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1.
PLoS One ; 15(4): e0231239, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294121

RESUMO

BACKGROUND: Chorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms. OBJECTIVE: The purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis. STUDY DESIGN: Maternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28-41 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma. RESULTS: Microbial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls (12/14 microorganisms, p = 0.02). Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis (Mycoplasma hominis p = 0.0001; Ureaplasma parvum p = 0.002; Streptococcus mitis p = 0.007). These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma. CONCLUSION: This is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications.


Assuntos
Ácidos Nucleicos Livres/sangue , Corioamnionite/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Cordão Umbilical/microbiologia , Adulto , Corioamnionite/microbiologia , Estudos de Coortes , Feminino , Sangue Fetal/química , Sangue Fetal/metabolismo , Sangue Fetal/microbiologia , Idade Gestacional , Humanos , Recém-Nascido , Mycoplasma/genética , Mycoplasma/patogenicidade , Sepse Neonatal/sangue , Sepse Neonatal/diagnóstico , Sepse Neonatal/microbiologia , Gravidez , Streptococcus mitis/genética , Streptococcus mitis/patogenicidade , Cordão Umbilical/patologia , Ureaplasma/genética , Ureaplasma/patogenicidade , Adulto Jovem
2.
Int J Syst Evol Microbiol ; 70(4): 2369-2381, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32068526

RESUMO

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S-23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02-99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00-96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma. Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis. Based on the genetic data, we propose a novel species of the genus Mycoplasma, for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.


Assuntos
Gansos/microbiologia , Mycoplasma/classificação , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Hungria , Mycoplasma/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA
4.
Parasitol Res ; 119(4): 1423-1427, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32107621

RESUMO

We report two cases of bovine babesiosis caused by Babesia divergens in a region of central Bosnia and Herzegovina. The cases were detected in June 2017 and July 2018 from two small backyard farms. Routine clinical assessments, including physical examination and haematology, revealed lethargy, fever, anaemia, leukopenia and haemoglobinuria in the affected animals. Serum alterations included an elevation of aspartate aminotransferase and a decrease of serum phosphate or hypophosphatemia. Thrombocytopenia was detected in the first clinical case. Microscopic examination of blood smears revealed intracytoplasmic protozoan parasites from the genus Babesia. Molecular screening of both animals confirmed the presence of Babesia divergens, the causative agent of bovine babesiosis. B. divergens DNA was also detected in two engorged female Ixodes ricinus ticks removed from these animals. In addition, Mycoplasma wenyonii DNA was identified by molecular screening in the animal examined in June 2017, and in I. ricinus ticks feeding on this animal. This study provides molecular confirmation of B. divergens as a cause of piroplasmosis in cattle in South-East Europe. The detection of M. wenyonii DNA ain I. ricinus also provides the first evidence of this bacterium in ticks in Europe.


Assuntos
Babesia/genética , Babesiose/diagnóstico , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/parasitologia , Infecções por Mycoplasma/veterinária , Mycoplasma/genética , Animais , Aspartato Aminotransferases/sangue , Babesia/isolamento & purificação , Babesiose/parasitologia , Bósnia e Herzegóvina , Bovinos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Europa (Continente) , Fazendas , Feminino , Hipofosfatemia/sangue , Ixodes/microbiologia , Ixodes/parasitologia , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/diagnóstico , Trombocitopenia/sangue
6.
Epidemiol Infect ; 148: e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31933451

RESUMO

Cervids represent a mammal group which plays an important role in the maintenance of ecological balance. Recent studies have highlighted the role of these species as reservoirs for several arthropods-borne pathogens. Globally, hemotropic mycoplasmas (haemoplasmas) are emerging or remerging bacteria that attach to red blood cells of several mammals species causing hemolytic anaemia. Therefore, the aim of this study was to investigate the occurrence and assess the phylogenetic positioning of Mycoplasma ovis in free-ranging deer from Brazil. Using a polymerase chain reaction targeting the 16S rRNA region, 18 (40%) out of 45 sampled deer were positive to M. ovis. Among the nine sequences analysed, four distinct genotypes were identified. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA. On the other hand, the Neighbour-Net network analysis showed that the human-associated M. ovis genotypes were related to genotypes detected in sheep and goats. The present study shows, for the first time, the occurrence of M. ovis in Mazama gouazoubira and Mazama bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. Once several deer species have your population in decline, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations around the world and assess its potential as reservoir hosts to human infections.


