Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry

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 μg/mL to tylosin and with MIC ≥ 1.25 μg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides

The mysterious orphans of Mycoplasmataceae

Background: The length of a protein sequence is largely determined by its function, i.e. each functional group is associated with an optimal size. However, comparative genomics revealed that proteins length may be affected by additional factors. In 2002 it was shown that in bacterium Escherichia coli and the archaeon Archaeoglobus fulgidus, protein sequences with no homologs are, on average, shorter than those with homologs. Most experts now agree that the length distributions are distinctly different between protein sequences with and without homologs in bacterial and archaeal genomes. In this study, we examine this postulate by a comprehensive analysis of all annotated prokaryotic genomes and focusing on certain exceptions. Results: We compared lengths distributions of having homologs proteins (HHPs) and non-having homologs proteins (orphans or ORFans) in all currently annotated completely sequenced prokaryotic genomes. As expected, the HHPs and ORFans have strikingly different length distributions in almost all genomes. As previously established, the HHPs, indeed, are, on average, longer than the ORFans, and the length distributions for the ORFans have a relatively narrow peak, in contrast to the HHPs, whose lengths spread over a wider range of values. However, about thirty genomes do not obey these rules. Practically all genomes of Mycoplasma and Ureaplasma have atypical ORFans distributions, with the mean lengths of ORFan larger than the mean lengths of HHPs. These genera constitute over 80% of atypical genomes. Conclusions: We confirmed on a ubiquitous set of genomes the previous observation that HHPs and ORFans have different gene length distributions. We also showed that Mycoplasmataceae genomes have distinctive distributions of ORFans lengths. We offer several possible biological explanations of this phenomenon

Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and timeconsuming isolation of M. synoviae strains

Development of mismatch amplification mutation assay (MAMA) for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics

The aadE*-sat4-aphA-3 Gene Cluster of Mycoplasma bovirhinis HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence

The 54 kb GC-rich prophage region of Mycoplasma bovirhinis HAZ141_2 contains three structural ‘compartments’, one of which is a highly transmittable cluster of three genes, aadE-like (aadE*), sat4, and aphA-3. In this study, we characterized recombination events and their consequences occurred within the aadE*-sat4-aphA-3 containing region. Analysis of this region revealed direct repeats (DRs) of 155 and invert repeats (IRs) of 197 base pairs (bps) each, flanking and overlapping with the primary promoter P* located upstream of the aadE*. Two recombination events, including inversions via both 197 and 155-bp IRs (the latter become inverted after the initial 197-bp IRs associated inversion) and the excision of the aadE*-sat4-aphA-3 cluster, were confirmed. Inversion via 155-IRs results in changes within the P* promoter region. Using Escherichia coli JM109 carrying plasmids containing derivatives of the aadE*-sat4-aphA-3 cluster, we validated the expression of those genes from different promoters. Our results showed no difference in the minimal inhibitory concentrations (MICs) to kanamycin and neomycin and only 2-fold decrease in MIC (from 512 to 256 μg/mL) to nourseothricin between the wild type and a P* derivative promoter. However, the MICs to kanamycin and neomycin were at least 4-fold lower in the construct where aphA-3 expressed under its P2 promoter (128 µg/mL) in comparison to the construct where aphA-3 expressed under P1″ promoter located within the sat4 gene (512–1024 µg/mL). PCR confirmed the excision of the aadE*-sat4-aphA-3 cluster via 197- and 155-bp DRs, but no selection of antibiotic-sensitive M. bovirhinis were obtained after 100 passages in kanamycin-free medium

Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.

An innovative approach for inactivation of Mycoplasma gallisepticum using the hydrophobic photoinduced alkylating probe 1, 5-iodonaphthylazide (INA) is described. Treatment of washed M. gallisepticum mid-exponential culture (0.2 mg cell protein /mL) with INA followed by irradiation with far-ultraviolet light (310-380 nm) completely abolished viability. Transmission electron microscopy showed that the majority of the inactivated M. gallisepticum were comparable in size to intact cells, but that part of the INA-treated M. gallisepticum preparation also contained low density cells and membrane vesicles. Confocal microscopy revealed that untreated M. gallisepticum cells were internalized by chicken red blood cells (c-RBCs), whereas the INA-inactivated cells remained attached to the outer surface of the c-RBCs. INA treatment of M. gallisepticum resulted in a complete inactivation of F0F1 -ATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot analysis of the lipoprotein fraction showed that the INA-treated M. gallisepticum retained their lipoproteins. Following subcutaneous injection of M. gallisepticum INA-bacterin, 100% and 68.8% of chickens were positive by the rapid serum agglutination test and enzyme-linked immunosorbent assay respectively, 2 weeks post-injection. These data suggest that the photoinducible alkylating agent INA inactivates M. gallisepticum but preserves its surface lipoproteins and thus has the potential to be used as a general approach for the inactivation of mycoplasmas for vaccine development

The effect of INA treatment on ³H-<i>L</i>-arginine uptake by <i>M</i>. <i>gallisepticum</i>.

<p><i>M</i>. <i>gallisepticum</i> Rlow cells (0.2 mg cell protein/mL) were treated with INA followed by UV irradiation and tested for ³H-<i>L</i>-arginine uptake. Open symbols, INA treated cells; closed symbols, control of untreated cells.</p