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Antimicrobial Agents and Chemotherapy, June 2006, p. 1959-1966, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01527-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Persistence of Mycoplasma hyopneumoniae in Experimentally Infected Pigs after Marbofloxacin Treatment and Detection of Mutations in the parC Gene

J. Le Carrou,¹ M. Laurentie,² M. Kobisch,¹ and A. V. Gautier-Bouchardon^1*

Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches Avicoles et Porcines, Unité de Mycoplasmologie-Bactériologie, BP 53, 22440 Ploufragan, France,¹ Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, Unité de Pharmacocinétique-Pharmacodynamie, BP 90203, 35302 Fougères Cedex, France²

Received 30 November 2005/ Returned for modification 12 January 2006/ Accepted 20 March 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability of Mycoplasma hyopneumoniae to persist despite fluoroquinolone treatments was investigated with pigs. Groups of specific-pathogen-free pigs were experimentally infected with M. hyopneumoniae strain 116 and treated with marbofloxacin at the therapeutic dose (TD) or half of the therapeutic dose (TD/2) for 3 days. Results showed that, despite tissue penetration of marbofloxacin, particularly in the trachea and the tracheal secretions, the treatments did not have any influence on M. hyopneumoniae recovery from tracheal swabs. Mycoplasmas were also isolated from inner organs and tissues such as liver, spleen, kidneys, and bronchial lymph nodes. Recontamination of pigs via environment could not explain mycoplasma persistence after medication, as decontamination of pigs and allocation to a new disinfected environment did not have any significant effect on the phenomenon. A significant decrease in the susceptibility level to marbofloxacin of 12 mycoplasma clones reisolated after the treatments (TD/2 and TD) was observed. Two point mutations were found in the ParC quinolone resistance-determining region (QRDR) of DNA topoisomerase IV (Ser80Phe and Asp84Asn), and one point mutation was observed just behind the QRDR of ParC (Ala116Glu). This is the first time that mutations in a gene coding for topoisomerase IV have been described for M. hyopneumoniae after in vivo marbofloxacin treatments in experimentally infected pigs. However, development of resistance is not sufficient to explain M. hyopneumoniae persistence in vivo since (i) marbofloxacin concentrations were above the marbofloxacin MIC of the wild-type strain and (ii) mycoplasmas reisolated after a single injection of marbofloxacin did not display an increased marbofloxacin MIC.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mycoplasma hyopneumoniae is one of the primary pathogens associated with the porcine respiratory disease complex, one of the most common and economically important diseases for swine producers worldwide (43). M. hyopneumoniae is the etiological agent of enzootic pneumonia in swine, a chronic respiratory disease characterized by high morbidity and low mortality rates (15). The principal clinical but not constant sign is a chronic cough. Mycoplasmal pneumonia is located mainly in the anterior lobes of the lung. In the acute phase of the disease, catarrhal pneumonia is observed, with exudates in the airways. The bronchial and mediastinal lymph nodes are often enlarged. In the chronic stage of the disease, recovering lesions, consisting of fissures of collapsed alveoli adjoining areas of alveolar emphysema, are observed (26). M. hyopneumoniae is a very contagious bacterium and may be transmitted via direct contact between pigs (43) or via environment (14, 48).

In vitro, M. hyopneumoniae is susceptible to various antibiotics, including fluoroquinolones, tetracyclines, spiramycin, and tiamulin (17, 18, 19, 25, 42, 49). However, although antibiotic treatments are usually able to control the disease (49), persistence is observed under field conditions (43) and experimental infections (16, 25). Fluoroquinolones, particularly marbofloxacin and enrofloxacin, are used to treat enzootic pneumonia under field conditions.

The main targets of fluoroquinolones are replication and transcription enzymes, i.e., DNA gyrase and topoisomerase IV, which are both essential for bacterial viability (32). Most reported mutations involved in fluoroquinolone resistance are concentrated in the quinolone resistance-determining regions (QRDRs) of the gyrAB and parCE genes of DNA gyrase and topoisomerase IV, respectively (37, 39, 40).

