. | n.a. | 1.25 | 10 | 0.312 | 0.625 | 0.5 | 4 | 8 | 64 | >64 | >64 | 0.5 | 2 |
| MYCAV415 | ++ | n.a. | n.a. | n.a. | n.a. | 1.25 | 10 | 0.625 | 2.5 | n.a. | n.a. | n.a. | n.a. | >64 | >64 | n.a. | n.a. |
| MYCAV668 | ++ | 0.312 | 0.625 | 0.25 | 2 | 0.078 | 0.312 | 0.156 | 0.312 | 0.25 | 1 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV177 | + | 2.5 | >10 | >64 | >64 | n.a. | 10 | n.a. | 0.625 | 2 | 4 | n.a. | >64 | n.a. | >64 | n.a. | 4 |
| MYCAV178 | + | 5 | 5 | >64 | >64 | 1.25 | 5 | n.a. | 0.312 | n.a. | 2 | 0.25 | 4 | 0.25 | >64 | n.a. | 0.5 |
| MYCAV271 | + | n.a. | n.a. | n.a. | n.a. | 1.25 | 10 | 0.625 | n.a. | 1 | 4 | n.a. | 64 | >64 | >64 | 0.5 | 8 |
| MYCAV494 | + | 5 | 10 | n.a. | n.a. | 0.078 | 0.312 | 0.312 | 1.25 | 0.25 | 1 | 2 | 4 | >64 | >64 | 0.25 | 0.25 |
| MYCAV671 | + | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |
| MYCAV675 | + | 0.156 | 1.25 | 0.25 | 1 | 0.039 | 0.312 | 0.312 | 1.25 | 0.5 | 4 | 8 | 64 | >64 | >64 | 0.5 | 2 |
| MYCAV929 | + | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |
| MYCAV55 | − | 2.5 | 10 | 1 | 8 | 0.078 | 0.312 | 0.156 | 0.625 | 1 | 4 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV61 | − | 5 | 5 | 0.5 | 2 | 0.039 | 0.078 | 0.312 | 0.132 | 2 | 2 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV63 | − | 0.625 | 1.25 | 0.5 | 4 | 0.039 | 0.312 | n.a. | 0.156 | 0.5 | 2 | n.a. | 4 | n.a. | 64 | 0.25 | 0.25 |
| MYCAV66 | − | 1.25 | 5 | >64 | >64 | >10 | >10 | 0.156 | 0.625 | 1 | 4 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV68 | − | 2.5 | 5 | 64 | >64 | 0.625 | 10 | n.a. | 5 | >64 | >64 | n.a. | >64 | n.a. | >64 | 8 | 16 |
| MYCAV75 | − | n.a. | 5 | 32 | >64 | n.a. | 10 | 0.156 | 0.625 | 2 | 4 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV94 | − | 2.5 | 2.5 | >64 | >64 | 5 | >10 | n.a. | 0.625 | 2 | 4 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| MYCAV176 | − | n.a. | 10 | >64 | >64 | n.a. | 5 | n.a. | 0.625 | 2 | 4 | n.a. | 64 | n.a. | >64 | n.a. | 4 |
| MYCAV179 | − | n.a. | 10 | 1 | 4 | 0.156 | 0.312 | 0.312 | 1.25 | 4 | 4 | n.a. | 4 | 1 | 4 | 0.25 | 0.5 |
| MYCAV180 | − | 5 | 5 | n.a. | 4 | 0.039 | 0.312 | 0.625 | 1.25 | n.a. | 4 | n.a. | 32 | 64 | >64 | 0.25 | 1 |
| MYCAV688 | − | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |
| MYCAV967 | − | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. |
| BAA-2147 | − | 0.312 | 1.25 | 16 | >64 | 5 | >10 | 0.312 | 1.25 | 1 | 2 | 0.25 | 0.5 | 0.25 | 0.25 | 0.25 | 0.25 |
a no staining (−), slight staining (+), or strong staining (++) in the crystal violet assay; n.a.: data not available.
Initial minimal inhibitory concentration values (MICi, µg mL−1) of different antibiotics against biofilm and planktonic cell cultures of five Mycoplasma anserisalpingitidis strains
| Strain ID | Culture type | enro | oxy | doxy | tiam | linco | tylo | tilm | tylv |
| MYCAV47 | culture B | 5 | 64 | 0.312 | 0.625 | >64 | 2 | 64 | 0.25 |
| culture P | 2.5 | 64 | 0.312 | 0.625 | >64 | 4 | 64 | 0.25 | |
| MYCAV67 | culture B | 5 | >64 | 0.312 | 0.312 | >64 | >64 | >64 | 8 |
| culture P | 5 | >64 | 0.312 | 0.625 | >64 | >64 | >64 | 8 | |
| MYCAV70 | culture B | 10 | 8 | 0.312 | 0.312 | 2 | 4 | 64 | 0.25 |
| culture P | 10 | 16 | 0.312 | 0.312 | 2 | 8 | 64 | 0.25 | |
| MYCAV178 | culture B | 5 | 64 | 1.25 | 0.156 | 1 | 0.25 | 0.25 | 0.25 |
| culture P | 5 | 32 | 1.25 | 0.156 | 1 | 0.25 | 0.25 | 0.5 | |
| MYCAV271 | culture B | 5 | 64 | 1.25 | 0.625 | 1 | 32 | >64 | 0.5 |
| culture P | 5 | 64 | 1.25 | 0.312 | 1 | 32 | >64 | 0.5 |
enro: enrofloxacin; oxy: oxytetracycline; doxy: doxycycline; tiam: tiamulin; linco: lincomycin; tylo: tylosin; tilm: tilmicosin; tylv: tylvalosin; culture B: biofilm-forming culture; culture P: planktonic cell form culture.
