Biological characterization, sequence type distribution and drug resistance profiling of Mycoplasma hyorhinis field isolates from pigs in Chongqing, China

Mycoplasma hyorhinis is a ubiquitous pathogen of swine that causes polyserositis and polyarthritis and is also associated with conjunctivitis, meningitis, pneumonia, and abortions. This microorganism is a high prevalence pathogen in Chinese swine herds. However, few studies on M. hyorhinis have been reported in Chongqing, China. The overuse of antimicrobials has led to an increased risk of antimicrobial resistance, but a series of Chinese herbal monomers exhibited antibacterial activity to drug-resistant bacteria, including mycoplasmas. The aim of the study was to determine the prevalence, sequence types, growth kinetics, susceptibility to antimicrobials and Chinese herbal monomers, and relationships between the phenotypes and genotypes in terms of the resistance of M. hyorhinis to fluoroquinolones, macrolides and lincomycin. A total of 28 M. hyorhinis strains were recovered from the lungs of 404 slaughtered pigs. The isolates belonging to 11 novel STs, ST226, ST227, ST228, ST229, ST230, ST260, ST261, ST262, ST263, ST264 and ST265, were clustered separately from other reference isolates in the database. The growth kinetic of each isolate was generated, and the maximum color changing unit (CCU) values of isolates varied from 10¹² to 10²⁰ CCU/ml. In vitro susceptibility testing showed that the isolates were inhibited by low concentrations of tiamulin (MIC: ≤ 0.25 μg/ml), doxycycline (MIC: ≤ 0.25–1 μg/ml), ciprofloxacin (MIC: ≤ 0.25–2 μg/ml), florfenicol (MIC: 0.5–2 μg/ml), kanamycin (MIC: ≤ 0.25–4 μg/ml), enrofloxacin (MIC: 0.5–4 μg/ml) and berberine hydrochloride (MIC range: 1–8 μg/ml). However, the MICs of erythromycin were high (MIC: 32–≥128 μg/ml) for all isolates. The MICs of tylosin, tilmicosin and lincomycin for 50%, 60.7% and 46.4% of isolates were equal to or >16, 32 and 8 μg/ml, respectively. No correlation was detected between resistance to fluoroquinolones and QRDRs. However, the high MICs of tylosin, tilmicosin and lincomycin were most likely attributed to an A1553G mutation in domain V of the 23S rRNA. Our findings demonstrated the diversity of STs among M. hyorhinis isolates in Chongqing. The high MICs of M. hyorhinis isolates to macrolides and lincomycin suggested that the use of lincomycin for the treatment of M. hyorhinis infections should be carefully evaluated.

1 Introduction

Mycoplasma hyorhinis, a species belonging to the Mollicutes class, is a small, fastidious, and cell wall-free bacterium that was first isolated in 1953 (1). For decades, it was widely regarded as a commensal bacterium that inhabits the ciliated upper respiratory tract of swine, with rare manifestation of overt clinical signs (2). However, under certain conditions, polyserositis and polyarthritis, are most frequently observed in infected pigs (3–5). The pathogen has also been implicated in conjunctivitis, otitis, meningitis, pneumonia and abortions (6, 7). Notably, M. hyorhinis has further been linked to multiple human malignancies, such as gastric, esophageal, lung, breast, glioma and colon cancers (8). In contrast to well-established research on other porcine Mycoplasma species such as M. hyopneumoniae, few studies of have employed molecular typing to characterize isolate heterogeneity, trace infection sources, or resolve phylogenetic relationships among M. hyorhinis strains.

A commercial vaccine against M. hyorhinis infection (Ingelvac MycoMAX™, Boehringer Ingelheim Animal Health, USA, Inc., Duluth, USA) is available. However, to date, its use is only authorized in the United States. Thus, beyond optimizing husbandry conditions for prevention, antimicrobial therapy remains the sole strategy for controlling active infections in affected animals. Due to their lack of a cell wall, Mycoplasma species are inherently resistant to antibiotics targeting cell wall biosynthesis, such as β-lactams, glycopeptides, and fosfomycin (9, 10). Additionally, the absence of key enzymes in the folate metabolic pathway renders sulfonamides and trimethoprim—agents that target this pathway—ineffective (11, 12). Despite these limitations, several antimicrobial classes are clinically useful for treating M. hyorhinis infections, though field isolates exhibit variable susceptibility profiles across studies (13–15).

Macrolides (e.g., tylosin, tilmicosin, tylvalosin) and fluoroquinolones (e.g., enrofloxacin, difloxacin) are the most widely prescribed antimicrobial families for metaphylaxis and treatment of M. hyorhinis infections globally, including in China (16). However, previous studies have documented a marked increase in minimal inhibitory concentrations (MICs) of field isolates to these agents (13–15, 17). Acquired resistance to macrolides and lincosamides arises via multiple mechanisms, including enzymatic inactivation of macrolides, enhanced efflux pump activity, and methylation or structural alteration of macrolide-binding sites on the ribosome (18).

Point mutations in domain V of the 23S rRNA gene—a critical macrolide/lincosamide-binding region—have been well-documented in resistant M. hyorhinis strains (19–22). Furthermore, mutations in domain II of the 23S rRNA gene, as well as in rplD and rplV (which encode ribosomal proteins L4 and L22, respectively), represent major mechanisms of macrolide resistance across Mycoplasma species (19–22). Molecular characterization of the quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV in M. hyorhinis isolates with different levels of susceptibility to fluoroquinolones revealed that enrofloxacin-resistant isolates harbored amino acid substitutions in GyrA, ParC and ParE of the QRDRs (15). Importantly, mutation hotspots in these resistance-associated genes vary across drug-resistant strains, and numerous uncharacterized mutations may also contribute to antimicrobial resistance (19–22).

To date, few report of M. hyorhinis have been published in Chongqing Municipality, China. To address this knowledge gap, we isolated M. hyorhinis from swine lung tissues collected at local slaughterhouses and characterized the biological properties of these isolates. Specifically, we analyzed sequence type (ST) distribution, generated growth curves, and evaluated susceptibility to antimicrobials and Chinese herbal monomers. Additionally, we established correlations between phenotypic resistance profiles and underlying genotypic alterations for macrolides and fluoroquinolones.

