Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.

Occurrence and Characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Quarter Milk Samples From Dairy Cows in Germany.

The aim of this study was to identify and characterize LA-MRSA in a very recent collection of staphylococci isolated from bovine quarter milk samples. All milk samples (n = 14,924) sent to the MBFG in March 2017 were included into this study. The samples originated from 3,887 cows with 3,367 samples from 2,280 animals being positive for bacteria, prototheca and/or yeast. The second most common infectious agent was Staphylococcus and 659 isolates were investigated. Staphylococcus aureus was confirmed by PCR for the spa gene. A CC398-specific PCR was performed for all S. aureus isolates. Susceptibility to penicillin was tested for all isolates by agar disk diffusion. All oxacillin resistant isolates were analyzed by microarray and tested for their susceptibility to 30 antimicrobial agents. Of the isolates 372 were S. aureus from Germany with 214 isolates being not epidemiologically related. Among the independent isolates nine were identified as oxacillin resistant. In addition five isolates epidemiologically related to these nine were MRSA. One of them showed differences to the other MRSA isolate from the same farm resulting in altogether ten different MRSA isolates. All ten belonged to the clonal complex CC398. These ten LA-MRSA isolates had three to six antimicrobial resistance genes. The gene mecA was in all cases located on a SCCmec V element. Among the remaining S. aureus seven independent isolates belonged to CC398. In conclusion this study showed a high detection rate of staphylococci in bovine quarter milk samples. In contrast MRSA was rarely detected and belonged in all cases to CC398. Only 7/214 MSSA (3.3%) belonged to this CC.

Comparative erythromycin and tylosin susceptibility testing of streptococci from bovine mastitis.

Tylosin, a 16-membered macrolide, is - besides other indications - used for the treatment of bovine mastitis. So far, there is only limited information available on the tylosin susceptibility of streptococci isolated from mastitis. The aim of the present study was to comparatively investigate 303 streptococci from bovine mastitis, including 101 Streptococcus agalactiae, 100 Streptococcus dysgalactiae and 102 Streptococcus uberis, for their tylosin and erythromycin susceptibility by broth microdilution and agar disk diffusion. Both tests followed the recommendations of the Clinical and Laboratory Standards Institute (CLSI). For erythromycin, the results were interpreted using the CLSI-approved clinical breakpoints. Moreover, erythromycin-resistant isolates were tested for the presence of macrolide resistance genes and for inducible macrolide resistance. In general, both testing methods showed a good correlation for the three streptococcal species, although for the erythromycin susceptibility testing 11 S. uberis isolates fell into the very major error category. All but one of the erythromycin-resistant isolates harbored at least one macrolide resistance gene, with the erm(B) gene being most common. Moreover, single isolates of S. agalactiae and S. dysgalactiae proved to be inducibly macrolide-resistant. Since inducible macrolide resistance can easily switch to constitutive resistance, tylosin should not be used for the treatment of infections caused by inducibly resistant streptococci.

Tylosin susceptibility of Staphylococci from bovine mastitis.

Although the 16-membered macrolide tylosin is commonly used for the treatment of bovine mastitis, little information is currently available about the susceptibility of mastitis pathogens to tylosin. In the present study, 112 Staphylococcus aureus and 110 coagulase-negative Staphylococcus (CoNS) spp. isolates from cases of bovine mastitis were tested by broth microdilution and agar disk diffusion with 30 µg tylosin disks. Susceptibility to erythromycin was tested by broth microdilution and disk diffusion using 15 µg disks. Both test populations showed bimodal distributions of minimal inhibitory concentrations (MICs) and zone diameters with eleven S. aureus and eight CoNS isolates showing tylosin MICs of ≥ 256 µg/ml and no zones of growth inhibition around the tylosin 30 µg disks. All 19 isolates with tylosin MICs of ≥ 256 µg/ml were also resistant to erythromycin. For six additional erythromycin-resistant isolates, tylosin MICs of 1-8 µg/ml were observed. One S. aureus and two CoNS isolates showed inducible macrolide resistance. PCR analysis of the 25 erythromycin-resistant staphylococcal isolates identified the resistance genes erm(A), erm(B), erm(C), erm(T), mph(C) and msr(A) alone or in different combinations. An excellent correlation between the results of the different tylosin susceptibility tests (broth microdilution versus disk diffusion) was seen for S. aureus and CoNS isolates. Since tylosin does not induce the expression of the aforementioned erm genes, isolates with an inducible resistance phenotype may - if only tylosin is tested - be falsely classified as tylosin-susceptible. Thus, erythromycin should be tested in parallel and tylosin should only be used for the treatment of infections caused by erythromycin-susceptible staphylococci.

[Antimicrobial resistance, ESBL and MRSA--definitions and laboratory diagnostics]. / Antimikrobielle Resistenz, ESBL und MRSA--Definitionen und Labordiagnose.

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms "antimicrobial resistance", "ESBL", and "MRSA" are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria--clinical breakpoints and epidemiological cut-off values--including their applications are explained in detail. Furthermore, currently valid diagnostic procedures--as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI)--for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and--as a consequence--for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions.