PLSDB: advancing a comprehensive database of bacterial plasmids.

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb.

Microbial communities of poultry house dust, excreta and litter are partially representative of microbiota of chicken caecum and ileum.

Traditional sampling methods for the study of poultry gut microbiota preclude longitudinal studies as they require euthanasia of birds for the collection of caecal and ileal contents. Some recent research has investigated alternative sampling methods to overcome this issue. The main goal of this study was to assess to what extent the microbial composition of non-invasive samples (excreta, litter and poultry dust) are representative of invasive samples (caecal and ileal contents). The microbiota of excreta, dust, litter, caecal and ileal contents (n = 110) was assessed using 16S ribosomal RNA gene amplicon sequencing. Of the operational taxonomic units (OTUs) detected in caecal contents, 99.7% were also detected in dust, 98.6% in litter and 100% in excreta. Of the OTUs detected in ileal contents, 99.8% were detected in dust, 99.3% in litter and 95.3% in excreta. Although the majority of the OTUs found in invasive samples were detected in non-invasive samples, the relative abundance of members of the microbial communities of these groups were different, as shown by beta diversity measures. Under the conditions of this study, correlation analysis showed that dust could be used as a proxy for ileal and caecal contents to detect the abundance of the phylum Firmicutes, and excreta as a proxy of caecal contents for the detection of Tenericutes. Similarly, litter could be used as a proxy for caecal contents to detect the abundance of Firmicutes and Tenericutes. However, none of the non-invasive samples could be used to infer the overall abundance of OTUs observed in invasive samples. In conclusion, non-invasive samples could be used to detect the presence and absence of the majority of the OTUs found in invasive samples, but could not accurately reflect the microbial community structure of invasive samples.

Phylogenomics of expanding uncultured environmental Tenericutes provides insights into their pathogenicity and evolutionary relationship with Bacilli.

BACKGROUND: The metabolic capacity, stress response and evolution of uncultured environmental Tenericutes have remained elusive, since previous studies have been largely focused on pathogenic species. In this study, we expanded analyses on Tenericutes lineages that inhabit various environments using a collection of 840 genomes. RESULTS: Several environmental lineages were discovered inhabiting the human gut, ground water, bioreactors and hypersaline lake and spanning the Haloplasmatales and Mycoplasmatales orders. A phylogenomics analysis of Bacilli and Tenericutes genomes revealed that some uncultured Tenericutes are affiliated with novel clades in Bacilli, such as RF39, RFN20 and ML615. Erysipelotrichales and two major gut lineages, RF39 and RFN20, were found to be neighboring clades of Mycoplasmatales. We detected habitat-specific functional patterns between the pathogenic, gut and the environmental Tenericutes, where genes involved in carbohydrate storage, carbon fixation, mutation repair, environmental response and amino acid cleavage are overrepresented in the genomes of environmental lineages, perhaps as a result of environmental adaptation. We hypothesize that the two major gut lineages, namely RF39 and RFN20, are probably acetate and hydrogen producers. Furthermore, deteriorating capacity of bactoprenol synthesis for cell wall peptidoglycan precursors secretion is a potential adaptive strategy employed by these lineages in response to the gut environment. CONCLUSIONS: This study uncovers the characteristic functions of environmental Tenericutes and their relationships with Bacilli, which sheds new light onto the pathogenicity and evolutionary processes of Mycoplasmatales.

Evaluation of Mollicutes Microorganisms in Respiratory Disease of Cattle and Their Relationship to Clinical Signs.

BACKGROUND: Bovine respiratory disease (BRD) is an important problem in cattle production that is responsible for economic losses in dairy herds. Mycoplasma spp. are described as an important etiological agent of BRD. HYPOTHESIS: To evaluate the occurrence of the most important mycoplasmas in the lower respiratory tract of healthy and BRD cattle in relationship to clinical signs of BRD. ANIMALS: Sixty young dairy cattle were classified as healthy (n = 32) or cattle showing clinical signs of BRD (n = 28). METHODS: Tracheal lavage samples were collected and added to tubes containing Hayflick media. Mycoplasma spp. were identified by the presence of "fried egg" like colonies, biochemical tests and polymerase chain reaction (PCR). Occurrence of Mollicutes, M. bovis, M. mycoides subsp. mycoides SC and M. dispar was evaluated. The association between clinical signs of BRD and the presence of Mycoplasma spp. also was evaluated. RESULTS: Colonies were obtained from a 1-year-old BRD calf only. However, species identification was not possible. Mollicutes (P = .035) and M. dispar (P = .036) were more common in BRD cattle. The relationship between Mollicutes and crackle (P = .057) was not significant. M. dispar was associated to tachypnea (P = .045) and mixed dyspnea (P = .003). Relationships to heart rate (P = .062) and crackle (P = .062) were not significant. CONCLUSIONS AND CLINICAL IMPORTANCE: The results confirmed the importance of mycoplasma as an etiologic agent of BRD and suggested M. dispar as part of the respiratory microbiota and its possible role in the development of BRD.