2 Materials and methods

2.1 Sample collection and M. hyorhinis isolation

From March 30th to May 23rd, 2024, 404 lung specimens with confirmed pathological lesions were collected from slaughtered pigs at a slaughterhouse in Chongqing. These pigs were sourced from multiple distinct pig farms (Table 1). Prior to sample processing, the lung surface was disinfected with 3% hydrogen peroxide, after which a rice-grain-sized internal tissue fragment was homogenized in 1 ml of complete KM2 medium (KM2 broth medium containing 20% porcine serum and 0.01% NAD, pH 7.5) (23) supplemented with 100 μg/ml ampicillin. Homogenates were incubated at 37 °C for 7–14 days, with a red-to-yellow color change indicating medium acidification. After three passages, cultures were plated on KM2 agar and incubated at 37 °C for 5–10 days. Fried egg-like colonies were selected, expanded in complete KM2 broth, and re-plated on KM2 agar; this purification was repeated a minimum of three times to ensure monoclonality.

Table 1

Table 1. Samples and isolates data.

DNA was extracted from the purified final culture using rapid boiling DNA extraction. Each broth sample was centrifuged at 13,500 × g for 10 min and discarded the supernatant. The bacterial pellet was resuspended in 50 μl of sterile double-distilled water. The suspended cells were treated with dry heat block at 105 °C for 5 min, placed on ice for 10 min, and centrifuged at 13,500 × g for 5 min. The supernatant containing the DNA template was collected. A real-time TaqMan PCR assay was conducted by amplification of p37 gene of M. hyorhinis (24) using ApexHF HS DNA Polymerase FS Master Mix (Accurate Biology, Changsha, Hunan, China). Moreover, a partial sequence of the 16S rRNA gene of purified final culture was amplified by PCR using a primer pair (forward:5′-ACCCGAGAACGTATTCAC3′; reverse:5′-GCAAGCGTTATCCGGA3′). The PCR was performed in a 25 μl reaction volume consisting of 0.5 μl of DNA template, 0.5 μl of each primer (10 μm), 12.5 μl of ApexHF HS DNA Polymerase FS Master Mix, and 11 μl of ddH₂O. The amplification protocol consisted of an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of amplification at 98 °C for 10 s, annealing at 55 °C for 10 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 5 min. The PCR products were subsequently sequenced in both directions by Sangon Biotech Co., Ltd.

2.2 Multilocus sequence typing (MLST) assay

MLST primers for six housekeeping genes were synthesized according to published methods (Supplementary Table S1) (25). PCR was performed in 25 μl reactions with ApexHF HS DNA Polymerase FS Master Mix, following the method of Trüeb et al. (25), and products were sequenced by Sangon Biotech (Shanghai, China). The DNA sequences of the six housekeeping genes were submitted to the PubMLST database (https://pubmlst.org/organisms/mycoplasma-hyorhinis) for allele IDs assignment, and an allelic profile (six loci) was generated for each strain to determine STs. STs were compared with 176 reference strains (from 9 countries across Asia, Europe, North America) in the database. BURST analysis (26), in which allelic profiles matched at 4 loci, was applied to divide the strains into various lineage groups. A phylogenetic tree was constructed by MEGA 12.0 and visualized and annotated using iTOL v7.2 (https://itol.embl.de/).

2.3 Growth kinetics of M. hyorhinis isolates

M. hyorhinis strains were inoculated into complete KM2 medium at a ratio of 1:9, followed by incubation at 37 °C. When the medium turned yellow, the culture was inoculated into two portions of fresh complete KM2 medium portion (1:9 ratio) and incubated at 37 °C. At 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, 200 μl and 100 μl aliquots were collected from each culture. The optical density at 600 nm (OD₆₀₀) of each 200 μl sample was measured using an ELISA plate reader (Thermo Fisher Scientific, Ratastie 2, FI-01620 Vantaa, Finland). Serial tenfold dilutions were performed on 100 μl samples: 100 μl sample was added to 900 μl complete KM2 medium, then 100 μl of this suspension was transferred to a second tube with 900 μl medium. Dilutions were continued from the 2nd to the 22nd tube, with the 23rd tube as a negative control. Tubes were incubated at 37 °C for 14 days; no visible growth (no red to yellow color change) was confirmed. The color changing unit (CCU) the bacterial fluid at each time point was the average of three repeats. Three growth-related curves were plotted for each isolate: (1) h-OD₆₀₀ curve (X-axis: time points; Y-axis: average OD₆₀₀ values), (2) h-lgCCU curve (X-axis: time points; Y-axis: lgCCU values), and (3) growth curve (X-axis: average OD₆₀₀ values; Y-axis: lgCCU values).

2.4 In vitro susceptibility testing

The minimum inhibitory concentration (MIC) of M. hyorhinis isolates for 15 antimicrobials and 12 Chinese herbal monomers (Supplementary Table S2) was determined via broth microdilution (27). Kanamycin, gentamicin, amikacin, apramycin, chlortetracycline, doxycycline, tylosin, tylvalosin, lincomycin, tiamulin, enrofloxacin and ciprofloxacin were diluted with ddH₂O at room temperature. Berberine hydrochloride was diluted with ddH₂O at 40 °C. Florfenicol, erythromycin, tilmicosin, allicin, gingerenone A, baicalin, trans-cinnamic acid, cinnamaldehyde, gallic acid, glycyrrhizic acid, quercetin, curcumin, sodium houttuyfonate and ferulic acid were diluted with DMSO. The broth microdilution test was performed in 96-well microliter plates (31121, Labselect, Beijing, China) with two-fold serial dilutions (antimicrobials: 0.25–128 μg/ml; herbal monomers: 0.25–256 μg/ml) in 100 μl complete KM2 medium with sterility, pH and growth control. M. hyorhinis cultures (105 CCU/ml, 100 μl) were inoculated into wells and incubated at 35 ± 2 °C; MICs were read after 1 week (no further growth). All tests were run in triplicate. Notably, official breakpoint criteria for in vitro susceptibility testing of animal mycoplasmas are currently lacking. The M. hyorhinis reference strain BTS7 (ATCC 17981) was used as a quality control for MIC determination. MIC50 and MIC90 values were defined as the lowest concentrations inhibiting the growth of 50% and 90% of the tested strains, respectively (27).

