Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

Identification of a Novel Lincomycin Resistance Mutation Associated with Activation of Antibiotic Production in Streptomyces coelicolor A3(2)

ABSTRACT Comparative genome sequencing analysis of a lincomycin-resistant strain of Streptomyces coelicolor A3(2) and the wild-type strain identified a novel mutation conferring a high level of lincomycin resistance. Surprisingly, the new mutation was an in-frame DNA deletion in the genes SCO4597 and SCO4598, resulting in formation of the hybrid gene linR. SCO4597 and SCO4598 encode two histidine kinases, which together with SCO4596, encoding a response regulator, constitute a unique two-component system. Sequence analysis indicated that these three genes and their arrangement patterns are ubiquitous among all Streptomyces genomes sequenced to date, suggesting these genes play important regulatory roles. Gene replacement showed that this mutation was responsible for the high level of lincomycin resistance, the overproduction of the antibiotic actinorhodin, and the enhanced morphological differentiation of this strain. Moreover, heterologous expression of the hybrid gene linR in Escherichia coli conferred resistance to lincomycin in this organism. Introduction of the hybrid gene linR in various Streptomyces strains by gene engineering technology may widely activate and/or enhance antibiotic production.

LmrS Is a Multidrug Efflux Pump of the Major Facilitator Superfamily from Staphylococcus aureus

ABSTRACT A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp.

tISCpe8, an IS1595-Family Lincomycin Resistance Element Located on a Conjugative Plasmid in Clostridium perfringens

ABSTRACT Clostridium perfringens is a normal gastrointestinal organism that is a reservoir for antibiotic resistance genes and can potentially act as a source from which mobile elements and their associated resistance determinants can be transferred to other bacterial pathogens. Lincomycin resistance in C. perfringens is common and is usually encoded by erm genes that confer macrolide-lincosamide-streptogramin B resistance. In this study we identified strains that are lincomycin resistant but erythromycin sensitive and showed that the lincomycin resistance determinant was plasmid borne and could be transferred to other C. perfringens isolates by conjugation. The plasmid, pJIR2774, is the first conjugative C. perfringens R-plasmid to be identified that does not confer tetracycline resistance. Further analysis showed that resistance was encoded by the lnuP gene, which encoded a putative lincosamide nucleotidyltransferase and was located on tISCpe8, a functional transposable genetic element that was a member of the IS1595 family of transposon-like insertion sequences. This element had significant similarity to the mobilizable lincomycin resistance element tISSag10 from Streptococcus agalactiae. Like tISSag10, tISCpe8 carries a functional origin of transfer within the resistance gene, allowing the element to be mobilized by the conjugative transposon Tn916. The similarity of these elements and the finding that they both contain an oriT-like region support the hypothesis that conjugation may result in the movement of DNA modules that are not obviously mobile since they are not linked to conjugation or mobilization functions. This process likely plays a significant role in bacterial adaptation and evolution.

Lincomycin Resistance Gene lnu(D) in Streptococcus uberis

ABSTRACT Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 μg/ml) while remaining susceptible to clindamycin (MIC = 0.06 μg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinAN2, and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2′′) was identified.