This study describes the effects of marbofloxacin treatments on M. hyopneumoniae recovery in experimentally infected pigs and emergence of mutations in the parC gene of topoisomerase IV.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mycoplasma strains and culture conditions. A field strain of M. hyopneumoniae, strain 116, isolated from an outbreak of enzootic pneumonia in France (2003), was used for experimental infections. This strain was susceptible to marbofloxacin (MIC = 0.03 µg/ml), enrofloxacin (MIC 0.03 µg/ml), and oxytetracycline (MIC = 0.25 µg/ml). M. hyopneumoniae strain 137, another field strain, isolated from an outbreak of enzootic pneumonia in France (2004), and two reference strains, M. hyopneumoniae ATCC 25934 and ATCC 25617, were used for the sequencing analysis. Strains were grown in Friis broth medium (37°C) until an acid color change was observed and plated on Friis agar medium (37°C, 5% CO₂) as previously described (10). The titer of M. hyopneumoniae cultures was expressed as color-changing units (CCU)/ml.

Effect of marbofloxacin treatment on M. hyopneumoniae recovery. Twenty-four specific-pathogen-free (SPF), hysterectomy-derived piglets were obtained from the Porcine Experimental Unit of the French Food Safety Agency of Ploufragan, France. Animal experiments were performed according to the law and followed ethical and welfare recommendations (agreement B-22-745-1). Very strict biosecurity measures were implemented in order to avoid undesirable contamination of the pigs: an air filtration system and airlocks for each unit, unit-specific clothes, and compulsory showering before and after visiting the pigs (3). Pigs were weaned at 3 weeks of age, randomly allocated to four separated animal rooms (treatment groups 1 to 4), containing six pigs each, and allowed to acclimatize for 3 weeks. Pigs from the first group were not inoculated and were used as uninfected controls. At 6 weeks of age, pigs from groups 2 to 4 were inoculated intratracheally on two consecutive days (days 0 and 1) with 5 x 10⁸ CCU of M. hyopneumoniae strain 116. On day 23 postinfection, pigs from groups 2 to 4 were reinoculated intratracheally with 7 x 10⁸ CCU of M. hyopneumoniae strain 116. From day 27 to day 29, pigs from groups 3 and 4 were treated for three consecutive days by intramuscular injection of marbofloxacin (Marbocyl 2%; Vétoquinol SA, Lure, France) at half of the therapeutic dose (TD/2) (1 mg/kg of body weight/day) and at the therapeutic dose (TD) (2 mg/kg of body weight/day), respectively. Antibiotic doses were based on the body weights of pigs on day 27. Pigs from group 2 were infected untreated controls. From day 27 to day 30, blood samples were collected from three randomly chosen pigs from each group to determine plasma marbofloxacin concentrations. Tracheal swabs were collected weekly from day 0 (before inoculation) to day 76 and cultured for M. hyopneumoniae recovery. On day 27, tracheal swabs were collected before the marbofloxacin injection. Two infected pigs from each group were killed on day 34, and all remaining pigs were sacrificed from day 76 to day 80. Swabs were collected from tracheas, livers, spleens, kidneys, and bronchial lymph nodes and cultured for M. hyopneumoniae recovery.

Marbofloxacin dosage in the respiratory tracts of infected pigs. Eight SPF piglets were weaned at 3 weeks of age and allowed to acclimatize for 3 weeks in the experimental room. At 6 weeks of age, pigs were inoculated intratracheally on two consecutive days (days 0 and 1) with 5 x 10⁹ CCU of M. hyopneumoniae strain 116. On day 27, six pigs were treated once by an intramuscular injection of marbofloxacin at the therapeutic dose. The two remaining pigs were used as infected untreated controls. Two marbofloxacin-treated pigs were killed 4 h, 8 h, and 24 h after the start of the medication. The two untreated pigs were sacrificed on day 28. Tracheal swabs were performed, and plasma, tracheas, tracheal secretions, and lungs were collected to determine marbofloxacin concentrations.