2.5 PCR and nucleotide sequence analysis of QRDRs and domains II and V of the 23S rRNA genes

PCR was used to detect the mutations within four QRDRs of gyrA, gyrB, parC and parE, and domains II and V of the 23S rRNA genes to analyze the resistance mechanisms. The primer sequences and corresponding PCR amplification conditions are presented in Supplementary Table S1, as described in previous studies (15, 22). The PCR products were subjected to electrophoresis on a 1.5% agarose gel, and positive amplicons were bidirectionally sequenced. The sequencing results were aligned and analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The resulting DNA sequences and the corresponding amino acid sequences of all the PCR products were compared with the gyrA, parC and parE sequences (accession number: NZ_KB911491) and the 23S rRNA sequence (accession number: AB182581) of the M. hyorhinis BTS7 (ATCC 17981) strain and the gyrB sequence of the M. hyorhinis HUB-1 strain (accession number: CP002170).

3 Results

3.1 Isolation frequency of M. hyorhinis and MLST analysis

A total of 28 M. hyorhinis isolates were obtained from 404 lung samples (6.9%). M. hyorhinis isolates were confirmed by single colony selection, amplification of the M. hyorhinis-specific p37 gene, and sequence alignment between the 16S rRNA of the isolates and that of M. hyorhinis strains. After the nucleotide sequences were submitted to the PubMLST database for homology alignment, which revealed that none of the STs of the 28 isolates matched any entries in the database. Nucleotide sequences of the isolates were submitted to the PubMLST database for homology alignment, which revealed that none of the STs of the 28 isolates matched any entries in the database. These results indicated that the isolates identified in this study represent novel STs that have not been previously recorded in the database. Following reannotation by the database curator, the 28 isolates were classified into 11 distinct ST patterns (Tables 2, 3). Among these, ST226 and ST229 were the most prevalent, each accounting for 25.0% (7/28) of the total isolates. Three isolates (10.7%, 3/28) were assigned to ST227, while ST230, ST260, and ST262 each comprised two strains (7.1%, 2/28). Additionally, five isolates were distributed across five unique STs, namely ST228, ST261, ST263, ST264, and ST265.

Table 2

Table 2. Sequence types of 28 M. hyorhinis isolates in this study.

Table 3

Table 3. Sequence types of M. hyorhinis isolates.

3.2 Alleles for housekeeping genes

Three novel alleles were identified among the isolates, designated as dnaA-38 (5), rpoB-31 (4), and gyrB-18 (n). Specifically, three strains carried two novel gene sequences, whereas four strains harbored a single novel allelic variant. As summarized in Table 4, the dnaA gene exhibited five allelic variants: dnaA-1 was the dominant allele, detected in 39.3% (11/28) of isolates, followed by dnaA-10 (35.7%, 10/28), dnaA-38 (17.9%, 5/28), dnaA-2 (3.6%, 1/28), and dnaA-26 (3.6%, 1/28). The rpoB gene was represented by three alleles: rpoB-1 (46.4%, 13/28), rpoB-3 (39.3%, 11/28), and rpoB-31 (14.3%, 4/28). For the gyrB gene, five alleles were identified across the 28 strains: gyrB-1 (28.6%, 8/28), gyrB-3 (28.6%, 8/28), gyrB-4 (25.0%, 7/28), gyrB-11 (14.3%, 4/28), and gyrB-18 (3.6%, 1/28). Notably, gltX-4 was the sole allelic variant of the gltX gene detected in all isolates. Regarding the adk gene, adk-2 was the most abundant allele (78.6%, 22/28), followed by adk-8 (14.3%, 4/28), adk-1 (3.6%, 1/28), and adk-15 (3.6%, 1/28). For the gmk gene, gmk-3 was the predominant allele (42.9%, 12/28), with gmk-1 (25.0%, 7/28) and gmk-5 (32.1%, 9/28) as the other two variants.

Table 4

Table 4. Alleles of the 6 loci and their distribution.

3.3 BURST analysis

To investigate the genetic clustering of the isolates, BURST analysis was performed with a threshold of 4 shared allelic loci among group members. A total of 204 STs were included in the analysis, consisting of the 28 STs identified in this study and 176 reference STs from domestic and international sources. Notably, all STs were clustered into one group (data not shown). ST40 was identified as the central genotype, with ST13, ST17, ST24, ST30, ST32, ST41, ST88, and ST171 forming eight distinct subgroups (Figure 1). Sequence divergence analysis showed that ST40 differed from ST265 at 3 loci and from ST227 and ST228 at 4 loci. ST13 differed from ST226 at 3 loci. Seven additional STs were not visualized in Figure 1, as these did not cluster within any of the eight established subgroups. The inability of these isolates to form independent subgroups separate from other isolates is likely due to the insufficient data present in the current database.

Figure 1

Figure 1. Graphic representations of the BURST analysis of group 1. The center genotype is located in the center circle. The single-locus variants (SLVs) are indicated by a red circle surrounding the central profile. The double-locus variants (DLVs) are shown as blue circles. The most distant profiles (triple-locus variants) are linked with a line.

Furthermore, a phylogenetic tree was constructed based on the concatenated nucleotide sequences of six housekeeping genes using the neighbor-joining method (Figure 2). The 28 isolates from this study, marked with green pentagrams, formed a distinct clade that was separated from all reference isolates in the database.

Figure 2

Figure 2. MLST neighbor-joining tree of 204 M. hyorhinis isolates belonging to 158 STs. The phylogenetic tree was developed with the MEGA 12.0 program and visualized via the iTOL v7.2 program (https://itol.embl.de/). The rings (starting from the innermost) contain information on isolate identifiers; the green pentagrams indicate the strains isolated in this study, and the different background colors of the isolates represent different groups. The three outer rings from inside to outside are country, year and ST type.

