Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin

Abstract

Mycoplasma bovis isolates belonging to the sequence type 5 (ST5) group, the dominant group in Japan since 1999, were low susceptible to 16-membered macrolides and tetracyclines and were confirmed to have a guanine-to-adenine transition mutation at position 748 in the 23S rRNA gene (rrl) and adenine-to-thymine transversion mutations at positions 965 and 967 in the 16S rRNA gene (rrs) (Escherichia coli numbering). Moreover, isolates of ST93 and ST155, members of the ST5 group, were low susceptible to lincosamides and azithromycin and showed an adenine-to-guanine transition mutation at position 2059 of rrl. Isolates of ST93 were additionally low susceptible to spectinomycin and showed a cytosine-to-adenine transversion mutation at position 1192 of rrs. Strains of the ST5 group seem to spread to Japan and Europe from North America with imported cows, while strains of ST93 and ST155 originated in Japan. Melting curve analysis using hybridization probes revealed the existence of point mutations involved in decreased susceptibility to macrolides, lincosamides, and spectinomycin, as demonstrated by changes in the melting curve shape and/or decreases in the melting peak temperature, so the susceptibility to these antimicrobials can be assessed on the same day. For decreased susceptibility to fluoroquinolones to exist, nonsynonymous mutations in the DNA gyrase gene (gyrA) and topoisomerase IV gene (parC) had to coexist. The combination of amino acid substitutions of serine at position 83 in gyrA and serine at position 80 in parC resulted in particularly low susceptibility to fluoroquinolones. IMPORTANCE Mycoplasma bovis is the main causal species of bovine mycoplasmal disease and leads to significant economic losses because of its severe symptoms, strong infectivity, and refractoriness. As for mastitis, culling cows with intramammary infections is a general countermeasure to prevent spreading. The conventional antimicrobial susceptibility test for mycoplasma is time-consuming and troublesome, but no quick and easy method for grasping the antimicrobial susceptibility of the causal strain exists at present. Treatment without antimicrobial susceptibility information may be one reason why M. bovis infection is refractory. Detecting a mutation involved in decreased susceptibility to antimicrobial agents of the causal strain makes it possible to easily select suitable antimicrobials for treatment, and this technique will help improve the cure rate and prevent the overuse of ineffective antimicrobial agents. In this study, we developed a technique to quickly and easily assess antimicrobial susceptibility based on the genetic characteristics of M. bovis strains in Japan. KEYWORDS Mycoplasma bovis, antimicrobial resistance, hybridization probe, multilocus sequence typing Citation Hata E, Harada T, Itoh M. 2019. Relationship between antimicrobial susceptibility and multilocus sequence type of Mycoplasma bovis isolates and development of a method for rapid detection of point mutations involved in decreased susceptibility to macrolide...

Phylogenomic analysis of Mycoplasma bovis from Belgian veal, dairy and beef herds

“…The persistence of strains within a country or herd has been described before [16,24,41]. Furthermore, shifts between dominant lineages from older to new strains have been reported before as well [13,15,46,47]. Also, we did not observe an association between cluster and affected organ, which is in line with previous studies [14,23,24].…”

Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle

“…This study was unique in that it assessed the concordance between the genotypes and phenotypes of M. bovis for antimicrobial resistance (AMR) to five macrolides used to control and treat bovine respiratory disease in feedlot cattle. Of note was the inclusion of TUL, which is the most commonly used antimicrobial for BRD prophylaxis, but a pharmaceutical that has not been assessed in previous genotype-phenotype AMR studies [13,31,32,47]. This is salient because even though macrolides have a similar antibacterial mode of action, they differ in the size of the macrocyclic lactone ring and associated side-chains [48].…”

“…Mutations within 23S rRNA and the L4 and L22 ribosomal proteins are associated with macrolide resistance [25,30]. This mechanism of resistance is not unique to M. bovis [13,31,32], having been reported in a variety of bacterial species, including other Mycoplasma spp. [33,34], Neisseria gonorrhoeae [30], Streptococcus spp.…”

“…As there are currently no effective vaccines for M. bovis, antimicrobials remain the primary means for the prevention and control of mycoplasmosis [2,7]. This has led to a number of M. bovis antimicrobial susceptibility studies in Canada [8][9][10][11], United States [12], Japan [13] and Europe [7,[14][15][16][17][18][19]. These studies suggest that M. bovis will continue to become increasingly resistant to antimicrobials.…”

“…Another challenge with performing AST on M. bovis is its very fastidious culture requirements, which is related to its reduced genome and limited biosynthetic capacity [44]. These requirements, coupled with relatively slow nonprolific growth, have encouraged the development of rapid molecular testing techniques for predicting antimicrobial susceptibility for M. bovis [13,45]. Utilization of a genotypic approach to assess antimicrobial susceptibility of M. bovis could allow for more expeditious evaluation of antimicrobial efficacy and evidence-based selection of antimicrobials to enable judicious use of antimicrobials, which are all principles of antimicrobial stewardship.…”