The Phenotypic Divergence and Potential Microevolution of a Dominant Mycoplasmopsis bovis ST-52 Clone Within a Closed Dairy Herd in China

TL;DR

A study in China found that a single Mycoplasmopsis bovis clone can evolve phenotypically within a closed dairy herd, affecting biofilm formation, antibiotic resistance, and cell invasion.

Contribution

The study provides evidence of microevolution in a dominant M. bovis clone within a closed herd, highlighting adaptive phenotypic changes.

Findings

A single ST-52 clone of M. bovis was found to exhibit significant phenotypic diversity in biofilm formation and antibiotic resistance.

Isolates showed varying abilities to invade different bovine-derived cell types.

Genetic identity was maintained despite phenotypic divergence, suggesting microevolution.

Abstract

Mycoplasmopsis bovis is a significant pathogen causing substantial economic losses in cattle, yet its within-herd microevolution remains poorly understood. This study aimed to characterize phenotypic and genomic variations within a dominant ST-52 clone circulating in a closed dairy herd. We isolated M. bovis from respiratory (n = 11) and milk (n = 5) samples. Phenotypic characterization included biofilm formation, antimicrobial susceptibility testing, and cellular invasion assays. Whole-genome sequencing was performed on four representative isolates to identify genetic variations. All isolates were genetically identical according to MLST (ST-52). However, significant phenotypic diversity was observed. Biofilm formation capacity varied significantly (OD595 from 0.25 to 1.10), and resistance to doxycycline was higher in nose swabs (100%) than milk isolates (20%). Cellular invasion assays demonstrated that all isolates could invade bovine-derived cells (MDBK, MAC-T, EBL, and PBMC), but the invasion efficiency differed by strain and cell type. These findings confirm the circulation of a single genetic lineage within a closed herd while highlighting significant phenotypic diversification in biofilm formation, antibiotic resistance, and cellular invasiveness. The results provide evidence consistent with microevolution and underscore the adaptive potential of M. bovis. This study underscores the adaptive potential of M. bovis during within-host colonization and cross-tissue transmission, providing critical insights for optimizing herd management and treatment strategies.

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Full text

1. Introduction

Mycoplasmopsis bovis (M. bovis) is a major pathogen responsible for bovine respiratory disease (BRD), mastitis, arthritis, and reproductive disorders in cattle, leading to substantial economic losses in the global cattle industry [1,2,3]. The pathogen transmits through multiple routes, including milk, aerosols, genital tract secretions, and semen, exhibiting strong host adaptability and the ability to cause long-term latent infections [4,5]. Due to the absence of a cell wall, M. bovis is inherently resistant to β-lactam antibiotics, and clinical isolates increasingly demonstrate resistance to commonly used drugs such as tetracyclines and macrolides, complicating control measures [6,7,8].

Current research primarily focuses on cross-regional epidemiological investigations and molecular typing of M. bovis. Multilocus sequence typing (MLST) analyses reveal that sequence type 52 (ST-52) is dominant in countries including China, Israel, Australia, and the United States, with particular association with elevated rates of mastitis in China [9,10,11]. However, most epidemiological studies compare M. bovis populations across different geographic regions or host groups, leaving a gap in systematic research on transmission dynamics, microevolution, and tissue adaptation-associated phenotypic variations within a closed cattle herd [12,13]. Within the same herd, M. bovis can cross barriers to infect different tissues (e.g., respiratory tract and mammary gland), and phenotypic differentiation may occur due to niche adaptation. This adaptation likely contributes to variations in treatment efficacy and the challenge of achieving eradication through therapy.

To address this knowledge gap, we conducted a study within a closed, self-replenishing dairy farm experiencing persistent M. bovis infections. We simultaneously collected respiratory swabs from calves and milk samples from lactating cows to isolate, identify, and compare M. bovis strains from different tissues. The comparative analysis included molecular characteristics, biofilm-forming capacity, antimicrobial susceptibility, and the ability to invade and survive in bovine-derived cells. This study aims to elucidate the genetic identity and phenotypic diversity of M. bovis within a single host population, exploring its microevolution under varying pressures. The findings are expected to provide a theoretical basis for designing more effective treatment protocols and eradication strategies in similarly managed herds.