3.4 Growth curves of the isolates

The h-OD₆₀₀ curves, h-lgCCU curves, and growth curves of all isolates are presented in Table 5, Figure 3, Supplementary Figures S1, S2. Overall, the OD600 values of isolates CQ001–CQ024, CQ026, and BTS07 exhibited a gradual decline with prolonged cultivation time. In contrast, isolates CQ025, CQ027, and CQ028 showed an increase in OD600 values up to 24 h of cultivation, followed by a gradual decrease thereafter. With respect to lgCCU, all isolates displayed a phase of increase until a specific time point, after which lgCCU values declined. The peak lgCCU was observed at 24 h for isolates CQ001, CQ003, CQ010, CQ012, CQ016, CQ021, and CQ026–CQ028; at 36 h for isolates CQ004–CQ006, CQ008, CQ009, CQ011, CQ013–CQ015, CQ017, CQ020, CQ023–CQ025, and BTS07; and at 48 h for isolates CQ002, CQ007, CQ018, CQ019, and CQ022. Growth curves were plotted with the average OD600 values at 0 h, 12 h, 24 h, 36 h, and 48 h on the X-axis and the corresponding lgCCU values on the Y-axis. As shown in Figure 3, Supplementary Figures S1, S2, a negative correlation between OD600 and lgCCU values was observed for isolates CQ001–CQ024, CQ026, and BTS07. In contrast, isolates CQ025, CQ027, and CQ028 exhibited a positive correlation between these two parameters during the 0–24 h cultivation period. Significant inter-isolate variability was observed in the maximum CCU achieved in complete KM2 medium, with values ranging from 10²² to 10²0 CCU/ml (Table 5).

Table 5

Table 5. Growth curves of M. hyorhinis isolates.

Figure 3

Figure 3. The h-OD₆₀₀ curves, h-lgCCU curves and growth curves of M. hyorhinis isolates CQ004 and CQ027.

3.5 Susceptibility profiles of M. hyorhinis isolates to antimicrobial agents and Chinese herbal monomers

The MIC values, as well as the MIC50 and MIC90 values, for all tested antimicrobials and Chinese herbal monomers are summarized in Table 6 and Supplementary Table S3. The isolates demonstrated high susceptibility to tiamulin (MIC ≤ 0.25 μg/ml), doxycycline (MIC ≤ 0.25–1 μg/ml), ciprofloxacin (MIC ≤ 0.25–2 μg/ml), florfenicol (MIC 0.5–2 μg/ml), kanamycin (MIC ≤ 0.25–4 μg/ml), and enrofloxacin (MIC 0.5–4 μg/ml). The MICs of gentamicin (96.4%, 27/28), chlortetracycline (92.9%, 26/28), tylvalosin (85.7%, 24/28) and apramycin (82.1%, 23/28) for most of the isolates were lower than 8 μg/ml. In contrast, all isolates exhibited high MIC values (32–≥128 μg/ml) to erythromycin. A bimodal distribution of MIC values was observed for tylosin and tilmicosin: 50.0% (14/28) of isolates had a tylosin MIC ≤ 1 μg/ml, and 39.3% (11/28) of isolates had a tilmicosin MIC ≤ 2 μg/ml.

Table 6

Table 6. Summary of the MIC range and MIC₅₀ and MIC₉₀ values (μg/ml) of the M. hyorhinis isolates for various antimicrobial agents and Chinese herbal monomers.

Overall, compared with antimicrobials, most Chinese herbal monomers exhibited higher MIC values against M. hyorhinis isolates (Table 6, Supplementary Table S3). The MIC ranges of the tested herbal monomers were as follows: ferulic acid (128 to ≥256 μg/ml); cinnamaldehyde, gallic acid, glycyrrhizic acid, quercetin, curcumin, and sodium houttuyfonate (64 to ≥256 μg/ml); trans-cinnamic acid (32 to ≥256 μg/ml); baicalin (64–128 μg/ml); gingerenone A (16–128 μg/ml); and allicin (16– 64 μg/ml). Notably, the MICs of 85.7% (24/28) of the isolates for berberine hydrochloride were < 8 μg/ml.

3.6 Molecular characterization of the QRDRs of M. hyorhinis isolates

All isolates were phenotypically susceptible to the two tested fluoroquinolones, although the MIC values of enrofloxacin for four isolates were equal to 4 μg/ml. To explore the potential association between QRDR mutations and MIC values, we amplified four genes encoding QRDRs by using the primers designed by Li et al. (15). PCR amplification results showed that gyrA and parE were successfully amplified in all 28 isolates, whereas gyrB and parC were detected in only 8 and 23 isolates, respectively.

Nucleotide sequence alignment was performed using the gyrA, parC, and parE sequences of the BTS7 strain and the gyrB sequence of the HUB-1 strain as references. Several nonsynonymous mutations were identified in one or two QRDR genes across the isolates (Table 7, Supplementary Table S3). A conserved nucleotide substitution at position 185 (A185G) in the gyrA gene resulted in an I62S amino acid substitution in all the strains. A nucleotide mutation at position 274 (A274G) in the parC gene led to an I92V amino acid change in 5 strains. For the parE gene, a nonsynonymous mutation at position 1098 (C1098A) caused an E366D amino acid substitution in 6 strains; additionally, isolate CQ005 carried two mutations in parE (T873A and C1098A), which resulted in amino acid substitutions N291K and E366D, respectively. No mutations were detected in the amplified gyrB gene fragments.

Table 7

Table 7. Prevalence of QRDRs in M. hyorhinis isolates.

Notably, none of the mutations identified in this study matched those previously reported by Li et al. (15). To further explore genetic variations in gyrB, we aligned the gyrB sequences used for MLST analysis (25) against the HUB-1 reference sequence. A nonsynonymous mutation at position 1708 (G1708A) was detected in isolates CQ021, CQ022, and CQ023, leading to a V570I substitution. Collectively, these results indicate that no key QRDR mutations in gyrA, gyrB, parC, or parE were associated with elevated enrofloxacin or ciprofloxacin MIC values in the tested isolates.

3.7 Mutational analysis of domains II and V of the 23S rRNA in M. hyorhinis isolates

Nucleotide sequence alignment of the 23S rRNA gene against the BTS7 reference strain revealed no mutations in domain II among the 28 isolates. In contrast, two distinct mutations were identified in domain V: an A1553G substitution was detected in 15 isolates, and a C2014T substitution was uniquely present in isolate CQ013 (Supplementary Table S3). The A1553G mutation appeared to be strongly correlated with high MIC values for tylosin, tylvalosin, tilmicosin, and lincomycin, despite the fact that all isolates exhibited high MICs of erythromycin (MIC: 32–≥128 μg/ml). Specifically, all isolates carrying the A1553G mutation displayed elevated MIC values for tylosin (≥16 μg/ml), tylvalosin (≥2 μg/ml), tilmicosin (≥32 μg/ml), and lincomycin (≥32 μg/ml), with three exceptions: isolate CQ017 showed low tylosin susceptibility (MIC ≤ 0.25 μg/ml), while isolates CQ003 and CQ004 exhibited low tylvalosin MIC values (0.5 and 1 μg/ml, respectively).