Influence of the environment on M. hyopneumoniae persistence. Fifteen SPF piglets were weaned at 3 weeks of age and randomly allocated to two separated animal rooms, A1 and A2, containing 5 and 10 pigs, respectively. At 6 weeks of age, pigs were inoculated intratracheally on two consecutive days (days 0 and 1) with 5 x 10⁹ CCU of M. hyopneumoniae strain 116. From days 27 to 29, pigs from room A2 were treated daily by intramuscular injections of marbofloxacin at the therapeutic dose. On the last day of treatment (day 29), five pigs from room A2 were washed with a disinfectant (Rogé Cavaillès, Courbevoie, France) and placed in a disinfected animal room (room A3). Pigs from room A1 were infected untreated controls. Tracheal swabs were collected weekly from all pigs (15 samples) from day 0 to day 48 and cultured for M. hyopneumoniae recovery. All pigs were killed on days 48 to 50. Swabs were collected from tracheas, livers, spleens, and kidneys and cultured for M. hyopneumoniae recovery.

Determination of marbofloxacin concentrations. The marbofloxacin concentrations were determined by a high-performance liquid chromatography method adapted from that of Schneider et al. (46). For pig plasma, the method was validated between 20 and 500 ng/ml, with a recovery of 93.6% ± 5.03%. The repeatability (intraday precision) and the intermediate precision (interday precision) were below 10%. The limit of quantification was established to be 20 ng/ml. For lungs, tracheas, and tracheal secretions, the validated method was slightly modified. The limit of quantification was 50 ng/g for all matrices. The recovery was within 90 to 110%. Repeatability and intermediate precision were below 15%.

Clinical observations and pneumonia assessment. Coughing was counted daily for 15 min. Postmortem examinations were carried out on each animal. The lung lesions were scored as previously described (28). Tissue samples (cardiac lobes) were collected from sections with lesions typical of M. hyopneumoniae infection or from healthy tissue without macroscopic lesions. Samples were fixed in 10% buffered formalin; paraffin-embedded sections were cut at 5 µm, stained by a trichrome coloration (hematoxylin, eosin, and saffron), and examined by light microscopy.

Cultures. Swabs were placed in 2 ml of transport medium (2% buffered peptone water containing glycerol [1.2%, vol/vol]; amphotericin B, 2.5 µg/ml; ampicillin, 100 µg/ml; and colistin, 7.5 µg/ml). Mycoplasmas were cultured directly from swabs by dilution of 100 µl of transport medium from each swab in 900 µl of Friis broth (containing bacitracin, 150 µg/ml; amphotericin B, 2.5 µg/ml; ampicillin, 100 µg/ml; and colistin, 7.5 µg/ml), and the cultures were incubated at 37°C until they developed an acid color change or up to 30 days. When a color change of the broth medium was observed, cultures were stored at –70°C in 20% glycerol. Mycoplasma cultures were cloned on Friis agar medium (containing 0.002% DNA calf thymus; Sigma-Aldrich, Lyon, France), grown in Friis broth, aliquoted, and stored at –70°C in 20% glycerol. Cultures were confirmed by an M. hyopneumoniae-specific PCR.

Susceptibility level determination. The marbofloxacin, enrofloxacin, and oxytetracycline MICs of the M. hyopneumoniae clones isolated from tracheal swabs were determined by a broth microdilution method as previously described (2), with antibiotic concentrations ranging from 0.03 to 32 µg/ml.

Time-kill kinetic study. The M. hyopneumoniae 116 wild-type strain (marbofloxacin MIC [MIC_marbo] = 0.03 µg/ml) and one clone, isolated on day 34 from an M. hyopneumoniae 116-infected pig treated at the therapeutic dose of marbofloxacin for 3 days (from day 27 to day 30) and displaying an increased resistance level to marbofloxacin (MIC_marbo = 0.5 µg/ml), were evaluated. Each strain was grown in Friis broth medium for 24 h and then diluted 10-fold in Friis broth medium and incubated at 4°C for 18 h in order to obtain a stationary-phase culture.

The ranges of antibiotic concentrations to be tested were determined for the two strains in order to observe the effects of seven different concentrations, including three concentrations below and three concentrations above the MIC.

