PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Persistence of antibiotic resistance: evaluation of a probiotic approach using antibiotic-sensitive Megasphaera elsdenii strains to prevent colonization of swine by antibiotic-resistant strains

Megasphaera elsdenii is a lactate-fermenting, obligately anaerobic bacterium commonly present in the gastrointestinal tracts of mammals, including humans. Swine M. elsdenii strains were previously shown to have high levels of tetracycline resistance (MIC=64 to >256 μg/ml) and to carry mosaic (recombinant) tetracycline resistance genes. Baby pigs inherit intestinal microbiota from the mother sow. In these investigations we addressed two questions. When do M. elsdenii strains from the sow colonize baby pigs? Can five antibiotic-sensitive M. elsdenii strains administered intragastrically to newborn pigs affect natural colonization of the piglets by antibiotic-resistant (AR) M. elsdenii strains from the mother? M. elsdenii natural colonization of newborn pigs was undetectable (<10⁴ CFU/g [wet weight] of feces) prior to weaning (20 days after birth). After weaning, all pigs became colonized (4 × 10⁵ to 2 × 10⁸ CFU/g feces). In a separate study, 61% (76/125) of M. elsdenii isolates from a gravid sow never exposed to antibiotics were resistant to chlortetracycline, ampicillin, or tylosin. The inoculation of the sow''s offspring with mixtures of M. elsdenii antibiotic-sensitive strains prevented colonization of the offspring by maternal AR strains until at least 11 days postweaning. At 25 and 53 days postweaning, however, AR strains predominated. Antibiotic susceptibility phenotypes and single nucleotide polymorphism (SNP)-based identities of M. elsdenii isolated from sow and offspring were unexpectedly diverse. These results suggest that dosing newborn piglets with M. elsdenii antibiotic-sensitive strains delays but does not prevent colonization by maternal resistant strains. M. elsdenii subspecies diversity offers an explanation for the persistence of resistant strains in the absence of antibiotic selection.     

In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.     

In Vitro Susceptibility of Atypical Mycobacteria To Rifampin

Atypical mycobacteria (209 strains) were examined for susceptibility to rifampin by the proportion method by using Middlebrook 7H-10 agar. All strains of Mycobacterium kansasii and tap-water scotochromogens were inhibited by 0.25 to 1 μg of the drug per ml. Seventy-six per cent of M. scrofulaceum and 61% of M. intracellulare strains were susceptible to 4 μg/ml or less; 5% of the former and 8% of the latter were resistant to 16 μg/ml. All strains of M. gastri and M. triviale and most strains of M. terrae were sensitive to 1 to 4 μg/ml. Two strains of M. borstelense were both inhibited by 8 μg/ml. Nearly all strains of M. fortuitum were resistant to the drug. The results of this study suggest that rifampin may be a valuable agent for the treatment of many atypical mycobacterial infections.     

Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species

The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.     

Pectobacterium zantedeschiae sp. nov. a new species of a soft rot pathogen isolated from Calla lily (Zantedeschia spp.)

Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily.The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA–DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9M^T (PCM2893 = DSM105717 = IFB9009) as the type strain.     

Thermal Tolerance of Mycobacterium paratuberculosis

D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) of Mycobacterium paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71°C). Viable M. paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for the M. paratuberculosis strains tested were generally linear, with R² of ≥0.90, but a few curves (R², 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similar D values in milk and in lactate solution. However, D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. D values for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains of M. paratuberculosis in milk at 71°C (D_71°C) was 11.67 s. Pooled D_62°C, D_65°C, and D_68°C of clinical M. paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11°C. The D values of clumped and single M. paratuberculosis cells were not significantly different. The D values of all M. paratuberculosis strains tested were considerably higher than those published for Listeria, Salmonella, and Coxiella spp. and estimated for Mycobacterium bovis, indicating that M. paratuberculosis is more thermally tolerant. This study supports the premise that M. paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 10¹ cells/ml.     

Intraspecific variability in response to phosphorus depleted conditions in the cyanobacteria Microcystis aeruginosa and Raphidiopsis raciborskii

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken together, these results do not support the idea that R. raciborskii, as a species, can withstand phosphorus limitation better than M. aeruginosa and also point that the level of intraspecific variation may preclude generalizations based on studies that use only one or few strains.     