The mutation at A1553G of the 23S rRNA in M. hyorhinis was equivalent to the mutation at A2058G on the basis of 23S rRNA of E. coli (22). The C2014T mutation in domain V of the 23S rRNA in the CQ013 strain was related at least to the high MICs to tilmicosin, as the MIC for CQ013 (MIC: 32 μg/ml) was greater than that for strains without mutation in domain V of the 23S rRNA (MIC: ≤ 0.25 μg/ml) to tilmicosin.

4 Discussion

M. hyorhinis is distributed worldwide, with a high estimated prevalence (28, 29). On one hand, the pathogen can induce serious systemic inflammation, resulting in substantial economic losses to the pig industry (30); on the other hand, infected pigs often lack obvious clinical manifestations, and subclinical infections can may lead to growth retardation (29). Additionally, M. hyorhinis frequently coinfects with other swine pathogens, which exacerbates disease progression, increases mortality rates, and ultimately causes greater economic losses (7, 31). Therefore, the economic impact of M. hyorhinis infection should not be underestimated. Despite its significance, research on M. hyorhinis remains limited compared to that on M. hyopneumoniae.

M. hyorhinis typically exists as a commensal bacterium in the respiratory tract of pigs. In this study, we aimed to isolate M. hyorhinis from healthy pigs awaiting slaughter, therefore selecting porcine lung tissues as the isolation source. A total of 28 M. hyorhinis strains were successfully isolated using KM2 medium, instead of Friis broth—a conventional culture medium for mycoplasmas (32). KM2 broth is composed of lactalbumin hydrolysate (5 g/L), fresh yeast extract (10 g/L), Eagle's medium (5.975 g/L), Dulbecco's phosphate-buffered saline (2.925 g/L), and phenol red (0.007 g/L), and is widely used for the cultivation of M. hyopneumoniae (23, 33). Previous studies have also confirmed its applicability for M. hyorhinis culture (15, 34). Similar to other mycoplasma culture media, phenol red serves as a pH indicator in KM2 medium (35). During M. hyorhinis cultivation, the medium color changes from red to yellow, reflecting a shift in pH from weakly alkaline to acidic due to bacterial metabolic activities.

To establish a quantitative correlation between culture chromaticity and bacterial cell density, we characterized the growth kinetics of all isolates. The obtained specific growth curves enable accurate inference of bacterial load in cultures from the OD values of bacterial suspensions, thereby facilitating various downstream experiments. For instance, in vitro susceptibility testing, predefined growth curves allowed efficient harvesting of bacteria at a standardized concentration. Notably, the OD600 values of isolates CQ025, CQ027 and CQ028 increased from 0 to 24 h of cultivation and then gradually decreased, whereas the OD600 values of other strains consistently declined with prolonged culture time. This decrease in OD600 values may be attributed to nutrient consumption and metabolic changes that affect medium turbidity. After 24–48 h of cultivation, the lgCCU values of all strains decreased, indicating depletion of essential nutrients in the medium. To avoid the impact of nutrient exhaustion on growth curve characterization, we constructed growth curves for different time windows (0–24 h, 0–36 h, or 0–48 h) based on the growth characteristics of individual isolates.

To date, the M. hyorhinis PubMLST database has accumulated over 200 isolates and more than 150 STs. Among these, 17 strains from Chinese mainland are assigned to ST27, ST146, and ST148–ST162. In the present study, the combination of 6 MLST housekeeping gene alleles from each isolate did not match any existing STs in the database. Eventually, 11 novel ST patterns identified from the 28 isolates were submitted to and validated by the PubMLST database curators. Furthermore, significant variations were observed in the allelic diversity of the target housekeeping genes: 5 distinct alleles were detected for both dnaA and gyrB, while only one allele (gltX-4) was identified for gltX. In contrast, the reference database currently documents 38, 18, and 26 established alleles for dnaA, gyrB, and gltX, respectively. Notably, among the 6 MLST housekeeping genes, gyrB—which has the lowest number of known alleles in the PubMLST database—exhibited a relatively high allelic diversity (13.1%) in our isolates. Conversely, gltX, which has the largest number of curated alleles in the database, showed remarkably low allelic coverage (3.8%) in the tested strains. These findings collectively indicate that M. hyorhinis strains isolated from different geographic regions exhibit substantial genetic heterogeneity.

Previous studies from European countries have reported that M. hyorhinis strains have developed resistance to several macrolides and lincosamides, such as tylosin, tilmicosin, and lincomycin (13, 14, 36). Additionally, strains isolated from Italy showed reduced susceptibility to tiamulin (36). A domestic study in China also reported high MIC values of tylosin, tilmicosin, and lincomycin for 25 field isolates of M. hyorhinis (15). In the present research, we determined the MIC values of 14 veterinary antimicrobials (approved for swine use) against the isolates to screen for potential effective agents. Our results showed that most isolates exhibited high MICs for tylosin and tilmicosin, which may be associated with the widespread use of these antimicrobials in the pig industry. However, all tested isolates were susceptible to low concentrations of tiamulin (MIC ≤ 0.25 μg/ml), which contradicts the findings of Rosales et al. (36). This discrepancy may be attributed to differences in geographic origin, isolation time, or local antimicrobial use practices among the tested strains.