2. Materials and Methods

2.1. Farm Background and Clinical Sample Collection

This study was conducted on a large-scale commercial dairy farm in China, which maintained a closed, self-replenishing herd with no history of cattle introduction from external sources, yet reported persistent M. bovis infections over time. The farm employed therapeutic interventions only, with no vaccination program, segregation of M. bovis-positive animals, or specific eradication measures in place. The farm’s therapeutic protocols for the reported conditions involved the use of antimicrobials. For bovine respiratory disease in calves, common treatments included antibiotics such as ceftiofur, penicillin, oxytetracycline, and kanamycin sulfate, alongside antipyretics like metamizole sodium and antipyrine and aminopyrine caffeine citrate. The traditional Chinese herbal formulation Maxingshigan San was also used for treatment. For clinical mastitis in lactating cows, treatments included cephalosporins such as ceftiofur and cephapirin. In December 2024, calves aged 1–4 months presented with clinical signs indicative of bovine respiratory disease, including depression, dyspnea, tachypnea, and coughing. Concurrently, lactating cows displayed mastitis-like symptoms, with affected quarters producing abnormal milk secretions that were thin, translucent, or pale in appearance, often containing fine flocculent material. A total of 28 nasal swabs from symptomatic calves and 53 milk samples from mastitic cows were aseptically collected. All samples were promptly transported under refrigerated conditions (4 °C) and arrived at the laboratory within 48 h post-collection for subsequent bacteriological analysis.

2.2. Isolation and Identification of Pathogens

Mycoplasmopsis bovis was isolated from clinical samples according to established methods with minor modifications [14]. Briefly, samples were initially processed and filtered through 0.45 μm pore-size membranes to remove contaminating bacteria. The filtrates were inoculated into PPLO (pleuropneumonia-like organism) broth (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 20% horse serum and incubated at 37 °C for 3–5 days. Bacterial growth was indicated by a color change in the medium from red to yellow, which was monitored daily. Subsequently, the cultures were streaked onto PPLO agar plates and incubated inverted at 37 °C under 5% CO_2_ for 3–5 days. After incubation, colonies were examined under an optical microscope at 40× magnification; those exhibiting the characteristic “fried-egg” morphology were selectively picked for purification. Additionally, Dienes staining was carried out according to the standard method [15], wherein blue-stained colonies were identified as Mycoplasma-positive. DNA was extracted using a SteadyPure Bacterial Genomic DNA Extraction Kit (Accurate Biology, Changsha, China) and stored at −20 °C until PCR testing. The isolates were further confirmed as M. bovis by M. bovis-specific real-time PCR using a commercial kit (Shandong Xinda Gene Technology Co., Ltd., Shangdong). The primers and probe sequences were designed in the 5′→3′ direction as follows:

• P59-F: 5′-GGAACAAACCACGGAAACTCTT-3′.
• P59-R: 5′-ACAATCGCTTTTTGTTGGTTACC-3′.
• P59-Probe: FAM-CCAGATCGCTTTCTG-MGB.

Amplification was performed in a 20 μL reaction volume containing 5 μL of DNA template, using a LightCycler 480 real-time PCR system (Roche, Basel, Switzerland).

2.3. Multilocus Sequence Typing (MLST)

Multilocus sequence typing (MLST) was performed on all 16 M. bovis isolates to determine their sequence types (STs) and investigate genetic relationships, following the standardized scheme available in the PubMLST database (http://pubmlst.org/mbovis/; accessed on 18 June 2025) [16]. This scheme targets internal fragments of seven housekeeping genes (dnaA, gltX, gpsA, gyrB, pta-2, tdk, tkt). Each gene was amplified by PCR using published primers and conditions [17]. The PCR products were purified and sequenced commercially by Qingdao Vland Biotech Co., Ltd. The obtained sequences for each locus were compared against the PubMLST allele database to assign allele numbers, and the combination of alleles defined the sequence type (ST) for each isolate. These sequences of external isolates were obtained from the PubMLST M. bovis database. Selection was designed to encompass the genetic diversity of M. bovis with a specific focus on the Chinese epidemic population. To assess phylogenetic relationships, the sequences of the seven loci were concatenated for each isolate. A phylogenetic tree was constructed from the concatenated alignment using the Maximum Likelihood method implemented in MEGA software (version 11) [18]. The robustness of the tree topology was evaluated using 1000 bootstrap replicates. The evolutionary relationships were further visualized and annotated using the Interactive Tree of Life (iTOL) platform (version 7) [19].