Mollicutes species and Mycoplasma bovis prevalence and association with health outcomes in beef feeder calves at arrival and initial treatment for bovine respiratory disease.

Mollicutes nasal swab culture status and potential associations with health outcomes were determined in beef feeder calves. Mollicutes culture was positive in 7.6% (22/291) of calves at arrival and in 26.2% (34/130) of calves at first disease treatment. Positive Mollicutes culture at first treatment was associated with increased odds for subsequent retreatment or death.

[Physiological-biochemical and taxonomic studies in mollicutology].

From the beginning of researches in the field of molliculotology and for all the time of existence of special Mycoplasmology Department in the Institute of Microbiology and Virology numerous original results were obtained which are of priority for this science. The collection of strains of various representatives of the class of Mollicutes was formed. Phytopathogenic mollicute strains were first cultived in the elaborated artificial medium and their nature and specific pathogen factors were investigated. Fundamental principles of realization of pathogenic potencies by Mollicutes-agents of plant "yellows" were researched. A number of enzymes of nucleic metabolism and the proteinases, their part as the aggression factors to host organisms was distinguished for the first time, the enzymes localization was studied by cytochemical methods. The composition of carbohydrate part from glycocalix of the mollicutes and microorganisms related to them was studied, and the model of their interaction with the cells of affected organisms was elaborated. The theory and the basis for the practical use of antisignature olygodeoxyribonucleotides as the universal antimicrobial means was formulated. Properties of DNA from mollicutes genome was researched. The system position of these microorganisms and the phylogenetic relations with the representatives of affined taxones was specified. The artificial model system for studying the interaction of phytopathogenic mollicutes with plant cells was created which use helps investigate the particuliarities of the signal and metabolic relations ofmollicutes and cells of host macroorganism. In the course of done researches the changes in the fatty acids composition of the common lipids, in the activity oflectins and enzymes and in the amount of some proteins by the action of mollicute infection were stated.

Regulation of carbon metabolism in the mollicutes and its relation to virulence.

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes.

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.

[Pathogenicity factors of mollicutes and pathogenesis of some human mycoplasmoses]. / Faktory patohennosti molikutiv i patohenez deiakykh mikoplazmosiv liudyny.

The data of pathogenicity factors of mollicutes and mechanisms of the effect of these microorganisms on the organisms they have infected and, first of all, on people occur in countless literary sources. Such data have been accumulated for above 100 year of mycoplasmology existence as science and remain unachievable for most interested specialists. An attempt has been made to generalize in maximum complete volume everything known about pathogenic potential of human and other mollicutes, their pathogenicity factors and mechanisms of their realization on the dramatic changes occurring in the affected organism under the effect of mycoplasmic infection.

Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

A plasmid from a non-insect-transmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line.

Two novel rolling circle replication (RCR) plasmids, pOYM (3932 nt) and pOYNIM (3062 nt), were isolated from a mildly pathogenic variant line (OY-M) and a mildly pathogenic plus non-insect-transmissible line (OY-NIM), respectively, of onion yellows (OY) phytoplasma, a plant and insect endocellular mollicute. OY-M was isolated from an original wild-type line (OY-W) after regular maintenance using alternate plant/insect infections, while OY-NIM was further isolated from OY-M after maintenance by plant grafting without insect vectors. The RCR-initiator proteins (Rep) of both plasmids, which have a characteristic structure with both plasmid- and virus-like domains, were highly homologous to that of a previously described OY-W plasmid, pOYW (3933 nt), and were expressed in OY-M- and OY-NIM-infected plants, indicating that this replicon is stably maintained in the phytoplasma. Interestingly, pOYNIM lacked two ORFs that exist in both pOYW and pOYM, which encode a single-stranded DNA binding protein (SSB) and an uncharacterized putative membrane protein, indicating that these two proteins are not necessary for the phytoplasma to live in plant cells. These are the first candidates as phytoplasma proteins possibly related to host specificity.

In planta expression of a protein encoded by the extrachromosomal DNA of a phytoplasma and related to geminivirus replication proteins.

