No change in avilamycin (Surmax® Premix) minimum inhibitory concentration for Clostridium perfringens isolates recovered from poultry up to 7 years post-approval in Canada.

BACKGROUND: Clostridium perfringens, the causative agent of necrotic enteritis (NE) in chickens, has an enormous economic impact on global broiler production. The non-medically important antibiotic avilamycin was approved in Canada in 2014 to prevent and control NE in broiler chickens. OBJECTIVES: To compare avilamycin susceptibility in C. perfringens isolates collected pre- and 7 years post-avilamycin approval in Canada and determine the avilamycin resistance mutation frequency rate in C. perfringens. METHODS: The MICs of avilamycin were determined for 89 strains of C. perfringens recovered from clinically relevant NE field cases pre-avilamycin approval between 2003 and 2013 (n = 50) and post-avilamycin approval between 2014 and 2021 (n = 39) across Canada. For determining the mutant prevention concentration (MPC) of avilamycin for C. perfringens strains, a strain with avilamycin MIC of 1 mg/L was randomly selected. RESULTS: MIC studies showed no difference in avilamycin susceptibility in pre-avilamycin and post-avilamycin isolates (MIC50/90: pre-avilamycin approval 2/2 mg/L and post-avilamycin approval 1/2 mg/L). The MPC was 8 × MIC (8 mg/L) for the selected strain. CONCLUSIONS: These findings suggest that the susceptibility of C. perfringens strains to avilamycin was not impacted by its continued use in the 7 years following its approval in Canada. Avilamycin, a non-medically important antibiotic, poses no threat to human health regarding cross-resistance or co-selection of other medically important antibiotics. These factors make avilamycin an appropriate choice for continued use in broiler chickens to prevent and control NE without increased antimicrobial resistance concerns.

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae isolated from seven European countries during 2015-2016.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease, causing significant economic losses. Results from the 2015-2016 MycoPath pan-European antimicrobial susceptibility monitoring survey of M. hyopneumoniae are presented. In total, 147 M. hyopneumoniae porcine isolates from Belgium, France, Germany, Great Britain, Hungary, Italy, and Spain were tested. One isolate per farm was retained from pigs that had not been recently treated with antimicrobial agents. The minimal inhibitory concentration (MIC) of 13 antimicrobial agents was determined in a central laboratory using a broth microdilution method, with Friis Medium, incubated at 35 ± 1 °C for 5-12 days. M. hyopneumoniae NCTC 10110 was used as Quality Control. MIC50/MIC90 (mg/L) values were: enrofloxacin 0.06/1; marbofloxacin 0.06/2; spiramycin 0.06/0.25; tulathromycin ≤0.001/0.004; gamithromycin 0.06/0.5; tylosin 0.016/0.06; tilmicosin 0.06/0.5; florfenicol 0.5/1; doxycycline 0.25/1; oxytetracycline 0.25/2; lincomycin 0.06/0.25; tiamulin 0.016/0.06 and valnemulin ≤0.001/0.004. Compared with the data from 2010 to 2012 MycoPath study (50 isolates), MIC50/90 results were similar and the majority were within ± two dilution steps, except for the MIC50 of oxytetracycline which is more than two dilution steps higher in the present study. Between-country comparisons show some differences in the MIC values for the fluoroquinolones, tulathromycin and tylosin, but the limited sample size per country precludes performing meaningful country comparisons for several countries. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy.

Minimal inhibitory concentration of seven antimicrobials to Mycoplasma gallisepticum and Mycoplasma synoviae isolates from six European countries.

Mycoplasma gallisepticum and Mycoplasma synoviae are bacterial pathogens that cause disease in poultry, adversely affecting their health and welfare, and are a financial burden on producers. This manuscript describes the results of the MycoPath project that is the first international antimicrobial susceptibility programme for mycoplasma pathogens isolated from poultry. Improved comparative analysis of minimal inhibitory concentration (MIC) results from participating countries was facilitated by using one laboratory determining all MICs. Chicken and turkey isolates were obtained from France, Germany, Great Britain, Hungary, Italy and Spain during 2014-2016. One isolate per farm was retained. The MIC of seven antimicrobial agents was determined using a broth microdilution method, with Friis Medium (M. gallisepticum) or Modified Chanock's Medium (M. synoviae). Of the 222 isolates recovered, 82 were M. gallisepticum and 130 were M. synoviae. M. gallisepticum MIC50/90 values were 0.12/0.5, 2/8, 0.5/4, 0.12/>64, 0.008/0.062, 0.008/32, 0.062/4 mg/l for doxycycline, enrofloxacin, oxytetracycline, spiramycin, tiamulin, tilmicosin and tylosin, respectively. For M. synoviae, the values were 0.5/1, 8/16, 0.5/1, 0.5/8, 0.25/0.5, 0.062/2 and 0.062/16 mg/l respectively. A bimodal MIC distribution for the fluoroquinolone (enrofloxacin) and the macrolides (spiramycin, tilmicosin and tylosin) indicate that both species have sub-populations that are less susceptible in vitro to those antimicrobials. Some differences in susceptibilities were observed according to host species, Mycoplasma species, and country of origin. This study provides a baseline of novel data for future monitoring of antimicrobial resistance in poultry Mycoplasma species. Additionally, this information will facilitate the selection of the antimicrobial agents most likely to be effective, thus ensuring their minimal use with targeted and correct therapeutic treatments.Highlights First large-scale pan-European collection of representative Mg and Ms isolates.MIC values assessed in central laboratory for Mg and Ms from chickens and turkeys.Range of MIC values for 82 Mg and 130 Ms isolates to seven licenced antibiotics shown.Data can be used to help determine Mg and Ms veterinary-specific breakpoints.

New antimicrobial susceptibility data from monitoring of Mycoplasma bovis isolated in Europe.

Mycoplasma bovis is an important respiratory pathogen of cattle across Europe and is included in the MycoPath pan-European antimicrobial susceptibility monitoring programme. M. bovis strains (232) were isolated from cattle, not recently treated with antimicrobials, at diverse geographical locations in France, Great Britain, Hungary, Italy and Spain during 2014 to 2016. Only one isolate per farm and per outbreak was retained. For each isolate, the MICs of ten antimicrobials were determined in a central laboratory using a broth microdilution method with modified Eaton's medium and incubation at 35 °C ± 1 °C for 24 ± 6 h. MIC50/MIC90 (mg/L) values for the 232 strains were: danofloxacin 0.25/1; enrofloxacin 0.5/8; marbofloxacin 1/4; gamithromycin >64/>64; spiramycin 8/16; tilmicosin >64/>64; tulathromycin >64/>64; tylosin 64/>64; florfenicol 4/8; oxytetracycline 8/32. Minor between-country differences in the MIC90 values were observed for the fluoroquinolones, spiramycin and oxytetracycline, whilst the MIC values for the other compounds were similar. Spain and Italy had the higher MIC90 values for the fluoroquinolones. Compared with the 2010-2012 study (156 isolates) results are similar, with an overall MIC50 increase of at most one doubling dilution for enrofloxacin, spiramycin, tylosin, florfenicol and oxytetracycline. In contrast, the MIC90 value for oxytetracycline decreased from >64 to 32 mg/L. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy.

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae and Mycoplasma bovis isolated in Europe.

Mycoplasma hyopneumoniae in pigs and Mycoplasma bovis in cattle are major pathogens affecting livestock across Europe and are the focus of the MycoPath pan-European antimicrobial susceptibility monitoring programme. Fifty M. hyopneumoniae isolates from Belgium, Spain and the United Kingdom (UK), and 156 M. bovis isolates from France, Hungary, Spain and the UK that met specific criteria were tested for antimicrobial susceptibility in a central laboratory by using a microbroth dilution method. Specific isolate criteria included recovery from animals not recently treated with antimicrobials, isolates from different locations within each country and retaining only one isolate per farm. MIC50/MIC90 values were 0.031/0.5, 0.031/0.5, 0.062/0.25, ≤0.001/0.004, 0.031/0.125, 0.25/0.5 and 0.062/0.25mg/L for enrofloxacin, marbofloxacin, spiramycin, tulathromycin, tylosin, florfenicol and oxytetracycline respectively against M. hyopneumoniae and 0.25/4, 1/4, 4/16, >64/ >64, 32/ >64, 2/4 and 4/64mg/L, respectively against M. bovis. MIC50/MIC90 values for tiamulin and valnemulin against M. hyopneumoniae were 0.016/0.062 and ≤0.001/ ≤0.001mg/L respectively. The MIC50/MIC90 values of danofloxacin and gamithromycin for M. bovis were 0.25/1 and >64/ >64mg/L respectively. The highest MIC90 values for M. hyopneumoniae were found in the UK at 1.0mg/L for enrofloxacin, marbofloxacin and florfenicol. In contrast, for M. bovis the lowest MIC90 value was 1.0mg/L, but ranged to >64mg/L. Specific laboratory standards and clinical breakpoints for veterinary Mycoplasma species are required as no independently validated clinical breakpoints are specified for veterinary Mycoplasma species, which makes data interpretation and correlation to in vivo efficacy difficult.

Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.

