Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle.
ABSTRACT: Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at ?748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (?748?2059 or ?748?2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a ?748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.
Project description:The 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.
Project description:The mechanisms of intrinsic resistance of Mycoplasma hominis to 14- and 15-membered macrolides were investigated in comparison with those of M. pneumoniae, which is naturally susceptible to macrolides. Radiolabeled erythromycin was not accumulated by M. hominis PG21, but addition of an ABC transporter inhibitor increased the level of erythromycin uptake more than two times, suggesting the existence of an active efflux process. The affinity of [(14)C]erythromycin to ribosomes isolated from M. hominis was dramatically reduced relative to that to ribosomes isolated from M. pneumoniae. The nucleotide sequences of 23S rRNA of both ribosomal operons rrnA and rrnB and ribosomal proteins L4 and L22 of M. hominis were obtained. Compared to the sequence of M. pneumoniae, M. hominis harbored a G2057A transition in its 23S rRNA sequence, as did M. fermentans, another mycoplasma that is erythromycin resistant. An additional C2610U change was also found in the sequence of M. hominis. Moreover, two M. hominis clinical isolates with acquired resistance to 16-membered macrolides were examined for mutations in domain II and domain V of 23S rRNA and in ribosomal proteins L4 and L22. Compared to the sequence of reference strain PG21, one isolate harbored a A2059G transition and a C2611U transition in one of the two rrn operons, while the other one was mutated only at position 2059, also on the same operon. No mutation was found in the two ribosomal protein sequences. Overall, the present study is an exhaustive characterization of the intrinsic resistance of M. hominis to 14- and 15-membered macrolides and the first description of mycoplasma clinical isolates resistant to macrolide, lincosamide, and streptogramin antibiotics harboring a mutation at position 2611 in the 23S rRNA.
Project description:Over a two-year period, Mannheimia haemolytica (MH; n = 113), Pasteurella multocida (PM; n = 47), Histophilus somni (HS; n = 41) and Mycoplasma bovis (MB; n = 227) were isolated from bovine lung tissue at necropsy from cattle raised conventionally (CON, n = 29 feedlots) or without antimicrobials [natural (NAT), n = 2 feedlots]. Excluding MB, isolates were assayed by PCR to detect the presence of 13 antimicrobial resistance (AMR) genes and five core genes associated with integrative and conjugative elements (ICEs). Antimicrobial susceptibility phenotypes and minimum inhibitory concentrations (MICs, µg/mL) were determined for a subset of isolates (MH, n = 104; PM, n = 45; HS, n = 23; and MB, n = 61) using Sensititre analyses. A subset of isolates (n = 21) was also evaluated by whole-genome sequencing (WGS) based on variation in AMR phenotype. All five ICE core genes were detected in PM and HS by PCR, but only 3/5 were present in MH. Presence of mco and tnpA ICE core genes in MH was associated with higher MICs (p < 0.05) for all tetracyclines, and 2/3 of all macrolides, aminoglycosides and fluoroquinolones evaluated. In contrast, association of ICE core genes with MICs was largely restricted to macrolides for PM and to individual tetracyclines and macrolides for HS. For MH, the average number of AMR genes markedly increased (p < 0.05) in year 2 of the study due to the emergence of a strain that was PCR positive for all 13 PCR-tested AMR genes as well as two additional AMR genes (aadA31 and blaROB-1) detected by WGS. Conventional management of cattle increased (p < 0.05) MICs of tilmicosin and tulathromycin for MH; neomycin and spectinomycin for PM; and gamithromycin and tulathromycin for MB. The average number of PCR-detected AMR genes in PM was also increased (p < 0.05) in CON mortalities. This study demonstrates increased AMR especially to macrolides by bovine respiratory disease organisms in CON as compared to NAT feedlots and a rapid increase in AMR following dissemination of strain(s) carrying ICE-associated multidrug resistance.