2.4. Biofilm Formation Assay

Biofilm formation is a critical virulence mechanism for M. bovis, contributing to persistence in the host. We quantitatively assessed this phenotype to evaluate phenotypic diversity among the isolates. The biofilm formation capacity of the M. bovis isolates was quantitatively evaluated using a standardized crystal violet staining assay in 96-well microtiter plates, as previously described with minor modifications [20]. Briefly, bacterial cultures were grown in PPLO broth to the mid-logarithmic phase, adjusted to a standardized optical density, and diluted in fresh PPLO broth supplemented with 0.08 g/mL salmon sperm DNA to enhance biofilm formation. Aliquots of 200 μL of the bacterial suspension were dispensed into the wells of a sterile 96-well flat-bottom polystyrene plate in triplicate, with uninoculated broth serving as the negative control. The plates were incubated statically for 72 h at 37 °C under a 5% CO_2_ atmosphere. After incubation, the planktonic cells were carefully removed, and each well was gently washed three times with phosphate-buffered saline (PBS) to remove non-adherent bacteria. The remaining adherent biofilms were heat-fixed at 65 °C for 30 min, stained with 200 μL of 0.1% (w/v) crystal violet solution for 20 min at room temperature, and then washed again with PBS to remove excess stain. After air-drying, the bound crystal violet was solubilized by adding 200 μL of 33% (v/v) glacial acetic acid to each well. The optical density of the resulting solution was measured at 595 nm (OD_595_) using a microplate reader. The OD values from the control wells were averaged and subtracted from the test wells. Based on the corrected OD_595_ values, the isolates were classified into one of four categories—non-biofilm former (OD < 0.22), weak former (0.22 ≤ OD < 0.44), moderate former (0.44 ≤ OD < 0.88), or strong former (OD ≥ 0.88)—according to the established criteria [21].

2.5. Antimicrobial Susceptibility Testing

The minimum inhibitory concentrations (MICs) of 14 antimicrobial agents against the Mycoplasmopsis bovis isolates were determined using the broth microdilution method, as adapted from previous studies [22] since no specific CLSI guidelines exist for Mycoplasmopsis bovis [23]. The panel of antimicrobials tested included valnemulin (VAL), spectinomycin (SPE), doxycycline (DOX), enrofloxacin (ENR), florfenicol (FLO), erythromycin (ERY), lincomycin (LIN), marbofloxacin (MAR), gamithromycin (GAM), tetracycline (TET), tildipirosin (TIL), tulathromycin (TUL), tiamulin (TIA), and tylvalosin (TYL). Bacterial suspensions were prepared from logarithmic-phase cultures, adjusted to approximately 10^5^ color-changing units (CCU)/mL, and inoculated into 96-well plates containing serial two-fold dilutions of each antimicrobial agent, achieving a final concentration range of 0.015625 to 512 μg/mL in a total volume of 200 μL per well. Each plate included growth control wells (inoculated broth without antimicrobials) and sterility control wells (uninoculated broth). The M. bovis type strain PG45 was included as a quality control organism in each assay run. The plates were incubated at 37 °C under appropriate atmospheric conditions and monitored until a distinct color change was observed in the growth control wells. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible bacterial growth. All MIC values were interpreted using published clinical breakpoints or epidemiological cut-off values where available, with minor modifications as noted in [22,24,25,26,27]. The entire experiment was independently performed in triplicate to ensure the reliability and reproducibility of the results.

2.6. Growth Kinetics Analysis

To characterize the in vitro growth dynamics of M. bovis isolates, growth curve analyses were performed as described previously [28] on representative strains isolated from the respiratory tract (H1, H6) and milk (R1, R4). For the growth curve inoculum, bacterial stocks were standardized using the color change unit (CCU) method, which is a more practical and commonly used method for initial standardization of fastidious microorganisms like Mycoplasmas. The bacterial stocks were inoculated into fresh PPLO broth supplemented with 20% horse serum and incubated at 37 °C until the mid-logarithmic phase. The cultures were then diluted to 100-fold CCU and used to inoculate fresh medium in 15 mL tubes, with uninoculated broth serving as a blank control.

The growth kinetics were monitored at 12 h intervals over a 96 h period. At each time point, aliquots from triplicate cultures were serially diluted and plated onto PPLO agar plates to determine the viable bacterial count (colony-forming units per milliliter (CFU/mL)) using the plate count method. Given the documented difficulty in enumerating M. bovis by classical colony counting due to its small colony size, colony identification and counting were handled with specific care. After serial dilution, cultures were plated onto PPLO agar and incubated for 5–7 days. Colonies exhibiting the characteristic “fried-egg” morphology under 40× microscopic examination were selectively counted. Counts were performed in triplicate for each dilution, and the mean CFU/mL was calculated. To assess reproducibility, the growth curve experiment was independently repeated three times, and the data shown are representative of consistent patterns observed across replicates. The resulting growth curves were plotted as log10 CFU/mL versus time. Key growth phases, including the lag phase, logarithmic (exponential) phase, stationary phase, and decline phase, were identified for each strain based on the plotted data. The time point corresponding to the onset of the stationary phase was defined as the optimal harvest time for subsequent experiments requiring maximum bacterial yield. The strains used in the subsequent experiments were collected during the logarithmic growth phase.