The killing curves were determined using a microdilution method with a final broth volume of 1 ml. A 100-µl aliquot of antibiotic was added to 900 µl of the bacterial suspension in a sterile microtube. All tubes were incubated at 37°C, and sampling for colony count was performed at 0, 2, 4, 6, 8, 24, and 48 h. For viable count, serial 10-fold dilutions were performed in Friis broth medium and 5 µl of each dilution was deposited on Friis agar medium. The counts were carried out under a binocular magnifying lens, after incubation of the agar plates at 37°C, 5% CO₂ for 7 days, by reading the first dilution that showed between 20 and 200 colonies. The log₁₀ of viable counts in CFU/ml was plotted against time for each concentration of marbofloxacin tested. A bactericidal effect was defined as a decrease of 3 log₁₀ (99.9%) in the number of viable mycoplasmas observed on Friis agar medium.

M. hyopneumoniae-specific PCR and amplification of the QRDRs. DNAs were prepared according to standard methods (23). Amplification of the I141-fragment of M. hyopneumoniae (accession no. U02537) was performed using HP4 and HP6 primers (Table 1). Amplification of the QRDRs of gyrA, gyrB, parC, and parE was performed with primers chosen from the alignment of known nucleotide sequences of M. hyopneumoniae strain 232 (33). PCR was performed with a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Courtaboeuf, France) in a total volume of 50 µl containing 0.5 µM of each primer (Table 1), 200 µM of each deoxynucleoside triphosphate, 5 µl of 10x EurobioTaq buffer (Eurobio, Les Ulis, France), and 1 U of EurobioTaq polymerase. Amplification was performed over 40 cycles, for 30 s at 94°C, 30 s at 60°C or 62°C (depending on the primer used), and 30 s at 72°C (Table 1). Specific PCR products obtained for QRDRs were purified with a Qiaquick PCR purification kit (QIAGEN, Courtaboeuf, France) following manufacturer recommendations. The amplified product for the Il41 gene was separated in a 2% agarose gel in Tris-borate-EDTA buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA [pH 8]) for 1 h at a constant voltage of 125 V. Amplified products were stained with ethidium bromide and detected by UV transillumination.

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TABLE 1. Primers, hybridization temperatures, and PCR fragment lengths expected for M. hyopneumoniae I141, gyrA, gyrB, parC, and parE QRDR amplification

DNA sequencing and sequence analyses. All purified PCR products were sequenced directly on both strands by using a BigDye Terminator v 3.1 cycle sequencing kit in a 3100Avant sequencer (Applied Biosystems, Courtaboeuf, France). The QRDR sequences obtained were compared to databanks by use of Network Protein Sequence Analysis software (6).

Statistical analyses. Kruskal-Wallis and Kolmogorov-Smirnov tests were used to compare mean lung lesion scores and MICs. A chi-square test was used to compare the percentages of animals with pneumonia and pig coughing. Frequencies of mycoplasma reisolations were compared with Fisher's exact test. The analyses were made using SAS software (44). Differences were estimated to be significant when P was <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clinical evaluation and postmortem examinations. No clinical signs or lesions were observed in noninfected pigs (data not shown). Coughing started 9 to 14 days after inoculation in infected pigs (data not shown). Pigs were treated upon observation of well-established pneumonia, with 89% to 100% of the infected pigs coughing 4 weeks after the first inoculation (data not shown). Macroscopic lesions typical of M. hyopneumoniae were observed in all infected groups. Pneumonia was observed in most of the infected untreated pigs, whereas recovering lesions were observed in most of the infected treated pigs. Recovering lesions observed during necropsy examination were confirmed by light microscopy (data not shown). No significant differences between the mean of coughing per pig or the average lung lesion scores in infected groups were observed (data not shown).

Persistence of M. hyopneumoniae despite a marbofloxacin treatment. No mycoplasma was isolated from pigs before inoculation (day 0) (data not shown). M. hyopneumoniae was recovered from 83% to 100% of the tracheas of infected pigs before treatment (day 27) (Fig. 1). No significant difference between the tracheal swab cultures from the different treatment groups was observed after the marbofloxacin treatment (from day 34 to the end of the study) (Fig. 1). M. hyopneumoniae was also reisolated from tracheal swabs at 4, 8, and 24 h postinjection of marbofloxacin (data not shown). M. hyopneumoniae was reisolated from inner organs (livers, spleens, or kidneys) or bronchial lymph nodes of untreated and TD-treated pigs on day 34 postinoculation (Table 2). Nevertheless, a significant difference was observed in the frequencies of mycoplasma reisolation from inner organs of untreated and TD-treated pigs (P < 0.05). Mycoplasmas were reisolated from bronchial lymph nodes, but not from other inner organs, of TD/2-treated pigs. At the end of the study (days 76 to 80), M. hyopneumoniae was recovered only from lymph nodes and respiratory tracts.