Project description:Mycoplasma bovis is a major cause of pneumonia, arthritis, and mastitis in cattle and can lead to significant economic losses. Antimicrobial resistance is a concern and further limits the already short list of drugs effective against mycoplasmas. The objective of this study was to examine changes in in vitro minimum inhibitory concentrations (MICs) of antimicrobials of aminoglycoside, fluoroquinolone, lincosamide, macrolide, pleuromutilin, phenicol, and tetracycline classes for 210 M. bovis isolates collected from 1978 to 2009. The MIC50 values of the various antimicrobials were also compared. The MIC50 levels for enrofloxacin and danofloxacin remained low (0.25 μg/mL) across all 3 decades. MIC50 levels for tetracyclines, tilmicosin, and tylosin tartrate were low in the 1980s, then increased in the 1990s and remained high. In the 1980s, MIC50 levels were low for clindamycin, spectinomycin, and tulathromycin, increased in the 1990s to 8 μg/mL (clindamycin) and 32 μg/mL (spectinomycin and tulathromycin), then decreased again in the 2000s. Members of the fluoroquinolone class of antimicrobials had the lowest MIC50 levels across all 3 decades, which suggests in vitro susceptibility of M. bovis to this class of antimicrobials. Statistically significant associations were observed between MIC values for chlortetracycline, oxytetracycline, tylosin tartrate, and tilmicosin; between clindamycin, tulathromycin, spectinomycin, and tiamulin; and between tylosin tartrate and clindamycin. Changes in MIC levels of various antimicrobials over time show the importance of monitoring the susceptibility of mycoplasmas to antimicrobials. The number of antimicrobials that showed elevated MIC50 levels, and therefore possibly reduced in vitro effectiveness against M. bovis, supports initiatives that promote prudent use of antimicrobials in agriculture.
Project description: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.
Project description:Streptococcus pneumoniae serotype 3, isolated from a penicillin-allergic patient and initially susceptible to fluoroquinolones, macrolides, lincosamides, quinupristin-dalfopristin, and telithromycin, became resistant to all these drugs during treatment. Mutations in the parC and gyrA and in the 23S rRNA and the ribosomal protein L22 genes were detected in the resistant isolates.
Project description:The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ? 0.63 ?g/mL to tylosin and with MIC ? 1.25 ?g/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.
Project description:A collection of 23 macrolide-resistant Campylobacter isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as Campylobacter jejuni or Campylobacter coli based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A-->G transition at Escherichia coli equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant Campylobacter isolates. Another macrolide-associated mutation (A-->C transversion), at E. coli equivalent base 2058, was detected in about 13% of the isolates. An A-->G transition at a position cognate with E. coli 23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in Helicobacter pylori, was also identified in one of the C. jejuni isolates examined. In the majority of C. jejuni isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant Campylobacter isolate to a susceptible Campylobacter isolate. Growth rates of the resulting transformants containing A-2058-->C or A-2059-->G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One C. jejuni isolate showing an A-2058-->G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of C. jejuni and C. coli demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.
Project description:Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones (enrofloxacin 2.5 ?g/ml; marbofloxacin 5 ?g/ml) were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin (tilmicosin, tulathromycin and lincomycin >64 ?g/ml; gamithromycin 64 ?g/ml; tylosin 32 ?g/ml and tylvalosin 2 ?g/ml) were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA (Gly81Ala; Ala83Val; Glu87Gly, according to Escherichia coli numbering) and parC (Ser80Phe/Tyr; Asp84Asn) correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy.
Project description:Pneumococcal macrolide resistance is usually expressed as one of two phenotypes: the M phenotype conferred by the mef gene or the MLSB phenotype caused by modification of ribosomal targets, most commonly mediated by an erm methylase. Target-site modification leading to antibiotic resistance can also occur due to sequence mutations within the 23S rRNA or the L4 and L22 riboproteins. We screened 4,535 invasive isolates resistant to erythromycin and 18 invasive isolates nonsusceptible to quinupristin-dalfopristin (Q-D) to deduce the potential mechanisms involved. Of 4,535 erythromycin-resistant isolates, 66.2% were polymerase chain reaction (PCR)-positive for mef alone, 17.8% for ermB alone, and 15.1% for both mef and ermB. Thirty-seven isolates (0.9%) were PCR negative for both determinants. Of these, 3 were positive for ermA (subclass ermTR) and 25 had chromosomal mutations. No chromosomal mutations (in 23S rRNA, rplD, or rplV) nor any of the macrolides/lincosamides/streptogramin (MLS) resistance genes screened for (ermT, ermA, cfr, lsaC, and vgaA) were found in the remaining nine isolates. Of 18 Q-D nonsusceptible isolates, 14 had chromosomal mutations and one carried both mef and ermB; no chromosomal mutations or other resistance genes were found in 3 isolates. Overall, we found 28 mutations, 13 of which have not been previously described in Streptococcus pneumoniae. The role of these mutations remains to be confirmed by transformation assays.