Enrofloxacin, a commonly used fluoroquinolone in swine production, is widely employed for the prevention and treatment of bacterial diseases, including Mycoplasma infections. Fifteen M. hyorhinis isolates collected in the United States between 1995 and 1998 showed a low MIC for enrofloxacin (37). In contrast, a study conducted across five European countries reported that moderate concentrations of enrofloxacin inhibited the growth of most of the 76 M. hyorhinis isolates collected between 2019 and 2021, with only one Hungarian isolate showing reduced susceptibility (14), suggesting a potential trend of increasing enrofloxacin MICs in more recently isolated strains. In our study, the MICs of enrofloxacin for the strains were relatively lower compared to those of other tested antimicrobials. Nevertheless, we investigated whether the high MIC value (4 μg/ml) of enrofloxacin observed in four isolates was associated with mutations in QRDRs. Li et al. (15) reported that mutations in ParC (Ser80Phe, Ser80Tyr, Phe80Tyr, Glu84Gly, and Glu84Lys), GyrA (Ala83Val, Ser84Pro, Asp87Tyr, and Asp87Asn), and ParE (Glu470Lys) were strongly associated with increased fluoroquinolone MICs. However, in our study, four strains whose MICs were 4 μg/ml for enrofloxacin showed mutations only in GyrA (I62S), while no mutations were detected in the other three proteins (or one or two genes could not be amplified). Mutations in GyrA were detected in all the strains. Considering that the MICs of enrofloxacin were low for the isolates, the mutations in the four PMQR proteins are not associated with enrofloxacin resistance in the tested strains.

Antimicrobial susceptibility testing revealed that all isolates exhibited high erythromycin MICs (32–≥128 μg/ml). Additionally, 50% (14/28) and 60.7% (17/28) of the isolates had tylosin and tilmicosin MICs ≥16 μg/ml and ≥32 μg/ml, respectively. A previous study indicated that a completely tylosin-resistant BTS7 mutant strain presented one point mutation at position A2059G, whereas the BTS7 mutant strain, which developed lincomycin resistance, presented two point mutations at positions G2597U and C2611U in domain V and the addition of an adenine at the pentameric adenine loop in domain II (22). Our results demonstrated that 15 isolates with an A1553G mutation in domain V of the 23S rRNA had high MICs for tylosin (MIC: ≥ 16 μg/ml), tylvalosin (MIC: ≥ 2 μg/ml), tilmicosin (MIC: ≥ 32 μg/ml) and lincomycin (MIC: ≥ 32 μg/ml), except for CQ017, whose MICs were low for tylosin ( ≤ 0.25 μg/ml), and CQ003 (0.5 μg/ml) and CQ004 (1 μg/ml) yielded low MICs for tylvalosin. Therefore, the A1553G mutation was closely related to high MICs to tylosin, tylvalosin, tilmicosin and lincomycin. These results broaden the understanding of the resistance mechanism of M. hyorhinis to macrolides and lincosamides.

With respect to aminoglycosides, 67.9% (19/28) and 50.0% (14/28) of the isolates had amikacin and apramycin MICs > 4 μg/ml and >2 μg/ml, respectively. However, the resistance mechanisms of mycoplasmas to aminoglycosides have not been reported in the literature, warranting further investigation to elucidate the underlying genetic and biochemical pathways.

The widespread use of antimicrobials in food-producing animals has become a global concern. In response, the Chinese government launched the “National Action Plan for the Reduction of Veterinary Antimicrobial Use (2021–2025)” in 2021, emphasizing the need to promote the safe, standardized, and scientific use of veterinary antimicrobials, curb bacterial resistance, and support the development of alternative products (Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2021). Several studies have reported the antibacterial or bacteriostatic effects of Chinese herbal monomers (38–41). In this study, we evaluated the antibacterial activity of several Chinese herbal monomers against M. hyorhinis isolates. Our results showed that most Chinese herbal monomers exhibited high MICs against the isolates, making them unsuitable as direct substitutes for antimicrobials. However, berberine hydrochloride—an extensively validated antibacterial Chinese herbal monomer—exhibited relatively low MICs (1–8 μg/ml) compared to other tested herbal monomers. These findings suggest that berberine hydrochloride holds great promise as a potential alternative to antimicrobials for controlling M. hyorhinis infections, aligning with the national strategy of reducing veterinary antimicrobial use.

5 Conclusion

In summary, we isolated 28 M. hyorhinis strains from the lungs of slaughtered pigs. These isolates belonged to 11 new STs. Tiamulin, doxycycline, ciprofloxacin, florfenicol, kanamycin, enrofloxacin and berberine hydrochloride were the most effective agents against the M. hyorhinis isolates in vitro, and erythromycin displayed high MICs for all the isolates. Moreover, the MICs of tylosin, tilmicosin and lincomycin for 50%, 60.7% and 46.4% of the isolates were equal to or >16 μg/ml, 32 μg/ml and 8 μg/ml, respectively. The high MICs of tylosin, tilmicosin and lincomycin were most likely attributed to an A1553G mutation in domain V of the 23S rRNA.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement

The animal study was approved by Institutional Animal Care and Use Committee of Southwest University. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

HL: Writing – original draft, Methodology. XL: Data curation, Methodology, Writing – original draft. YL: Methodology, Writing – original draft. RJ: Conceptualization, Data curation, Methodology, Writing – original draft. YM: Methodology, Writing – original draft. MZ: Resources, Writing – original draft. YW: Methodology, Writing – original draft. DY: Resources, Writing – original draft. HH: Resources, Writing – original draft. YY: Conceptualization, Writing – original draft. HD: Conceptualization, Data curation, Supervision, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Natural Science Foundation of China (32172870) and Chongqing Natural Science Foundation (CSTB2024NSCQ-MSX0763).

Acknowledgments

The authors would like to acknowledge all the study participants and individuals who contributed to this study.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2026.1732762/full#supplementary-material

Supplementary Figure S1 | The h-OD₆₀₀ curves, h-lgCCU curves and growth curves of 13 M. hyorhinis isolates CQ001–CQ003 and CQ005–CQ014.

Supplementary Figure S2 | The h-OD₆₀₀ curves, h-lgCCU curves and growth curves of 13 M. hyorhinis isolates CQ015–CQ026 and CQ028.

Supplementary Table S1 | Primers and PCR amplification protocols used in this study.

Supplementary Table S2 | Antimicrobials and Chinese herbal monomers used in this study.

Supplementary Table S3 | In vitro susceptibility to antimicrobials and Chinese herbal monomers and the molecular characterization of QRDRs, domains II and V of the 23S rRNA.

References

1. Carter CR, McKay KA. A pleuropneumonia-like organism associated with infectious atrophic rhinitis of swine. Can J Comp Med Vet Sci. (1953) 17:413–6.