2.7. Cellular Invasion and Intracellular Survival Assays

The capabilities of M. bovis isolates to invade and survive within host cells were evaluated using four bovine-derived cell lines: Madin–Darby bovine kidney (MDBK) epithelial cells, bovine mammary epithelial cells (MAC-T), bovine embryonic lung (EBL) cells, and peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from heparinized bovine venous blood using Ficoll density gradient centrifugation according to standard protocols [29]. Prior to infection, cells were seeded in 6-well cell culture plates and incubated until they reached 80–90% confluence. The cells were then infected with the M. bovis PG45 reference strain and selected isolates (H1 and H6 from the respiratory tract, n = 2; R1 and R4 from milk, n = 2) at a multiplicity of infection (MOI) of 100, as established in previous studies [30]. Following a 4 h incubation period at 37 °C with 5% CO_2_ to allow for bacterial invasion, the cell culture medium was replaced with fresh medium containing gentamicin (50 μg/mL) to kill any extracellular bacteria. After a 1 h gentamicin treatment, the cells were thoroughly washed three times with phosphate-buffered saline (PBS) to remove the antibiotic and any residual non-internalized bacteria. To quantify the invasion rate, the infected cells were subsequently trypsinized and lysed with sterile distilled water at 1, 3, and 5 h post-infection (hpi). The lysates were serially diluted and plated onto PPLO agar plates to determine the number of internalized viable bacteria via colony counting. The invasion rate was calculated as a percentage using the following formula: (Number of CFUs recovered from lysates / Initial CFU inoculum) × 100%. For the intracellular survival assay, a parallel set of infected cells were trypsinized and lysed as previously mentioned at later time points (0, 24, 48, and 72 hpi). The viability of the intracellular bacteria was similarly assessed by plating the lysates and counting the resultant colonies, allowing for the construction of a survival curve over time.

2.8. Whole-Genome Sequencing and Bioinformatic Analysis

To investigate the genetic basis for the observed phenotypic variations, whole-genome sequencing was performed on four representative M. bovis isolates, encompassing both respiratory (H1, H6) and mammary (R1, R4) origins. High-quality genomic DNA was extracted from bacterial cultures during the mid-logarithmic growth phase using a commercial DNA extraction kit (Fast Pure Viral DNA/RNA Mini Kit, Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), following the manufacturer’s instructions. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and qualified samples were submitted to Tsingke Biotechnology Co., Ltd. for draft genome sequencing on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), generating 150 bp paired-end reads. Raw sequencing data were subjected to quality control using Fastp (v0.23.2) [31] to remove adapter sequences and low-quality reads. The genome assembly process was performed using SPAdes (v3.15.5) [32] with default parameters for bacterial genomes, generating scaffold sequences. SNP/indel calling was performed with GATK v4.3.0 HaplotypeCaller (–ploidy 1) after duplicate removal (Picard 2.27) and base quality recalibration. Assemblies were compared against the M. bovis PG45 reference genome (NCBI accession: NC_014760).

The assembled genomes were annotated by comparing against the NCBI Non-Redundant (NR) protein database using BLASTP (2.14.0) (E-value < 1 × 10^−5^) for gene function prediction. To identify antibiotic resistance genes, the assembled scaffold sequences were aligned against the Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca/; accessed on 10 June 2025) using Resistance Gene Identifier (RGI) software (6.0.4). Furthermore, the nucleotide sequences of key drug target genes associated with resistance in M. bovis—specifically, the 16S rRNA (rrs), 23S rRNA (rrl), DNA gyrase (gyrA, gyrB), and topoisomerase IV (parC, parE) genes—were extracted from the annotated genomes. Mutations potentially conferring resistance were identified by multiple sequence alignment of the isolate sequences and those of the reference susceptible strain PG45, focusing on quinolone resistance-determining regions in gyrA and parC and specific positions in rRNA genes associated with macrolide and tetracycline resistance.