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FIG. 1. M. hyopneumoniae reisolation from tracheal swabs in infected untreated pigs and in infected pigs treated at TD and TD/2 for three consecutive days with marbofloxacin (from day 27 to day 29).

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TABLE 2. M. hyopneumoniae reisolation from inner organs and tissues of infected pigs

Determination of marbofloxacin concentrations. Plasma marbofloxacin concentrations in pigs were investigated during the marbofloxacin treatments (TD and TD/2). The mean plasmatic concentration of marbofloxacin increased proportionally with the dose administered intramuscularly to the pigs (data not shown). Plasma concentrations of marbofloxacin administered at the therapeutic dose peaked at 1.20 ± 0.04 µg/ml 1 h postadministration (data not shown). The marbofloxacin was slowly eliminated from plasma and tissues, as indicated by the mean resident time (7 to 9 h) and the apparent terminal elimination half-life (7 to 9 h) (Table 3). Ratios of tissue to plasma concentrations showed that marbofloxacin was concentrated in the tracheas (1.95 ± 0.15) and the tracheal secretions (1.55 ± 0.78) of infected pigs but faintly diffused to the lungs (0.54 ± 0.21) (Table 3). Nevertheless, in all tested tissues, the mean marbofloxacin concentrations maintained for 20 h (from 4 to 24 h postinjection) were 8- to 28-fold higher than the MIC_marbo of the M. hyopneumoniae 116 wild-type strain (MIC_marbo = 0.03 µg/ml).

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TABLE 3. Pharmacokinetic data from pigs administered marbofloxacin (2 mg/kg of body weight) by intramuscular injection

Influence of the environment on M. hyopneumoniae persistence. No mycoplasma was isolated from pigs before inoculation (day 0) (data not shown). M. hyopneumoniae was recovered from 70 to 80% of the tracheas before treatment (day 27) (Table 4). Four days after the end of the marbofloxacin treatment and the reallocation of five pigs from room A2 to a new room, A3, M. hyopneumoniae was reisolated from 60% (treated pigs) to 80% (untreated pigs) of the tracheas. At the end of the study, M. hyopneumoniae was reisolated from 100% of the tracheal swabs. M. hyopneumoniae could not be reisolated from inner organs (liver, spleen, or kidney) (day 48) (data not shown). No significant difference in frequency of mycoplasma reisolation was observed between pigs kept in rooms A1, A2, and A3.

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TABLE 4. Influence of the environment on M. hyopneumoniae recovery from pigs

Marbofloxacin, enrofloxacin, and oxytetracycline MICs. The results of MIC determinations did not show any significant difference between the susceptibilities to marbofloxacin and enrofloxacin of the M. hyopneumoniae clones recovered from untreated pigs and the M. hyopneumoniae 116 wild-type strain (clones 116-1 to 116-5) (Table 5). All M. hyopneumoniae clones reisolated from tracheal swabs during treatment were also as susceptible to marbofloxacin as the M. hyopneumoniae 116 wild-type strain (MIC_marbo = 0.03 µg/ml) (data not shown). However, a significant decrease in susceptibility level to marbofloxacin (MIC₉₀ = 0.5 µg/ml) and enrofloxacin (MIC₉₀ = 0.25 µg/ml), compared to the initial MICs (0.03 µg/ml) (P < 0.05), was observed 4 days after the end of the marbofloxacin treatment (clones from TD/2- and TD-treated pigs). M. hyopneumoniae clones recovered from tracheal swabs at the end of the study (day 76 to day 80) still had decreased susceptibility levels to marbofloxacin and enrofloxacin (data not shown). No significant difference in susceptibility level was observed between clones recovered from TD/2- and TD-treated pigs. No significant decrease in the susceptibility level to oxytetracycline was observed for M. hyopneumoniae clones recovered 4 days after the end of the marbofloxacin treatment (Table 5).