A new extrachromosomal DNA, EcOYW1, was cloned from the onion yellows phytoplasma (OY-W). Southern blot and PCR analysis showed that EcOYW1 is not present in the OY-M, a mild symptom line derived from OY-W. We determined the complete nucleotide sequence of EcOYW1; it is a circular dsDNA of 7.0 kbp in length, which contains seven ORFs. ORF1 encoded a homologue of the geminivirus Rep protein. Western immunoblot analysis revealed that this Rep homologue is expressed in OY-W infected plants, suggesting that EcOYW1 replicates via a geminivirus-like rolling-circle replication mechanism. EcOYW1 is the first phytoplasmal extrachromosomal DNA shown to express encoded genes.

A plasmid isolated from phytopathogenic onion yellows phytoplasma and its heterogeneity in the pathogenic phytoplasma mutant.

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

Detección por técnicas de biología molecular de Mycoplasma hominis y Ureaplasma urealyticum en muestras urogenitales / Detection by molecular biology techniques of Mycoplasma hominis and Ureaplasma urealyticum in urogenital samples

Mycoplasma homonis y Ureaplasma urealyticum están estrechamente relacionadas con enfermedades del tracto urogenital como pielonefritis, uretritis no-gonocóccica, cálculos urinarios, epididimitis, inflamaciones pélvicas, infertilidad, abortos y fiebre post-parto; en recién nacidos también pueden causar neumonías y meningitis. Estas bacterias pueden ser diagnosticadas por diferentes métodos. En este trabajo utilizamos la hibridación de ácidos nucléicos y la reacción en cadena de la polimerasa para analizar 22 muestras de pacientes con diferentes síntomas urogenitales, en busca de micoplasmas y ureaplasmas. Como resultado obtuvimos 10 muestras positivas y 12 negativas. Entre las muestras positivas se identificaron 2 como Mycoplasma hominis, 2 como Ureaplasma urealyticum y 6 con ambas especies. los resultados obtenidos por las técnicas moleculares fueron comparados con los métodos de referencia, encontrándose en 18 muestras resultados coincidentes, mientras que en 4 los resultados fueron discordantes, siendo esta diferencia estadísticamente no significativa

Detección por técnicas de biología molecular de Mycoplasma hominis y Ureaplasma urealyticum en muestras urogenitales / Detection by molecular biology techniques of Mycoplasma hominis and Ureaplasma urealyticum in urogenital samples

Mycoplasma homonis y Ureaplasma urealyticum están estrechamente relacionadas con enfermedades del tracto urogenital como pielonefritis, uretritis no-gonocóccica, cálculos urinarios, epididimitis, inflamaciones pélvicas, infertilidad, abortos y fiebre post-parto; en recién nacidos también pueden causar neumonías y meningitis. Estas bacterias pueden ser diagnosticadas por diferentes métodos. En este trabajo utilizamos la hibridación de ácidos nucléicos y la reacción en cadena de la polimerasa para analizar 22 muestras de pacientes con diferentes síntomas urogenitales, en busca de micoplasmas y ureaplasmas. Como resultado obtuvimos 10 muestras positivas y 12 negativas. Entre las muestras positivas se identificaron 2 como Mycoplasma hominis, 2 como Ureaplasma urealyticum y 6 con ambas especies. los resultados obtenidos por las técnicas moleculares fueron comparados con los métodos de referencia, encontrándose en 18 muestras resultados coincidentes, mientras que en 4 los resultados fueron discordantes, siendo esta diferencia estadísticamente no significativa (AU)

"Candidatus phytoplasma australiense," a new phytoplasma taxon associated with Australian grapevine yellows.

A phytoplasma was detected in naturally diseased 'Chardonnay' grapevines exhibiting symptoms of Australian grapevine yellows disease. The use of PCR designed to amplify phytoplasma DNA resulted in detection of phytoplasma DNA in all of the diseased plants examined; no phytoplasma DNA was detected in healthy seedling grapevines. The collective restriction fragment length polymorphism (RFLP) patterns of amplified 16S ribosomal DNA differed from the patterns described previously for other phytoplamas. On the basis of the RFLP patterns, Australian grapevine yellows phytoplasma was classified as a representative of a new subgroup, designated subgroup 16SrI-J, in phytoplasma 16S rRNA group 16SrI (aster yellows and related phytoplasmas). A phylogenetic analysis in which parsimony of 16S rRNA gene sequences from this and other group 16SrI phytoplasmas was used identified the Australian grapevine yellows phytoplasma as a member of a distinct subclade (subclade xii) in the phytoplasma clade of the class Mollicutes. A phylogenetic tree constructed on the basis of 16S rRNA gene sequences was consistent with the hypothesis that there was divergent evolution of Australian grapevine yellows phytoplasma and its closet known relative, European stolbur phytoplasma (subgroup 16SrI-G), from a common ancestor. The unique properties of the DNA from the Australian grapevine yellows phytoplasma clearly establish that it represents a new taxon, "Candidatus Phytoplasma australiense."