The specific virulence and adhesion genes were selected for analysis based on their well-characterized roles in M. bovis pathogenesis, as reported in the literature [33,34]. The adhesion genes (P30, P48, P37, P59, EF-Tu) are known to mediate attachment to host cells, a critical first step in infection [35,36,37,38,39]. The variable surface protein genes (vspX, vspY2, vpma) are implicated in antigenic variation, allowing the bacterium to evade the host immune system [40,41,42]. Genes such as P48 have also been associated with virulence. The aim of profiling these genes in the four sequenced isolates was to conduct a preliminary investigation into whether variations in their presence or absence could be linked to the observed phenotypic differences.

2.9. Statistical Analysis

All experiments included in this study were performed with a minimum of three independent biological replicates. Data are presented as the mean ± standard deviation (SD) unless otherwise specified. Prior to statistical testing, the normality of all data distributions was assessed using the Shapiro–Wilk test, and the homogeneity of variances was verified using Levene’s test. For comparisons between two independent groups meeting assumptions of normality and equal variance, an unpaired two-tailed Student’s t-test was used. For comparisons between three or more groups, a one-way analysis of variance (ANOVA) was performed. If the ANOVA result was statistically significant, Dunnett’s post hoc test was applied for multiple comparisons against a single control group. All statistical analyses were conducted using GraphPad Prism software (version 10.1.2; GraphPad Software, Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant for all tests.

3. Results

3.1. Isolation and Identification of Pathogens

A total of 16 M. bovis strains were successfully isolated from 28 nasal swabs collected from symptomatic calves and 53 milk samples collected from mastitic cows in a closed dairy farm with an overall isolation rate of 19.6% (16/81). All isolates cultured in PPLO broth supplemented with 20% horse serum exhibited a typical color change from red to yellow after 3–5 days of incubation. On PPLO agar plates, microscopic examination (40× magnification) revealed circular, transparent-edged colonies with dense, elevated centers, characteristic of “fried-egg” morphology (Figure 1a). Dienes staining confirmed the colonies as M. bovis-positive by displaying blue coloration (Figure 1b). All isolates were confirmed positive by qPCR with Ct values < 35. The data are shown in Table S1.

3.2. Multilocus Sequence Typing (MLST)

Multilocus sequence typing (MLST) based on seven housekeeping genes (dnaA, gltX, gpsA, gyrB, pta, tdk, tkt) revealed that all 16 Mycoplasmopsis bovis isolates (respiratory isolates, n = 11; milk-derived isolates, n = 5) shared an identical allelic profile (5, 3, 2, 3, 5, 3, 4), unanimously classifying them as sequence type 52 (ST-52). The phylogenetic analysis shown in Figure 2 further demonstrates that all isolates formed a monophyletic cluster within the ST-52 clade, which is recognized as the predominant epidemic lineage circulating in China. These molecular findings provide compelling evidence for the circulation of a single, genetically homogeneous clonal population of M. bovis within this closed dairy herd, indicating limited genomic diversification despite differences in tissue origin or clinical manifestation.

3.3. Biofilm Formation Assay

Quantitative assessment of biofilm formation revealed significant phenotypic diversity among the 16 genetically identical M. bovis isolates (one-way ANOVA). Based on the optical density (OD595) measurements (mean ± SD), the isolates were categorized as strong, moderate, or weak biofilm formers (Figure 3). Post hoc analysis (Tukey’s test) confirmed that the strong biofilm producers (H6, H11, R1) formed significantly more biofilm than the weak producers (H1, R4) (p < 0.01 for all comparisons). Notably, both strong and weak biofilm producers were identified among respiratory-derived and milk-derived isolates, indicating that biofilm formation capacity varied between isolates. However, with only 16 isolates, we cannot exclude a hidden niche association and therefore refrain from an ecological interpretation.

3.4. Antimicrobial Susceptibility Testing

The antibiotic susceptibility test results of the 16 Mycoplasmopsis bovis isolates are shown in Figure 4 as a heat map, and the detailed data are shown in Table S2. All 16 M. bovis isolates were uniformly susceptible to valnemulin and tiamulin. However, universal resistance was observed against macrolides, including erythromycin, gamithromycin, tilmicosin, tulathromycin, and tylosin, with minimum inhibitory concentrations (MICs) at or above their respective clinical resistance breakpoints. In contrast, all strains were susceptible to spectinomycin, enrofloxacin, florfenicol, and tetracycline, as their MICs fell below established breakpoints. Divergent resistance profiles emerged for other antibiotics: 75% (12/16) isolates were resistant to doxycycline, 12.5% (2/16) showed resistance to lincomycin, and 2 isolates (2/16) were resistant to marbofloxacin. A significant difference in resistance to doxycycline was observed between isolates from different origins. Specifically, 100% (11/11) of respiratory isolates were resistant, compared with 20% (1/5) of milk-derived isolates (Fisher’s exact test, p < 0.05). Notably, valnemulin demonstrated consistent activity against all isolates at low concentrations, suggesting its potential clinical utility despite the lack of a definitive resistance breakpoint. This phenotypic diversity implies potential microevolution under antibiotic selection pressure despite the genetic uniformity of the strains.