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TABLE 5. Susceptibility levels and mutations observed in M. hyopneumoniae clones recovered after marbofloxacin treatment (day 34)

Time-kill kinetic studies. The pharmacodynamic activity of marbofloxacin was evaluated against that of the M. hyopneumoniae 116 wild-type strain (MIC_marbo = 0.03 µg/ml) and that of one clone reisolated 4 days after a marbofloxacin treatment at the therapeutic dose (MIC_marbo = 0.5 µg/ml). Time-kill kinetic profiles were similar for the two tested clones, irrespective of marbofloxacin susceptibility level (Fig. 2). During the lag phase (0 to 8 h), increased marbofloxacin concentrations did not produce a mycoplasmacidal effect. During the exponential phase (8 to 48 h), marbofloxacin was mycoplasmacidal at twice the MIC, 0.06 µg/ml and 1 µg/ml, respectively, for the wild-type and mutant strains (Fig. 2). Nevertheless, in both cases, mycoplasmas were still detectable after 48 h at twice the MIC. The mutant clone was still detectable after 24 h at four and eight times the MIC, in contrast to the wild-type strain. No difference in bactericidal effect was observed between four and eight times the MIC.

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FIG. 2. Time-kill curves of marbofloxacin against (a) the M. hyopneumoniae 116 wild-type strain and (b) one clone isolated on day 34 from an M. hyopneumoniae 116-infected pig treated at the therapeutic dose of marbofloxacin for 3 days (from day 27 to day 29) (limit of detection, log10 CFU/ml = 3; 200 CFU/ml).

Analysis of the GyrA, GyrB, ParC, and ParE QRDRs of M. hyopneumoniae. The GyrA, ParC, and ParE QRDR amino acid sequences of M. hyopneumoniae strain 116 were found to share 100% homology with those of M. hyopneumoniae 137, M. hyopneumoniae 232, M. hyopneumoniae ATCC 25934, and M. hyopneumoniae ATCC 25617 (data not shown). The GyrB QRDR amino acid sequence of M. hyopneumoniae 116 shared 100% homology with M. hyopneumoniae 137 and 98.4% homology with M. hyopneumoniae 232, M. hyopneumoniae ATCC 25617, and M. hyopneumoniae ATCC 25934 (data not shown). The GyrA, GyrB, ParC, and ParE sequences of M. hyopneumoniae share a high degree of homology with those of Mycoplasma pulmonis and Mycoplasma pneumoniae (Table 6).

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TABLE 6. Sequence identities between GyrA, GyrB, ParC, and ParE QRDRsa

The gyrA, gyrB, parC, and parE QRDRs were characterized for 5 clones of the M. hyopneumoniae 116 wild-type strain and 18 clones obtained from tracheal swabs of six experimentally infected pigs per group (Table 5). No mutations were detected in the QRDRs of the gyrA, gyrB, parC, and parE genes of clones isolated from infected untreated pigs. Three different alterations of the ParC amino acid sequence were found in clones from pigs treated at TD/2 or TD of marbofloxacin (only one mutation per clone). Two mutations, Ser80Phe and Asp84Asn (Escherichia coli numbering), were found in the ParC QRDR of DNA topoisomerase IV, and one mutation, Ala116Glu, was found in the ParC sequence, 14 amino acids behind the QRDR. These mutations were associated with an 8- to 16-fold increase in the marbofloxacin MIC (0.25 to 0.5 µg/ml) (Table 5).