Discussion
Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains. Exploring the reason for the observed diversity within this species requires further investigations, with special attention to the genetic background. Mycoplasmas lack almost all genes commonly associated with biofilm formation in other bacterial species (McAuliffe et al., 2006). Nevertheless, in addition to participating in various attachment and binding processes, variable surface proteins have been found to be involved in biofilm formation in M. bovis (Thomas et al., 2003; Sachse et al., 1996, 2000). Likewise, polysaccharide capsule production may also influence biofilm formation and adhesion, and this phenotype may even differ among closely related mycoplasmas, such as Mycoplasma mycoides subsp. mycoides SC and LC (McAuliffe et al., 2006).
Biofilm-forming M. anserisalpingitidis cultures were found to be more resistant to heat at 50 °C than their cultures consisting of planktonic cells only. In line with the results of the heat assay, cultures B of the examined M. anserisalpingitidis strains were more resistant to desiccation than their cultures P. Mycoplasmas are usually considered to be of low resilience due to their reduced metabolic pathways and lack of cell wall. However, in agreement with the findings of McAuliffe et al. (2006) in ruminant-pathogenic Mycoplasma species, biofilm cultures of the tested M. anserisalpingitidis strains were found to be quite resistant to heat and desiccation. These results indicate that biofilm formation of M. anserisalpingitidis may contribute to the persistence of the organisms in the environment. This phenomenon is of great importance in the control of M. anserisalpingitidis infection, as increased environmental survival of their biofilms should be taken into account during the housing management and disinfection procedures.
Increased resistance of biofilms to antibiotics has been described previously in many studies (Costerton et al., 1999; Reid, 1999; Mah and O'Toole, 2001; Stewart, 2002; Sharma et al., 2019; Yan and Bassler, 2019; Daubenspeck et al., 2020). However, no correlation was shown in this study between biofilm-forming ability and the minimal inhibitory concentration values determined previously (Grózner et al., 2016, 2022) in the 32 examined M. anserisalpingitidis strains. In case of other bacteria, the nutrient constitution of the growth media was described to affect inhibitory antibiotic concentrations of sessile bacteria (Chen et al., 2020). This phenomenon may have contributed to the observed lack of correlation between biofilm-forming ability and antibiotic susceptibility in M. anserisalpingitidis strains, although propagation and examinations were carried out in the nutrient-rich medium which is also used for isolation from clinical samples. Nevertheless, MIC values are primarily affected by resistance-associated genes and mutations, and other resistance mechanisms, such as extracellular vesicles or efflux pumps may also play a role (Medvedeva et al., 2014; Antunes et al., 2015; Grózner et al., 2022). Antibiotic susceptibility of the five tested biofilm-forming M. anserisalpingitidis strains was not clearly affected by the experimental prevention of biofilm formation, in the same way as in the study of McAuliffe et al. (2006) with ruminant-pathogenic Mycoplasma species. However, it is important to note that these studies examined the effect of biofilm formation on the susceptibility to antibiotics under in vitro conditions, and the effect of the constitution of growth media was not evaluated.
At present, the control of mycoplasmosis in waterfowl consists of appropriate animal hygiene measures and antibiotic treatment, while elevated MIC values have been detected against this pathogen in many antimicrobials. Therefore, discovering the factors responsible for increased resistance to these actions is essential. This is the first study demonstrating the biofilm-forming ability of M. anserisalpingitidis and confirming the protective effect of this characteristic on environmental survival of the pathogen.
Acknowledgements
The Polish strains were kindly provided by Anna Sawicka. This work was supported by the KKP19 (129751) grant of the National Research, Development and Innovation Office, Hungary, the SA-27/2021 grant of the Eötvös Loránd Research Network, the Project no. RRF-2.3.1-21-2022-00001 which has been implemented with the support provided by the Recovery and Resilience Facility (RRF), financed under the National Recovery Fund budget estimate, RRF-2.3.1-21 funding scheme and the support provided by the Ministry of Innovation and Technology of Hungary (legal successor: Ministry of Culture and Innovation of Hungary) from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA-01 funding scheme of the National Research, Development and Innovation Office.
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