[The lectins of Mollicutes]. / Lektyny molikutiv.

Lectine-carbohydrate interactions take the fundamental part in the intercellular relations as well as in microbes pathogenicity. Pathogenic Mollicutes are characterized by the firm adhesion to the cells of the affected organs of people, animals, insects and plants. Proteins which are localized on the external side of the mollicute membrane and interact with carbohydrate residues on the surface of mucous membranes of the damaged organs and vice versa take part in the adhesion. High degree of specificity of protein-carbohydrate interactions determines the pathogenic specializations to the cells of one or another organ of the host. Since proteins which take part in the mollicutes adhesion are rich in proline and hydrophobic fields it is not excluded that the adhesion processes are combined with hydrophobic interrelations between the cells of the pathogen and host. Substances from the cells of Mollicutes which completely correspond to the definition "lectin", i.e., are in a pure form, the carbohydrate-binding proteins specific to certain carbohydrate residues are not still isolated. Thus the mollicute lectines should be called lectine-like substances rather than lectines. Mollicutes form a lot of such substances and they may be separated into the extracellular (soluble) substances which are found outside the cell of the nutritious medium; intermediate (half-soluble) substances detected in the nutritious medium and in the state integrate into the microorganism membrane, and membrane-related (insoluble) substances which occur only in the state rigidly adhered to the membrane. Carbohydrate composition of lectine-like substances in different mollicutes is different which is the reflection of heterogenicity of the Mollicutes class representatives.

Experimental infection of rat (Rattus norvigus, strain Wistar) mammary gland by Mollicutes from the bovine udders.

Seven isolates of Mollicutes, Mycoplasma F-38; M. mycoides var. capri; mixed isolates of M. bovigenitalium and M. bovirhinis; M. bovigenitalium, Mycoplasma F-38 and M. bovirhinis; M. bovis, M. bovigenitalium, Mycoplasma F-38 and A. laidlawii; A. axanthum; A. laidlawii from bovine udders and a M. bovis type strain (NCTC-10131) produced significant histopathological changes characterized by infiltration of neutrophils in lumen of acini, interlobular and intralobular ducts along with the hyperplasia of lining cells of acini, interlobular and intralobular ducts and infiltration of mononuclear cells and fibroblasts in interstitium in the mammary gland of rat suggestive of mastitis. A. laidlawii and A. axanthum produced only mild changes suggestive of their negligible role in the bovine mastitis. Rat mammary gland is recommended as a suitable in vivo experimental laboratory model to screen the mastitogenic potential of Mollicutes.

Current status of the mollicute flora of humans.

Of the more than 20 species of mollicutes reported to be present in human tissues, 15 have been isolated more than once and are currently thought to typify mollicutes of human origin. In the past decade a number of new and potentially significant mollicutes have been added to the list of species inhabiting humans. As our understanding of the human mollicute flora increases, diagnostic and clinical advances should permit the identification and control of those species that are significant pathogens in human disease.

[Soluble and semisoluble lectins from mollicutes and their possible functions]. / Rozchynni ta napivrozchynni lektyny molikutiv i ïkh mozhlyvi funktsiï.

When studying mollicute lectins it was established that Acholeplasma laidlawii PG-8 synthesizes two half-soluble lectins one of which is specific to N-acetyl-D-glucosamine, and the other--to D-glucosamine.HCl; phytopathogenic strain A. laidlawii var. granulum 118 produced 4 lectins one of which is soluble and specific in respect to fructose-1.6-diphosphate, the rest three lectins are half-soluble and specific to one of the sugars--D-galactosamine.HCl, rafinose and D-glucosamine.HCl. In Mycoplasma pneumoniae FH all the four lectins found in the culture liquid have been classified as half-soluble, specific to one of carbohydrates--D-galactosamine.HCl, talose, N-acetyl-neuramine acid and D-glucose; M. capricolum Cal. Kid. synthesizes four lectins; two of them being defined as soluble (one of the lectins is, respectively, specific to talose and D-glucosamine.HCl, two others, as half-soluble, specific to one of sugars--rafinose or D-glucose. The results obtained permit a conclusion to be made that the half-soluble lectins of mollicutes, on the one hand, are the factors of adhesion on the corresponding organs of macroorganism and, on the other hand take part in the transport of substances from without into the microorganism cell. Soluble lectins determine pathogenicity of mollicutes and form with half-soluble lectins a single chain to providing the mycoplasma cells with nutrients and to protect them from the action of the macroorganism immune system.