Top Abstract Introduction Materials and Methods Results Discussion References

 
A marbofloxacin treatment at the therapeutic dose did not eliminate M. hyopneumoniae, with 87.5 to 100% of the pigs still positive at the end of the assays. Moreover, antibiotic treatments were not effective in significantly reducing clinical signs. Nevertheless, marbofloxacin treatments (TD and TD/2) seemed to decrease the lung lesion scores. Persistence of M. hyopneumoniae is regularly observed under field conditions (43) and with experimental infections (16, 25). This persistence phenomenon has already been described for Mycoplasma gallisepticum (38) and Mycoplasma synoviae (27) with experimentally infected chickens. M. hyopneumoniae is susceptible in vitro to many antibiotics, including fluoroquinolones (17, 18, 19). Hypotheses to explain M. hyopneumoniae persistence in treated pigs include (i) possible development of resistance mechanisms, as described in vitro for other mycoplasmas (12, 39, 40); (ii) lack of bioavailability of marbofloxacin in M. hyopneumoniae-infected pigs; (iii) ability of some mycoplasma species to invade host cells for long periods and reach subcellular fractions where marbofloxacin would not be active, or to persist inside the cell (34, 52) in a fluoroquinolone-insensitive state, as described for Mycoplasma penetrans (7); and (iv) survival of mycoplasmas on materials in an animal environment (14, 29) and subsequent natural reinfection of pigs.

The development of resistance in M. synoviae and M. gallisepticum has already been described to occur in vitro (12). An increase in resistance after several in vivo treatments at the therapeutic dose has been described for M. synoviae (27). In the present study, three independent substitutions in the ParC QRDR of DNA topoisomerase IV were associated with a 16-fold increase in resistance. The Ser80Phe and Ala116Glu substitutions in ParC were also found in quinolone-resistant Staphylococcus aureus mutants (22) or in clinical isolates of S. aureus (45, 50). The Asp84Asn substitution has never been described for other bacterial species, but amino acids at position 84 are implicated in fluoroquinolone resistance (22). The parC gene may be the primary target of marbofloxacin in M. hyopneumoniae, as for Mycoplasma bovirhinis (21) and S. aureus (grlA) (8, 50). A second mechanism of resistance to fluoroquinolones, namely, active efflux of the drug, could have explained the decreased susceptibility to fluoroquinolones of the reisolated M. hyopneumoniae clones. Active drug export systems have never been described for M. hyopneumoniae. Nevertheless, the complete genome sequence of M. hyopneumoniae revealed several multidrug resistance protein homologs (33). Active efflux pumps are often nonspecific, and cross-resistance to unrelated antibiotics, notably ciprofloxacin and tetracycline, has already been described (13). M. hyopneumoniae clones reisolated after marbofloxacin treatment were as susceptible to oxytetracycline as the M. hyopneumoniae 116 wild-type strain. Even if different active efflux systems may be implicated in tetracycline and fluoroquinolone resistance, the results suggest that increased MICs in this study were correlated to topoisomerase alterations.

In pigs, marbofloxacin is highly and rapidly absorbed after intramuscular medication at the therapeutic dose, as indicated by a high bioavailability (100%) (4) and a high peak of plasma concentration of marbofloxacin (maximum concentration of drug in serum [C_max] = 1.20 ± 0.04 µg/ml) obtained 1 h after injection (time to C_max = 1 h). These results are similar to those indicated by Vétoquinol (C_max = 1.47 µg/ml; time to C_max = 0.8 h) (4). Marbofloxacin is distributed to the respiratory tract but the ratio of lung to plasma concentrations (0.54 ± 0.21) is not in accordance with results obtained by Vétoquinol (ratio of lung to plasma concentrations, 1.8) (4). Pneumonia could explain such a difference, as marbofloxacin dosages were performed by Vétoquinol on healthy pigs. Recovering lesions observed in diseased pigs may disrupt blood circulation and thus marbofloxacin diffusion to the lungs. Nevertheless, achieved marbofloxacin concentrations in plasma, tracheas, tracheal secretions, and lungs were above the marbofloxacin MIC for the M. hyopneumoniae 116 wild-type strain (MIC_marbo = 0.03 µg/ml). Time-kill studies showed that marbofloxacin had a mycoplasmacidal effect during the exponential phase (on dividing cells) but not during the lag phase. The presence of non- or slow-dividing subpopulations named "persisters," as previously described for different bacterial species (1, 24), may be linked with M. hyopneumoniae persistence. Results also showed that M. hyopneumoniae can be reisolated from tracheal secretions (one tracheal swab), 24 h after a single marbofloxacin injection at the TD, without a MIC increase and despite high marbofloxacin concentrations achieved (0.124 ± 0.002 µg/ml in tracheal secretions 24 h postinjection). Results of time-kill studies showed that treatment of the M. hyopneumoniae 116 wild-type strain with 0.125 µg/ml for 24 h was effective in killing mycoplasma. Possible inhibitory properties of tracheal secretions could explain the decrease in mycoplasmacidal activity of marbofloxacin, as was described for feces (36). Persistence after fluoroquinolone treatment, without MIC change, has also been described previously for M. gallisepticum (38), M. synoviae (27), and M. hyopneumoniae (16). All of these studies are consistent with the fact that antibiotic resistance is not the only event in mycoplasma persistence. M. hyopneumoniae clones with a decreased susceptibility level to marbofloxacin were probably selected by the three repeated injections of marbofloxacin. Treatment at TD/2 was expected to select more easily the mycoplasma clones with decreased susceptibility to marbofloxacin. However, no difference was observed, even if the Asp84Asn substitution was found only in the TD/2 treatment group. Nevertheless, in this study, classical pharmacokinetic-pharmacodynamic surrogate markers, area under the concentration-time curve (AUC)/MIC (149) and C_max/MIC (15), were predictive of a good efficacy of marbofloxacin, without mutant selection (30).