Bacillus strain selection with plant growth-promoting mechanisms as potential elicitors of systemic resistance to gray mold in pepper plants

Certain soil bacteria produce beneficial effects on the growth and health of plants; hence, their use is steadily increasing. Five strains of Bacillus with plant growth-promoting potential were selected in this study, which produced indole-3-acetic acid levels below 50 µg.mL⁻¹. On the other hand, while only strains M8 and M15 dissolved phosphorus, the latter was the only strain that did not produce siderophores. Only strains M8 and M16 significantly inhibited the in vitro growth of Botrytis cinerea and Fusarium solani phytopathogens, whose inhibition ranges fluctuated between 60% and 63% for strains M8 and M16 against B. cinerea and between 40% and 53% for strains M8 and M16 against F. solani. Based on these results, the need to implement resistance induction against gray mold on pepper plants was determined using strains M8 and M16. In this case, strain M16 inhibited the propagation of the necrotic spot by approximately 70%, whereas strain M8 significantly reduced the superoxide dismutase activity in systemic leaves, which substantially increased in plants inoculated with strain M8 and infected with the pathogen. Accordingly, the use of native rhizobacteria may entail biotechnological progress for the integrated management of crops in agriculture industry.     

Microvirga tunisiensis sp. nov., a root nodule symbiotic bacterium isolated from Lupinus micranthus and L. luteus grown in Northern Tunisia

Three bacterial strains, LmiM8^T, LmiE10 and LluTb3, isolated from nitrogen-fixing nodules of Lupinus micranthus (Lmi strains) and L. luteus (Llu strain) growing in Northern Tunisia were analysed using genetic, phenotypic and symbiotic approaches. Phylogenetic analyses based on rrs and concatenated gyrB and dnaK genes suggested that these Lupinus strains constitute a new Microvirga species with identities ranging from 95 to 83% to its closest relatives Microvirga makkahensis, M. vignae, M. zambiensis, M. ossetica, and M. lotononidis. The genome sequences of strains LmiM8^T and LmiE10 exhibited pairwise Average Nucleotide Identities (ANIb) above 99.5%, significantly distant (73–89% pairwise ANIb) from other Microvirga species sequenced (M. zambiensis and M. ossetica). A phylogenetic analysis based on the symbiosis-related gene nodA placed the sequences of the new species in a divergent clade close to Mesorhizobium, Microvirga and Bradyrhizobium strains, suggesting that the M. tunisiensis strains represent a new symbiovar different from the Bradyrhizobium symbiovars defined to date. In contrast, the phylogeny derived from another symbiosis-related gene, nifH, reproduced the housekeeping genes phylogenies. The study of morphological, phenotypical and physiological features, including cellular fatty acid composition of the novel isolates demonstrated their unique profile regarding close reference Microvirga strains. Strains LmiM8^T, LmiE10 and LluTb3 were able to nodulate several Lupinus spp. Based on genetic, genomic and phenotypic data presented in this study, these strains should be grouped within a new species for which the name Microvirga tunisiensis sp. nov. is proposed (type strain LmiM8^T = CECT 9163^T, LMG 29689^T).     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Natural diversity of nodular microsymbionts of Myrica rubra

Myrica is often considered a promiscuous actinorhizal genus. However, there are large differences in diversity among Myrica spp., and M. gale does not exhibit such promiscuity in its natural environment. In order to understand the diversity of nodular microsymbionts of M. rubra in natural environments and whether or not the M. rubra is a `promiscuous' host, we studied the natural diversity of nodular microsymbionts of different cultivars of M. rubra. 15 nodules from nine horticultural cultivars of M. rubra were collected in 7 sites of eastern, southeastern, central and northern part of Zhejiang province, China. Unisolated strains were compared by sequence analyses of their nifD-nifK intergenic spacers and PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing sequences of their nifD-nifK intergenic spacers and reference strains. There is a high degree of diversity among nodular Frankia symbionts of M. rubra. Frankia strains from cluster I and cluster III were found in nodules from many different cultivars of M. rubra. Furthermore, there were sometimes two strains which belong to different infective clusters of Frankia in the same nodule, and Frankia strains of cluster I were often dominant strains when there were two strains. M. rubra can thus be considered to be promiscuous in nature. Identical sequences in nodules from different plants at widely separated sites were commonly found, indicating that some strains are cosmopolitan. Geographic separation, host selectivity for Frankia symbionts and soil environment may account for the diversity of Frankia strains and differences in Frankia populations found in M. rubra nodules. Several very closely related local Frankia populations in M. rubra nodules could be distinguished from one another by our approach.     

Performance of Rhizopus,Rhizomucor, and Mucor in ethanol production from glucose,xylose, and wood hydrolyzates

In searching for ethanol producing microorganisms also capable of fermenting pentoses, nine zygomycetes strains including three strains of Rhizopus oryzae, Mucor corticolous, M. hiemalis, M. indicus, Rhizomucor pusillus, R. miehei, and zygomycete IT were examined. Each strain was cultivated on glucose, xylose or dilute-acid hydrolyzate (DAH) as carbon sources, and the production of ethanol, lactic acid, glycerol, xylitol, and succinic acid were investigated. Great similarities but also conspicuous differences were seen between the species, to some extent linked to the genera. All strains were capable of growing on glucose or xylose as single carbon source. With the exception of the two Rhizomucor strains, all produced ethanol. All the strains produced glycerol as by-product, while Rhizopus and Rhizomucor but not Mucor produced lactic acid in significant amounts. All Mucor and Rhizopus strains and one strain of Rhizomucor produced xylitol in the xylose medium, but no xylitol was detected after growth on DAH. All Mucor and two R. oryzae strains were capable of growing on DAH. Two Mucor species, M. hiemalis and M. indicus showed greater ethanol production than the other strains. The ethanol yields by M. hiemalis on glucose, xylose, and DAH were 0.39, 0.18, and 0.44 g/g, respectively, whereas the corresponding results for M. indicus were 0.39, 0.22, and 0.44 g/g. The strains also rapidly consumed hydroxymethyl furfural present in DAH.     

Mycoplasma pneumoniae Large DNA Repetitive Elements RepMP1 Show Type Specific Organization among Strains

Mycoplasma pneumoniae is the smallest self-replicating bacterium with a streamlined genome of 0.81 Mb. Complete genome analysis revealed the presence of multiple copies of four large repetitive elements (designated RepMP1, RepMP2/3, RepMP4 and RepMP5) that are implicated in creating sequence variations among individual strains. Recently, we described RepMP1-associated sequence variations between reference strain M129 and clinical isolate S1 that involved three RepMP1-genes (i.e. mpn130, mpn137 and mpn138). Using PCR and sequencing we analyze 28 additional M. pneumoniae strains and demonstrate the existence of S1-like sequence variants in nine strains and M129-like variants in the remaining nineteen strains. We propose a series of recombination steps that facilitates transition from M129- to S1-like sequence variants. Next we examined the remaining RepMP1-genes and observed no other rearrangements related to the repeat element. The only other detected difference was varying numbers of the 21-nucleotide tandem repeats within mpn127, mpn137, mpn501 and mpn524. Furthermore, typing of strains through analysis of large RepMPs localized within the adhesin P1 operon revealed that sequence divergence involving RepMP1-genes mpn130, mpn137 and mpn138 is strictly type-specific. Once more our analysis confirmed existence of two highly conserved groups of M. pneumoniae strains.     

Phylogeny of Microcystins: Evidence of a Biogeographical Trend?

Microcystins, the most prevalent cyanotoxins occurring worldwide, were first recorded in the species Microcystis aeruginosa. Its production has been reported in all continents; thus, we propose a comprehensive phylogenetic study to characterize M. aeruginosa microcystin-producing strains and establish whether or not the species has an historic biogeography. To accomplish this, we compared phylogenetically the nucleotide sequences of three genes of the mcy gene cluster (mcyA, mcyD and mcyG) from toxin producing M. aeruginosa strains across all the five continents. The obtained results provided valuable insight on the biogeography of M. aeruginosa produced microcystins: (i) the Asian strains showed to be distinct from the other continental groups indicating a genetically unique population and (ii) Asian strains were more related to European and North American strains. Moreover, the evidence of positive selection was determined in all the three mcy genes indicating that some functionality yet to be determined could be under selection for these genes.     

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.     

Definition of the novel symbiovar canariense within Mesorhizobium neociceri sp. nov., a new species of genus Mesorhizobium nodulating Cicer canariense in the “Caldera de Taburiente” National Park (La Palma,Canary Islands)

Cicer canariense is a highly promiscuous wild chickpea nodulated by Mesorhizobium strains in La Palma Island located at Canary archipelago. Four of these strains, CCANP34, CCANP35^T, CCANP38 and CCANP95 belong to a group phylogenetically close to Mesorhizobium caraganae with 100% similarity values in the 16S rRNA gene. However, the genomes of the strains CCANP35^T and M. caraganae LMG 24397^T obtained in this work showed ANIb and dDDH values of 90.02% and 44.1%, respectively. These values are lower than those currently accepted for different bacterial species showing that the Canarian strains do not belong to the species M. caraganae. The Canarian strains also differ from M. caraganae in the amounts of several fatty acids and in several phenotypic traits. Based on the obtained results the Canarian strains belong to a novel species for which we propose the name Mesorhizobium neociceri sp. nov. and whose type strain is CCANP35^T. The results of the phylogenetic analyses of nodC and nifH symbiotic genes showed that the Canarian strains represent a novel symbiovar within genus Mesorhizobium phylogenetically divergent to that encompassing M. caraganae. We propose the names canariense and caraganae for the symbiovars encompassing the strains of M. neociceri and M. caraganae, respectively.     

Integrative analysis of transcriptome and genome indicates two potential genomic islands are associated with pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) is a successful human pathogen and widely prevalent throughout the world. Genomic islands (GIs) are thought to be related to pathogenicity. In this study, we predicted two potential genomic islands in M.tb genome, respectively named as GI-1 and GI-2. It is indicated that the genes belong to PE_PGRS family in GI-1 and genes involved in sulfolipid-1 (SL-1) synthesis in GI-2 are strongly associated with M.tb pathogenesis. Sequence analysis revealed that the five PGRS genes are more polymorphic than other PGRS members in full virulence M.tb complex strains at significance level 0.01 but not in attenuated strains. Expression analysis of microarrays collected from literatures displayed that GI-1 genes, especially Rv3508 might be correlated with the response to the inhibition of aerobic respiration. Microarray analysis also showed that SL-1 cluster genes are drastically down-expressed in attenuated strains relative to full virulence strains. We speculated that the effect of SL-1 on M.tb pathogenicity could be associated with long-term survival and persistence establishment during infection. Additionally, the gene Rv3508 in GI-1 was under positive selection. Rv3508 may involve the response of M.tb to the inhibition of aerobic respiration by low oxygen or drug PA-824, and it may be a common feature of genes in GI-1. These findings may provide some novel insights into M.tb physiology and pathogenesis.     

Molecular Epidemiology of Mycoplasma conjunctivae in Caprinae: Transmission across Species in Natural Outbreaks

Mycoplasma conjunctivae is the etiological agent of infectious keratoconjunctivitis, a highly contagious ocular infection that affects both domestic and wild Caprinae species in the European Alps. In order to study the transmission and spread of M. conjunctivae across domestic and wild Caprinae populations, we developed a molecular method for subtyping and identifying strains of M. conjunctivae. This method is based on DNA sequence determination of a variable domain within the gene lppS, a gene that encodes an antigenic lipoprotein of M. conjunctivae. This domain of lppS shows variations among different strains but remains constant upon generations of individual strains on growth medium and thus allows identification of individual strains and estimation of their phylogenetic intercorrelations. The variable domain of lppS is amplified by PCR using primers that match conserved sequences of lppS flanking it. Sequence analysis of the amplified fragment enables fine subtyping of M. conjunctivae strains. The method is applicable both to isolated strains and to clinical samples directly without requiring the cultivation of the strain. Using this method, we show that M. conjunctivae was transmitted between domestic and wild animals that were grazing in proximate pastures. Certain animals also presented infections with two different strains simultaneously.     

Superantigenic Activity of emm3 Streptococcus pyogenes Is Abrogated by a Conserved, Naturally Occurring smeZ Mutation

Streptococcus pyogenes M/emm3 strains have been epidemiologically linked with enhanced infection severity and risk of streptococcal toxic shock syndrome (STSS), a syndrome triggered by superantigenic stimulation of T cells. Comparison of S. pyogenes strains causing STSS demonstrated that emm3 strains were surprisingly less mitogenic than other emm-types (emm1, emm12, emm18, emm28, emm87, emm89) both in vitro and in vivo, indicating poor superantigenic activity. We identified a 13 bp deletion in the superantigen smeZ gene of all emm3 strains tested. The deletion led to a premature stop codon in smeZ, and was not present in other major emm-types tested. Expression of a functional non-M3-smeZ gene successfully enhanced mitogenic activity in emm3 S. pyogenes and also restored mitogenic activity to emm1 and emm89 S. pyogenes strains where the smeZ gene had been disrupted. In contrast, the M3-smeZ gene with the 13 bp deletion could not enhance or restore mitogenicity in any of these S. pyogenes strains, confirming that M3-smeZ is non-functional regardless of strain background. The mutation in M3-smeZ reduced the potential for M3 S. pyogenes to induce cytokines in human tonsil, but not during invasive infection of superantigen-sensitive mice. Notwithstanding epidemiological associations with STSS and disease severity, emm3 strains have inherently poor superantigenicity that is explained by a conserved mutation in smeZ.