PubMed Abstract | Google Scholar

2. Friis NF, Feenstra AA. Mycoplasma hyorhinis in the etiology of serositis among piglets. Acta Vet Scand. (1994) 35:93–8. doi: 10.1186/BF03548359

Crossref Full Text | Google Scholar

3. Clavijo MJ, Murray D, Oliveira S, Rovira A. Infection dynamics of Mycoplasma hyorhinis in three commercial pig populations. Vet Rec. (2017) 181:68. doi: 10.1136/vr.104064

PubMed Abstract | Crossref Full Text | Google Scholar

4. Giménez-Lirola LG, Meiroz-De-Souza-Almeida H, Magtoto RL, McDaniel AJ, Merodio MM, Matias Ferreyra FS, et al. Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions. PLoS ONE. (2019) 14:e0223459. doi: 10.1371/journal.pone.0223459

PubMed Abstract | Crossref Full Text | Google Scholar

5. Pillman D, Surendran Nair M, Schwartz J, Pieters M. Detection of Mycoplasma hyorhinis and Mycoplasma hyosynoviae in oral fluids and correlation with pig lameness scores. Vet Microbiol. (2019) 239:108448. doi: 10.1016/j.vetmic.2019.108448

PubMed Abstract | Crossref Full Text | Google Scholar

6. Bünger M, Brunthaler R, Unterweger C, Loncaric I, Dippel M, Ruczizka U, et al. Mycoplasma hyorhinis as a possible cause of fibrinopurulent meningitis in pigs? - a case series. Porcine Health Manag. (2020) 6:38. doi: 10.1186/s40813-020-00178-8

Crossref Full Text | Google Scholar

7. Lee JA, Oh YR, Hwang MA, Lee JB, Park SY, Song CS, et al. Mycoplasma hyorhinis is a potential pathogen of porcine respiratory disease complex that aggravates pneumonia caused by porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol. (2016) 177:48–51. doi: 10.1016/j.vetimm.2016.06.008

Crossref Full Text | Google Scholar

8. Vande Voorde J, Balzarini J, Liekens S. Mycoplasmas and cancer: focus on nucleoside metabolism. EXCLI J. (2014) 13:300–322

Google Scholar

9. Kobayashi H, Morozumi T, Munthali G, Mitani K, Ito N, Yamamoto K. Macrolide susceptibility of Mycoplasma hyorhinis isolated from piglets. Antimicrob Agents Chemother. (1996) 40:1030–2. doi: 10.1128/AAC.40.4.1030

PubMed Abstract | Crossref Full Text | Google Scholar

10. Williams PP. In vitro susceptibility of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis to fifty-one antimicrobial agents. Antimicrob Agents Chemother. (1978) 14:210–3. doi: 10.1128/AAC.14.2.210

PubMed Abstract | Crossref Full Text | Google Scholar

11. Breuer M, Earnest TM, Merryman C, Wise KS, Sun L, Lynott MR, et al. Essential metabolism for a minimal cell. Elife. (2019) 8:e36842. doi: 10.7554/eLife.36842

PubMed Abstract | Crossref Full Text | Google Scholar

12. Ferrarini MG, Siqueira FM, Mucha SG, Palama TL, Jobard É, Elena-Herrmann B, et al. Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling. BMC Genomics. (2016) 17:353. doi: 10.1186/s12864-016-2644-z

PubMed Abstract | Crossref Full Text | Google Scholar

13. Beko K, Felde O, Sulyok KM, Kreizinger Z, Hrivnák V, Kiss K, et al. Antibiotic susceptibility profiles of Mycoplasma hyorhinis strains isolated from swine in Hungary. Vet Microbiol. (2019) 228:196–201. doi: 10.1016/j.vetmic.2018.11.027

PubMed Abstract | Crossref Full Text | Google Scholar

14. Klein U, Földi D, Belecz N, Hrivnák V, Somogyi Z, Gastaldelli M, et al. Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021. PLoS ONE. (2022) 17:e0272903. doi: 10.1371/journal.pone.0272903

PubMed Abstract | Crossref Full Text | Google Scholar

15. Li J, Wei Y, Wang J, Li Y, Shao G, Feng Z, et al. Characterization of mutations in DNA gyrase and topoisomerase IV in field strains and in vitro selected quinolone-resistant Mycoplasma hyorhinis mutants. Antibiotics. (2022) 11:494. doi: 10.3390/antibiotics11040494

PubMed Abstract | Crossref Full Text | Google Scholar

16. Gautier-Bouchardon AV. Antimicrobial resistance in Mycoplasma spp. Microbiol Spectr. (2018) 6:ARBA-0030-2018. doi: 10.1128/microbiolspec.ARBA-0030-2018

PubMed Abstract | Crossref Full Text | Google Scholar

17. Ustulin M, Rossi E, Vio D. A case of pericarditis caused by Mycoplasma hyorhinis in a weaned piglet. Porcine Health Manage. (2021) 7:32. doi: 10.1186/s40813-021-00211-4

PubMed Abstract | Crossref Full Text | Google Scholar

18. Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis. (2002) 34:482–92. doi: 10.1086/324626

PubMed Abstract | Crossref Full Text | Google Scholar

19. Beko K, Kreizinger Z, Kovács ÁB, Sulyok KM, Marton S, Bányai K, et al. Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae. Vet Microbiol. (2020) 248:108818. doi: 10.1016/j.vetmic.2020.108818

PubMed Abstract | Crossref Full Text | Google Scholar

20. Földi D, Kreizinger Z, Beko K, Belecz N, Bányai K, Kiss K, et al. Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis. Acta Vet Hung. (2021) 69:110–5. doi: 10.1556/004.2021.00026

PubMed Abstract | Crossref Full Text | Google Scholar

21. Gerchman I, Levisohn S, Mikula I, Manso-Silván L, Lysnyansky I. Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry. Vet Res. (2011) 42:90. doi: 10.1186/1297-9716-42-90

PubMed Abstract | Crossref Full Text | Google Scholar

22. Kobayashi H, Nakajima H, Shimizu Y, Eguchi M, Hata E, Yamamoto K. Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA. J Vet Med Sci. (2005) 67:795–800. doi: 10.1292/jvms.67.795

PubMed Abstract | Crossref Full Text | Google Scholar

23. Wen Y, Chen Z, Tian Y, Yang M, Dong Q, Yang Y, et al. Incomplete autophagy promotes the proliferation of Mycoplasma hyopneumoniae through the JNK and Akt pathways in porcine alveolar macrophages. Vet Res. (2022) 53:62. doi: 10.1186/s13567-022-01074-5

PubMed Abstract | Crossref Full Text | Google Scholar

24. Tocqueville V, Ferré S, Nguyen NH, Kempf I, Marois-Créhan C. Multilocus sequence typing of Mycoplasma hyorhinis strains identified by a real-time TaqMan PCR assay. J Clin Microbiol. (2014) 52:1664–71. doi: 10.1128/JCM.03437-13

PubMed Abstract | Crossref Full Text | Google Scholar

25. Trüeb B, Catelli E, Luehrs A, Nathues H, Kuhnert P. Genetic variability and limited clonality of Mycoplasma hyorhinis in pig herds. Vet Microbiol. (2016) 191:9–14. doi: 10.1016/j.vetmic.2016.05.015

PubMed Abstract | Crossref Full Text | Google Scholar

26. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol. (2004) 186:1518–30. doi: 10.1128/JB.186.5.1518-1530.2004

PubMed Abstract | Crossref Full Text | Google Scholar

27. Hannan PC. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. International Research Programme on Comparative Mycoplasmology. Vet Res. (2000) 31:373–95. doi: 10.1051/vetres:2000100

PubMed Abstract | Crossref Full Text | Google Scholar

28. Clavijo MJ, Sreevatsan S, Johnson TJ, Rovira A. Molecular epidemiology of Mycoplasma hyorhinis porcine field isolates in the United States. PLoS ONE. (2019) 14:e0223653. doi: 10.1371/journal.pone.0223653

PubMed Abstract | Crossref Full Text | Google Scholar

29. Wang J, Hua L, Gan Y, Yuan T, Li L, Yu Y, et al. Virulence and inoculation route influence the consequences of Mycoplasma hyorhinis infection in Bama miniature pigs. Microbiol Spectr. (2022) 10:e0249321. doi: 10.1128/spectrum.02493-21

PubMed Abstract | Crossref Full Text | Google Scholar

30. Martinson B, Minion FC, Jordan D. Development and optimization of a cell-associated challenge model for Mycoplasma hyorhinis in 7-week-old cesarean-derived, colostrum-deprived pigs. Can J Vet Res. (2018) 82:12–23.

PubMed Abstract | Google Scholar

31. Chen D, Wei Y, Huang L, Wang Y, Sun J, Du W, et al. Synergistic pathogenicity in sequential coinfection with Mycoplasma hyorhinis and porcine circovirus type 2. Vet Microbiol. (2016) 182:123–30. doi: 10.1016/j.vetmic.2015.11.003

PubMed Abstract | Crossref Full Text | Google Scholar

32. Käbisch L, Schink AK, Höltig D, Spergser J, Kehrenberg C, Schwarz S. Towards a standardized antimicrobial susceptibility testing method for Mycoplasma hyorhinis. Microorganisms. (2023) 11:994. doi: 10.3390/microorganisms11040994

PubMed Abstract | Crossref Full Text | Google Scholar

33. Xie M, Huang Z, Zhang Y, Gan Y, Li H, Li D, et al. The Mycoplasma hyopneumoniae protein Mhp274 elicits mucosal and systemic immune responses in mice. Front Cell Infect Microbiol. (2025) 15:1516944. doi: 10.3389/fcimb.2025.1516944

PubMed Abstract | Crossref Full Text | Google Scholar

34. Ishag HZ, Liu M, Yang R, Xiong Q, Feng Z, Shao G. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis. Springerplus. (2016) 5:769. doi: 10.1186/s40064-016-2358-3

PubMed Abstract | Crossref Full Text | Google Scholar

35. Stemke GW, Robertson JA. The growth response of Mycoplasma hyopneumoniae and Mycoplasma flocculare based upon ATP-dependent luminometry. Vet Microbiol. (1990) 24:135–42. doi: 10.1016/0378-1135(90)90061-Y

PubMed Abstract | Crossref Full Text | Google Scholar

36. Rosales RS, Ramírez AS, Tavío MM, Poveda C, Poveda JB. Antimicrobial susceptibility profiles of porcine mycoplasmas isolated from samples collected in southern Europe. BMC Vet Res. (2020) 16:324. doi: 10.1186/s12917-020-02512-2

PubMed Abstract | Crossref Full Text | Google Scholar

37. Wu CC, Shryock TR, Lin TL, Faderan M, Veenhuizen MF. Antimicrobial susceptibility of Mycoplasma hyorhinis. Vet Microbiol. (2000) 76:25–30. doi: 10.1016/S0378-1135(00)00221-2

PubMed Abstract | Crossref Full Text | Google Scholar

38. Herculano LS, Kalschne DL, Canan C, Reis TS, Marcon CT, Benetti VP, et al. Antimicrobial curcumin-mediated photodynamic inactivation of bacteria in natural bovine casing. Photodiagnosis Photodyn Ther. (2022) 40:103173. doi: 10.1016/j.pdpdt.2022.103173

PubMed Abstract | Crossref Full Text | Google Scholar

39. Kashi M, Farahani A, Ahmadi A, Shariati A, Akbari M. Antibacterial and antibiofilm efficacy of eugenol, carvacrol, and cinnamaldehyde against colistin-resistant Klebsiella pneumoniae. Mol Biol Rep. (2025) 52:480. doi: 10.1007/s11033-025-10564-6

PubMed Abstract | Crossref Full Text | Google Scholar

40. Nguyen TLA, Bhattacharya D. Antimicrobial activity of quercetin: an approach to its mechanistic principle. Molecules. (2022) 27:2494. doi: 10.3390/molecules27082494

PubMed Abstract | Crossref Full Text | Google Scholar

41. Zhong C, Lin S, Li Z, Yang X. Characterization of carbapenem-resistant Klebsiella pneumoniae in bloodstream infections: antibiotic resistance, virulence, and treatment strategies. Front Cell Infect Microbiol. (2025) 15:1541704. doi: 10.3389/fcimb.2025.1541704

PubMed Abstract | Crossref Full Text | Google Scholar