Multiplex PCR To Identify Macrolide Resistance Determinants in Mannheimia haemolytica and Pasteurella multocida

Abstract

bThe bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLS B (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.

Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

“…Measurement of radioisotope incorporation, as opposed to GFP fluorescence (23), enabled quantification of aborted and extended products formed by translational drop-off and stop codon read-through, respectively. None of the drugs tested here aborted peptide chains longer than 10 residues, consistent with observations for other macrolides blocking synthesis after addition of 2 to (4,19). E. coli ATCC 25922 also lacks any resistance determinants and is a wild-type strain with regard to cell wall/membrane structures.…”

“…We note that such rRNA mutations were introduced primarily to study drug interaction and are unlikely to arise in the field in bacteria with multiple rrn operons (25). However, methylation at nucleotide A2058 has been seen to confer macrolide resistance in P. multocida and M. haemolytica isolates and is added by the Pasteurellaceae-specific monomethyltransferase Erm(42) (3,10,14,19). A2058 monomethylation causes a similar resistance profile in E. coli, while dimethylation at A2058 confers high resistance to all macrolides, including tylosin (Table 2).…”

Susceptibility to tulathromycin in Mannheimia haemolytica isolated from feedlot cattle over a 3-year period

“…In addition, 13.2% were administered subtherapeutic concentrations of tylosin phosphate in feed. We included in our analyses all macrolides commonly used in feedlot production because some tulathromycin resistance genes have also been shown to confer resistance to tilmicosin and tylosin (Rose et al, 2012) and increase the MICs of tildipirosin and gamithromycin (Michael et al, 2012a). Theoretically, tilmicosin and tylosin could have therefore exerted a selection pressure for tulathromycin resistance.…”

“…Recent studies have characterized genes in M. haemolytica that provide cross-resistance to tulathromycin and other macrolides (Desmolaize et al, 2011; Michael et al, 2012a; Rose et al, 2012). Resistance to antibiotics commonly used for BRD poses animal health and economic concerns.…”

Antimicrobial resistance genes in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs

“…The tetracycline resistance genes, tet B, tet C and tet H (coding for efflux pumps), and tet L and tet O (coding for ribosomal protective proteins), have been reported in the tetracycline‐resistant pathogens A. pleuropneumoniae , H. parasuis , Mannheimia species and P. multocida . The macrolide resistance gene that codes for an efflux pump ( msr E), genes that cause methylation of the ribosomal target ( erm A, erm C and erm 42) and the gene that codes for a phosphorylase‐inactivating enzyme ( mph E) have been detected in members of the family Pasteurellaceae associated with porcine and bovine respiratory disease …”