3.5. Growth Kinetics

The in vitro growth kinetics of four representative isolates of M. bovis, two from the respiratory tract (H1, H6) and two from milk (R1, R4), were characterized over an 96 h period to determine whether tissue origin influenced bacterial proliferation (Figure 5). Quantitative analysis revealed consistent growth dynamics across all tested strains, irrespective of their isolation source. Each strain exhibited a short lag phase of less than 12 h, followed by a robust logarithmic growth phase that spanned from 12 to 48 h post-inoculation. The cultures entered the stationary phase at approximately 60 h, achieving a peak mean bacterial density of 10^8.6^ CFU/mL.

Notably, the growth curves of the respiratory isolates and the milk-derived isolates were virtually superimposable, reaching the stationary phase simultaneously. This observation indicates that the intrinsic capacity for in vitro proliferation is conserved among the ST-52 clones within this herd and is not a primary phenotype differentiated by microevolution in different host tissues.

3.6. Cellular Invasion and Intracellular Survival

The gentamicin protection assay demonstrated that four isolates and the PG45 strain could invade the four cell types, but with significantly different efficiencies (Figure 6; detailed data are provided in Table S3). Specifically, in MDBK cells (Figure 6a), all isolates except R4 exhibited their highest invasion rates among all cell lines. The time to peak invasion varied: H1, H6, and R1 reached their peak invasions at 5, 1, and 3 h, respectively, and their peak invasion rates were all significantly higher than that of R4 (3 h) (p < 0.01). For MAC-T cells (Figure 6b), the isolates from milk (R1 and R4) showed significantly higher invasion rates at 1 h than those from nasal swabs (H1 and H6) (p < 0.01), with R1 demonstrating the strongest invasive potential. However, the peak invasion rates among all isolates in MAC-T cells were not significantly different (p > 0.05). A different pattern was observed in EBL cells (Figure 6c), where the peak invasion rate of isolate H6 was significantly lower than those of H1, R1, and R4, all of which peaked at 5 h (p < 0.01).

The intracellular survival kinetics further highlighted stark differences between cell types. For the proliferation count in MDBK cells, H1 and H6 isolated from nose swabs reached the peak at 24 h (Figure 7a,e), while R1 and R4 isolated from milk reached the peak at 48 h (Figure 7i) and 72 h (Figure 7m), respectively. Despite the quantitative differences, the intracellular survival trends among the four isolates and the PG45 strain are similar in MAC-T (Figure 7b,f,j,n,r) and EBL (Figure 7c,g,k,o,s) cells. However, the titer of the four isolates peaked at 24 h and subsequently declined in PBMCs, but the titers of H6 and R4 remained above 10^8^ CFU/mL at 72 h (Figure 7h,p), which was significantly higher than those of H1 (Figure 7d), R1 (Figure 7l) and the PG45 strain (p < 0.05). Interestingly, the four isolates exhibited a stronger survival capacity at 72 hpi in PBMCs compared with the PG45 strain (Figure 7t).

3.7. Genomic Analysis of Antimicrobial Resistance and Virulence Determinants

To elucidate the genetic basis for the observed phenotypic diversity, draft genome sequencing was performed on four representative isolates (H1 and H6 from nose swabs; R1 and R4 from milk). The genomic characteristics of isolated M. bovis strains are described in Table S4 of the Supplementary Materials. To correlate phenotype with genotype, we screened the four genome-sequenced ST-52 isolates (H1, H6, R1, R4) for previously described resistance determinants (Table 1). A detailed overview of the identified resistance genes and their specific mutation sites is provided in Table S5. All four isolates harbored the 23S rRNA mutations A573T and G788A that have been associated with macrolide resistance; this perfectly matches the universal phenotypic resistance to erythromycin, gamithromycin, tulathromycin, and tylvalosin (Table S2). Mutations A952T and A954T in the 16S rRNA gene were detected in all four isolates, with an additional A1000G mutation identified in H6 and R4 (Table 1). These mutations, however, did not correlate with high-level tetracycline resistance in M. bovis (Table S2), which is consistent with the observed tetracycline-sensitive phenotype. The previously unreported mutations may contribute to the doxycycline resistance observed in isolates H1 and H6 (Table S2). No canonical mutations in gyrA (Ser83Phe) or parC (Asp84Asn, Ser80Ile) were identified in any of the four isolates (Table S5 and Table 1). All isolates remained susceptible to enrofloxacin and marbofloxacin (Table S2). This suggests that the detected mutations parC (Arg91Ser) and gyrA (A267G) are not associated with fluoroquinolone resistance in M. bovis (Table 1).