Another hypothesis to explain the mycoplasma persistence in treated pigs was the ability to invade cells (34, 52) and reach subcellular fractions where marbofloxacin would not be active. Recent publications have shown the intracellular activity of fluoroquinolones (ciprofloxacin, ofloxacin, and pefloxacin) in vitro (31, 35, 41). Nevertheless, Seral et al. (47) have shown that, intracellularly, fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin, and garenoxacin) are 100 times less potent against S. aureus. Mycoplasma accessibility remains to be determined in vivo.

This is the first time that M. hyopneumoniae was reisolated from inner organs like livers, spleens, or kidneys. M. hyopneumoniae is considered an exclusive respiratory pathogen (51). Nevertheless, M. hyopneumoniae has already been reisolated from brains of infected pigs (9). Other mycoplasmas with predilection for the respiratory tract, M. pneumoniae in human (11, 53) and M. gallisepticum in poultry (34), are able to cross the mucosal barrier and spread in the host. Nevertheless, the persistence observed in this study cannot be explained by dissemination of mycoplasmas in inner organs, as this phenomenon seems to be transitory: no mycoplasma could be reisolated from inner organs at the end of the studies.

Results also clearly demonstrated that, under these experimental conditions, therapeutic failure could not be explained by the persistence of M. hyopneumoniae in the environment. This observation is consistent with the fact that mycoplasma recovered after the marbofloxacin medication presented nucleotide changes in the topoisomerase IV gene. M. hyopneumoniae seems to be able to persist in the pig.

In conclusion, results showed that, under the experimental conditions described in this study, intramuscular injections of marbofloxacin at the therapeutic dose for three consecutive days were not effective for the eradication of M. hyopneumoniae. This persistence was associated with a decrease in susceptibility level of some reisolated clones, but this phenomenon is not sufficient to explain the whole of M. hyopneumoniae persistence. Persistence of M. hyopneumoniae in the environment and subsequent recontamination of pigs after antibiotic elimination cannot explain therapeutic failures.

Works are in progress to determine if M. hyopneumoniae is able to invade phagocytic (alveolar macrophages) and nonphagocytic cells and escape the fluoroquinolone action.

 
We thank R. Cariolet (French Food Safety Agency of Ploufragan, France), B. Beaurepaire (French Food Safety Agency of Ploufragan, France), and J. Manceau (French Food Safety Agency of Fougères, France) for useful advice and skilled technical assistance.

 
* Corresponding author. Mailing address: Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches Avicoles et Porcines, Unité de Mycoplasmologie-Bactériologie, BP 53, 22440 Ploufragan, France. Phone: 33-2-96-01-62-86. Fax: 33-2-96-01-62-73. E-mail: a.bouchardon{at}ploufragan.afssa.fr.

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Antimicrobial Agents and Chemotherapy, June 2006, p. 1959-1966, Vol. 50, No. 6
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