In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model.

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.

Phenolic constituents, antioxidant and preliminary antimycoplasmic activities of leaf skin and flowers of Aloe vera (L.) Burm. f. (syn. A. barbadensis Mill.) from the Canary Islands (Spain).

The methanol extracts of leaf skins and flowers of Aloe vera from the Canary Islands were analyzed for their phenolic profiles and screened for their antioxidant and antimycoplasmic activities. The use of reversed phase high performance liquid chromatography (RP-HPLC) allowed the identification of 18 phenolic constituents. Leaf skin extracts were characterized by the abundance of catechin, sinapic acid and quercitrin. Gentisic acid, epicatechin and quercitrin were the most prominent phenolic compounds of the flowers. The in vitro antioxidant activities determined by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric antioxidant reducing power (FRAP) assays revealed that both extracts exhibited antioxidant activity, being the leaf skin extract the most active fraction. The leaf skin extract was also found to be active against the microbial strains tested. Therefore, A. vera extracts from leaf skin and flowers can be considered as good natural antioxidant sources.

Critical role of dispensable genes in Mycoplasma agalactiae interaction with mammalian cells.

Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogen Mycoplasma agalactiae and used this library to identify mutants displaying a growth-deficient pheonotype in cell culture. M. agalactiae mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction of M. agalactiae with HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens.

Inferring Active Metabolic Pathways from Proteomics and Essentiality Data.

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways.

Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

Recovery of Mycoplasma agalactiae from the ears of goats experimentally infected by the intramammary route.

The role of inapparent carriers of Mycoplasma agalactiae and the strategies used to colonise the external ear canal in goats remain unclear. This study examined the ability of M. agalactiae to colonise the ears of goats infected experimentally by the intramammary route. The right mammary glands of 15 lactating goats were inoculated with 10(10) colony forming units (cfu) of M. agalactiae. The goats were randomly assigned to three groups of five animals each and sampled at slaughter at 5, 15 or 45 days post-infection (dpi). A further four goats served as uninfected controls. Right and left ear swabs were collected for detection of M. agalactiae by culture before and after sacrifice. M. agalactiae was detected in 19/20 (95%) ear swabs from goats sampled at 15 and 45dpi, whereas all ear swabs collected before inoculation, ear swabs collected from the group sampled at 5dpi and ear swabs from control goats at the time of sacrifice were negative for M. agalactiae. Blood samples collected at 6, 12, 24, 48 and 72h post-infection for detection of M. agalactiae by culture were also negative. There were differences in the antigenic profiles of isolates recovered from the ears compared to the 7MAG strain used to inoculate the animals and most isolates from the mammary gland, milk and supramammary lymph nodes.

Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in ear swabs taken from goats.

This study was designed to evaluate the validity of PCR for the direct detection of Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two species most frequently causing contagious agalactia (CA) in goats. The PCR method was compared with the traditional culture technique to determine which method was most efficient at identifying all auricular carriers present in herds. The samples analyzed were 307 ear swabs taken from goats reared in a CA endemic area. We assessed the validity of each technique to detect each species and agreement between both methods. For each species, the result was taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246 were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117 showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR-positive samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative culture results and 4 culture-positive samples tested negative using PCR (2 for each species). Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae) and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M. agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and sensitive method for the detection of mycoplasmas in the external ear of asymptomatic carriers. Tools such as this are needed to adopt efficient control measures against CA.

Field trial of two dual vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in goats.

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (LC type) were developed using inactivated strains selected in previous characterization studies. The vaccines differed in terms of the adjuvants used: aluminium hydroxide (vaccine A) or aluminium hydroxide plus purified saponin (vaccine B). These vaccines were tested on 60 pregnant goats and 60 seronegative kids that were challenged by placing in a herd with a history of caprine contagious agalactia (CCA). Our findings indicate the effectiveness of the vaccines in preventing the appearance of new clinical signs such as mastitis, abortion, pneumonia and polyarthritis in CCA affected herds.

Flow cytometric determination of the effects of antibacterial agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides large colony type.

Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.