All four ST-52 isolates carried the canonical adhesin genes P30, P48, P37, P59, and EF-Tu (Table 2), indicating that the basic toolkit required for host cell attachment is genetically conserved within this clonal lineage. In contrast, genes encoding variable surface lipoproteins showed isolate-specific patterns: vspX was uniformly present in all isolates, while vspY2 was present only in H1, whereas vpma was absent in H6 but present in H1, R1, and R4 (Table 2). These differentially distributed genes are candidate contributors to the observed differences in cellular invasion efficiency and cell tropism (Figure 6 and Figure 7).

4. Discussion

This study provides evidence consistent with microevolution by demonstrating significant phenotypic divergence within a clonal population of M. bovis. While the genomic analysis was limited to four isolates and thus cannot fully represent population-wide genomic dynamics, the observed phenotypic differences in biofilm formation, antimicrobial resistance, and cellular invasion across all 16 isolates suggest the occurrence of adaptive changes within the host.

Multilocus sequence typing (MLST) confirmed that all isolates belonged to ST-52, suggesting a single clonal origin and transmission within the herd. The growth curves of the respiratory isolates and the milk-derived isolates were virtually superimposable. Sequence type (ST) typing is a classification method based on housekeeping genes [17] which are closely associated with the proliferation of Mycoplasma but exhibit no direct link to its pathogenicity or other virulence-related traits. The significant phenotypic diversification observed within the clonal population, particularly the strong biofilm-forming capability exhibited by isolates H6 (respiratory) and R1 (mammary), underscores a key adaptive strategy that is critical for persistence within their respective host niches. Biofilms function as complex physical barriers that reduce antibiotic penetration and confer increased tolerance to host defenses, which likely explains the persistent infections and treatment failures associated with such strains [43]. For the respiratory isolate H6, forming a robust biofilm may be an essential adaptation to evade mucociliary clearance in the respiratory tract, while for the mammary isolate R1, this phenotype could be a strategy to withstand immune factors and antimicrobial compounds in the mammary gland. This strain-specific virulence trait, which is not a universal characteristic of the sequence type, highlights the limitation of relying solely on genetic typing for virulence assessment and emphasizes that functional phenotypes are paramount for understanding within-host adaptation. Consequently, optimizing treatment strategies requires not only considering antimicrobial susceptibility profiles but also targeting biofilm-forming strains with specific therapies aimed at disrupting biofilm integrity [44].

All isolates remained universally resistant to macrolides and highly resistant to deoxytetracycline, including 91.67% (11/12) from nose swabs and 8.33% (1/12) from milk. This numerical difference is compatible with differential antibiotic exposure between calves (frequently treated for BRD) and lactating cows (mastitis therapy often relies on β-lactams), indicating intense antibiotic selection pressure within the herd. Genomic analysis identified mutations in the 23S rRNA gene (A573T and G788A), which represent well-documented mechanisms associated with macrolide resistance. Interestingly, mutations in the 16S rRNA gene (A952T and A954T) and in parC and gyrA genes did not correlate with tetracycline or fluoroquinolone resistance, as reported previously. Current studies have established correlations between Mycoplasmopsis bovis resistance to different classes of antibiotics and specific mutations in target genes: reduced susceptibility to tetracyclines is associated with alterations such as A965T and A967T/C at the Tet-1 site of the 16S rRNA-encoding genes (rrs3, rrs4), with double or triple mutations mediating resistance [45]; resistance to macrolides (e.g., tylosin, tilmicosin) involves synergistic actions of the G748A mutation in 23S rRNA (rrl3, rrl4) and species-specific nucleotide changes in the central loop of domain II [46]; resistance to aminoglycosides and fluoroquinolones is linked to mutations in the rrs3/rrs4 genes and gyrA/parC genes, respectively, with dual mutations inducing high-level resistance phenotypes [47]. Our targeted search for known resistance-associated mutations in the four sequenced isolates identified several alterations in key genes. For instance, mutations such as A573T and G788A in the 23S rRNA gene were found in isolates exhibiting macrolide resistance. However, the expected mutations in DOX-resistant isolates H6 were absence, and there was a lack of canonical 16S rRNA mutations in the tetracycline-resistant isolate. The strongest biofilm former H6 (from nose swabs; OD_595_ = 1.10 ± 0.04) carried the full set of 16S rRNA mutations (A952T/A954T/A1000G) and exhibited high-level doxycycline resistance (MIC = 32 μg/mL), whereas the weakest biofilm former R4 (from milk; OD_595_ = 0.25 ± 0.02) lacked these mutations and remained doxycycline-susceptible (MIC = 0.5 μg/mL) (Figure 3 and Table S2). These preliminary observations underscore the need for future studies that employ whole-genome approaches on a larger scale to uncover novel mechanisms, such as efflux pumps or ribosomal protection proteins, which may be operating in these strains [25]. The variability in resistance profiles among isolates sharing an identical MLST profile (ST-52) further suggests microevolution in response to local selective pressures, possibly influenced by differential antibiotic exposure between respiratory and mammary niches.

