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1.  Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry 
Veterinary Research  2011;42(1):90.
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.
PMCID: PMC3166906  PMID: 21810258
2.  Evaluation of Amplified Fragment Length Polymorphism for Differentiation of Avian Mycoplasma Species 
Journal of Clinical Microbiology  2005;43(2):909-912.
Amplified fragment length polymorphism (AFLP) was used for typing avian mycoplasma species. Forty-four avian mycoplasma strains were successfully typed into eight distinct groups, with each representing a different species. Homology of AFLP patterns of 35% or less was used as a cutoff value to differentiate avian mycoplasma strains into different species.
PMCID: PMC548061  PMID: 15695703
3.  Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions  
Infection and Immunity  2003;71(7):3821-3830.
Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.
PMCID: PMC162021  PMID: 12819065
4.  Cytadherence-Deficient Mutants of Mycoplasma gallisepticum Generated by Transposon Mutagenesis  
Infection and Immunity  2003;71(7):3812-3820.
Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gmr HA-negative (HA−) colonies displaying a stable HA− phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA− mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA− mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.
PMCID: PMC162017  PMID: 12819064
5.  Extended Repertoire of Genes Encoding Variable Surface Lipoproteins in Mycoplasma bovis Strains  
Infection and Immunity  2002;70(4):2220-2225.
A genomic cluster of vsp genes was previously shown to mediate high-frequency phenotypic switching of surface lipoprotein antigens in the bovine pathogen Mycoplasma bovis. This study revealed that field strains of M. bovis possess modified versions of the vsp gene complex in which extensive sequence variations occur primarily in the reiterated coding sequences of the vsp structural genes. These findings demonstrate that there is a vastly expanded potential for antigenic variation within populations of this organism.
PMCID: PMC127842  PMID: 11895991
6.  The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features 
Journal of Bacteriology  1999;181(18):5734-5741.
Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.
PMCID: PMC94094  PMID: 10482515
7.  Isolation, ultrastructure and antigenicity of Mycoplasma galliscpticum membranes 
The Journal of Hygiene  1973;71(4):725-737.
The cell membrane of Mycoplasma gallisepticum was isolated by lysing the cells with digitonin. Chemical and density-gradient analyses and electron microscopy showed the isolated membranes to be relatively free of cytoplasmic contaminants. The density of the membranes exceeded that of other mycoplasma membranes, indicating a higher protein content. Small vesicular extensions seen in the sectioned membranes were interpreted as empty blebs.
The isolated membranes, but not the cytoplasmic fraction, elicited in chickens the production of growth-inhibiting, agglutinating and haemagglutination-inhibition antibodies to M. gallisepticum in titres resembling those obtained by injection of whole cells. The peak of the serological response varied with the serological test employed. The rapid slide-agglutination test became positive as early as 3 days after the first injection of only 50 μg. of membrane protein. The haemagglutination-inhibition antibody titre reached its peak at about 10 days after the first injection, while that of the growth-inhibiting antibodies was reached only at about 25 days. The addition of adjuvant to the membrane antigen did not improve the production of the growth-inhibiting antibodies in chickens, but it produced some improvement in rabbits. Our results support the thesis that the chief immunogens of M. gallisepticum reside in the cell membrane of this organism.
PMCID: PMC2130413  PMID: 4520511
8.  Regulation of Extracellular Protease Production in Bacillus cereus 
Journal of Bacteriology  1967;93(3):1023-1030.
Both sporulation and protease production can be inhibited by growing Bacillus cereus T in a medium containing a high concentration of a mixture of amino acids. Mutants selected for the ability to sporulate in this inhibitory medium were found to produce high levels of protease in the normal and inhibitory media. Comparison of the mutant and wild-type enzymes by gel electrophoresis and heat inactivation suggested that they were identical. One of the mutants proved to be a purine-requiring auxotroph. Reversion to prototrophy resulted in the loss of the capacity to sporulate in the inhibitory medium and loss of the ability to produce large amounts of protease. Mutants capable of producing high levels of protease and of sporulating in the inhibitory medium were also found when selecting for a purine, pyrimidine, or lysine requirement or for the capacity to sporulate in the presence of a high concentration of glucose. Protease production could be considerably delayed in the purine auxotrophs or completely inhibited in the pyrimidine auxotrophs by growing the cells in a medium containing the inhibitory mixture of amino acids plus hypoxanthine for the former or a pyrimidine for the latter. The fact that a variety of metabolic alterations could lead to excessive protease production suggested that a common catabolic or biosynthetic intermediate was involved in the control of the production of this enzyme(s).
PMCID: PMC276550  PMID: 4960917

Results 1-8 (8)