Bactericidal properties of pradofloxacin against veterinary pathogens.

Pradofloxacin is a new veterinary 8-cyano-fluoroquinolone developed for use against bacterial infections in dogs and cats involving both aerobic and anaerobic bacteria. The minimal bactericidal concentrations have been determined against clinical isolates of Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, Streptococcus canis, Proteus spp., Fusobacterium spp., Porphyromonas gingivalis and Prevotella species. A subset of these species was selected, and the in vitro rate of kill by pradofloxacin was determined. For 27 of the 30 tested aerobic strains the pradofloxacin MBC was within two doubling dilutions of the MIC. For the remaining strains, the MIC and MBC were within three to four doubling dilutions. Pradofloxacin also demonstrated bactericidal activity against all anaerobic strains, and the MBC was equal to the MIC for four of the strains, within 1 doubling dilution for three strains, within 2 dilutions for a further 3 strains and within 3 dilutions for the remaining five strains. As pradofloxacin concentration was increased, a faster rate of killing was observed; bactericidal effects were seen in all cases at concentrations ≤ 0.25 µg/mL. The bactericidal activity against the anaerobic strains was marked, of particular relevance was the complete absence of regrowth even at 48 h at concentrations as low as 0.125 µg/mL. In conclusion, pradofloxacin exhibits clear bactericidal activity in terms of MBC and kill kinetics against aerobic and anaerobic clinical isolates from dogs and cats at concentrations that are greatly exceeded within the systemic circulation after administration of the recommended therapeutic doses to the target animals. It is expected that such a rapid rate of kill will play a significant role in clinical efficacy. These data demonstrate the complete and rapid killing of anaerobic bacteria by a veterinary 8-cyano-fluoroquinolone.

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae.

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in 'Veterinary Fastidious Medium' (VFM) (supplemented Mueller-Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3-72 µg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.

Activity of pradofloxacin against Porphyromonas and Prevotella spp. Implicated in periodontal disease in dogs: susceptibility test data from a European multicenter study.

Collaborating veterinarians from five European countries collected subgingival bacterial samples from dogs exhibiting clinical periodontal disease. Sterile endodontic paper points were used for collection of the samples, which were transported to a central laboratory for susceptibility testing. Anaerobic bacteria were isolated and Porphyromonas and Prevotella isolates identified to the species level; susceptibility to pradofloxacin and metronidazole was determined using the CLSI agar dilution methodology. A total of 630 isolates, 310 of Porphyromonas spp. and 320 of Prevotella spp., were isolated. Pradofloxacin MIC data for all isolates were in the range of < or =0.016 to 1 microg/ml, the overall MIC(50) was 0.062, and the overall MIC(90) was 0.25 microg/ml. There were no differences in activity against Porphyromonas and Prevotella isolates or in the pradofloxacin susceptibility distributions from the different European countries. All isolates were within the wild-type distribution and were fully susceptible to pradofloxacin. Metronidazole was also highly active against these strains: 316 of 320 Prevotella strains (98.8%) and 309 of 310 Porphyromonas strains (99.7%) were susceptible (MICs of < or =8 microg/ml). However, three Prevotella strains had intermediate metronidazole susceptibility (MICs of 16 microg/ml), while one Prevotella and one Porphyromonas strain were metronidazole resistant (MICs of 128 and 256 microg/ml, respectively). Pradofloxacin, a novel broad-spectrum fluoroquinolone, demonstrates a high degree of antianaerobic activity against strains isolated from clinical cases of periodontal disease and shows activity against metronidazole-resistant isolates. The broad-spectrum activity of pradofloxacin makes it a suitable candidate for the treatment of periodontal disease in dogs.

Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats.

OBJECTIVES: To compare the intrinsic activity of pradofloxacin, a new fluoroquinolone developed for use in veterinary medicine, with other fluoroquinolones, against anaerobic bacteria isolated from dogs and cats. METHODS: One hundred and forty-one anaerobes were isolated from dogs and cats and comparative MICs of pradofloxacin, marbofloxacin, enrofloxacin, difloxacin and ibafloxacin were determined according to standardized agar dilution methodology. RESULTS: Pradofloxacin exerted the greatest antibacterial activity followed by marbofloxacin, enrofloxacin, difloxacin and ibafloxacin. Based on the distinctly lower MIC(50), MIC(90) and mode MIC values, pradofloxacin exhibited a higher in vitro activity than any of the comparator fluoroquinolones. CONCLUSIONS: Pradofloxacin, a novel third-generation fluoroquinolone, has broad-spectrum anti-anaerobe activity and offers utility as single-drug therapy for mixed aerobic/anaerobic infections.