To investigate host cell interactions in detail, a subset of four representative isolates—H1 and H6 (nose swabs) and R1 and R4 (milk)—were selected from the total collection (n = 16) for cellular invasion and intracellular survival assays, based on their distinct phenotypic profiles in biofilm formation and antimicrobial susceptibility. Analysis of invasion efficiencies revealed clear cell type and strain-specific dependencies. Specifically, the milk-derived isolate R4 demonstrated the highest invasion rate for PBMCs (36%), whereas the respiratory isolate R1 showed a preferential tropism for MAC-T cells (10%). All isolates carried core adhesin genes (e.g., P30, P48, P37), which mediate host cell attachment. The P48 protein, a highly conserved alkaline membrane protein family in M. bovis, has been reported to participate in the adhesion, proliferation, and virulence of M. bovis. However, the underlying mechanisms remain poorly understood [48]. EF-Tu, a highly conserved constitutively expressed protein, has previously been shown to prevent M. bovis infection [49]. However, differential expression of variable surface proteins (e.g., vspY2, vpma) may explain the observed tissue tropism [50]. These proteins encode surface lipoproteins that enable bacterial strains to evade host immune clearance through frequent antigenic variation [51]. The unique presence of vspY2 in H1 (Table 2) coincided with its superior invasion of embryonic bovine lung cells (15.1% at 5 h), supporting the notion that a variable surface protein repertoire, rather than core adhesin content, drives tissue-specific tropism within an otherwise clonal ST-52 population.

The phenotypic diversification within a clonal population underscores the need for tailored intervention strategies. For instance, biofilm-forming strains may require treatments that disrupt biofilm integrity, while antibiotic therapy should be guided by susceptibility testing to avoid ineffective drug use. The high resistance to macrolides and tetracyclines calls for restricted use of these classes and emphasizes the value of drug sensitive testing in clinical management. This study has several limitations that should be considered. First, the genomic analysis was restricted to only four isolates, which prevents a comprehensive assessment of the molecular basis for phenotypic diversification across the entire population. Second, the low number of milk-derived isolates (n = 5) limits robust statistical comparisons between tissue-specific niches. Third, the cross-sectional design, without longitudinal sampling, precludes direct observation of the temporal dynamics of microevolution. Finally, the focus on a single herd affects the generalizability of the findings to other cattle populations with different management practices. Future studies should expand to multiple herds and incorporate multi-omics approaches (e.g., transcriptomics and proteomics) to elucidate the molecular mechanisms driving phenotypic diversification. Additionally, longitudinal sampling would help track microevolution dynamics over time and in response to management changes.

5. Conclusions

In summary, this study demonstrates that Mycoplasmopsis bovis can undergo significant phenotypic diversification, including variations in biofilm formation, antibiotic resistance, and cellular invasion efficiency, within a genetically clonal ST-52 population in a closed dairy herd. Although the genomic basis of this microevolution requires further large-scale investigation, the observed niche-specific adaptations underscore the need to integrate phenotypic assessments with genetic data for designing targeted control strategies. These findings emphasize the importance of precision antibiotic use and biofilm disruption approaches to effectively manage M. bovis infections, while also highlighting the adaptive potential in response to host environmental pressures.