Assuntos
Doenças dos Animais/epidemiologia , Doenças dos Animais/microbiologia , Cervos/microbiologia , Variação Genética , Infecções por Mycoplasma/veterinária , Mycoplasma/classificação , Mycoplasma/isolamento & purificação , Animais , Brasil , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genótipo , Mycoplasma/genética , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
7.
PLoS One ; 15(1): e0227234, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923195

RESUMO

Hemangiosarcoma (HSA), a locally invasive and highly metastatic endothelial cell neoplasm, accounts for two-thirds of all cardiac and splenic neoplasms in dogs. Bartonella spp. infection has been reported in association with neoplastic and non-neoplastic vasoproliferative lesions in animals and humans. The objective of this study was to determine the prevalence of Bartonella spp. in conjunction with two other hemotropic pathogens, Babesia spp. and hemotropic Mycoplasma spp., in tissues and blood samples from 110 dogs with histopathologically diagnosed HSA from throughout the United States. This was a retrospective, observational study using clinical specimens from 110 dogs with HSA banked by the biospecimen repository of the Canine Comparative Oncology and Genomics Consortium. Samples provided for this study from each dog included: fresh frozen HSA tumor tissue (available from n = 100 of the 110 dogs), fresh frozen non-tumor tissue (n = 104), and whole blood and serum samples (n = 108 and 107 respectively). Blood and tissues were tested by qPCR for Bartonella, hemotropic Mycoplasma, and Babesia spp. DNA; serum was tested for Bartonella spp. antibodies. Bartonella spp. DNA was amplified and sequenced from 73% of dogs with HSA (80/110). In contrast, hemotropic Mycoplasma spp. DNA was amplified from a significantly smaller proportion (5%, p<0.0001) and Babesia spp. DNA was not amplified from any dog. Of the 100 HSA tumor samples submitted, 34% were Bartonella PCR positive (32% of splenic tumors, 57% of cardiac tumors, and 17% of other tumor locations). Of 104 non-tumor tissues, 63% were Bartonella PCR positive (56% of spleen samples, 93% of cardiac samples, and 63% of skin/subcutaneous samples). Of dogs with Bartonella positive HSA tumor, 76% were also positive in non-tumor tissue. Bartonella spp. DNA was not PCR amplified from whole blood. This study documented a high prevalence of Bartonella spp. DNA in dogs with HSA from geographically diverse regions of the United States. While 73% of all tissue samples from these dogs were PCR positive for Bartonella DNA, none of the blood samples were, indicating that whole blood samples do not reflect tissue presence of this pathogen. Future studies are needed to further investigate the role of Bartonella spp. in the development of HSA.


Assuntos
Babesia/genética , Babesiose/epidemiologia , Infecções por Bartonella/epidemiologia , Infecções por Bartonella/veterinária , Bartonella/genética , Doenças do Cão/epidemiologia , Hemangiossarcoma/epidemiologia , Hemangiossarcoma/veterinária , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/veterinária , Mycoplasma/genética , Animais , Babesiose/parasitologia , Infecções por Bartonella/microbiologia , DNA Bacteriano/genética , Doenças do Cão/microbiologia , Doenças do Cão/parasitologia , Cães , Feminino , Hemangiossarcoma/microbiologia , Hemangiossarcoma/parasitologia , Masculino , Reação em Cadeia da Polimerase , Prevalência , Estudos Retrospectivos , Estados Unidos/epidemiologia
8.
PLoS One ; 15(1): e0226817, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978082

RESUMO

BACKGROUND: A large proportion of neonates are treated for presumed bacterial sepsis with broad spectrum antibiotics even though their blood cultures subsequently show no growth. This study aimed to investigate PCR-based methods to identify pathogens not detected by conventional culture. METHODS: Whole blood samples of 208 neonates with suspected early onset sepsis were tested using a panel of multiplexed bacterial PCRs targeting Streptococcus pneumoniae, Streptococcus agalactiae (GBS), Staphylococcus aureus, Streptococcus pyogenes (GAS), Enterobacteriaceae, Enterococcus faecalis, Enterococcus faecium, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium, a 16S rRNA gene broad-range PCR and a multiplexed PCR for Candida spp. RESULTS: Two-hundred and eight samples were processed. In five of those samples, organisms were detected by conventional culture; all of those were also identified by PCR. PCR detected bacteria in 91 (45%) of the 203 samples that did not show bacterial growth in culture. S. aureus, Enterobacteriaceae and S. pneumoniae were the most frequently detected pathogens. A higher bacterial load detected by PCR was correlated positively with the number of clinical signs at presentation. CONCLUSION: Real-time PCR has the potential to be a valuable additional tool for the diagnosis of neonatal sepsis.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Candida/isolamento & purificação , Candidíase/diagnóstico , Sepse Neonatal/microbiologia , RNA Ribossômico 16S/genética , Idade de Início , Bactérias/genética , Candida/genética , DNA Ribossômico/genética , Diagnóstico Precoce , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Enterococcus/genética , Enterococcus/isolamento & purificação , Humanos , Lactente Extremamente Prematuro , Recém-Nascido , Recém-Nascido Prematuro , Reação em Cadeia da Polimerase Multiplex , Mycoplasma/genética , Mycoplasma/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Ureaplasma/genética , Ureaplasma/isolamento & purificação
9.
Toxicol Lett ; 319: 155-159, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706005

RESUMO

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50∼5 µM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.


Assuntos
Citocromo P-450 CYP1A2/genética , Vetores Genéticos/genética , Hepatoblastoma/enzimologia , Hepatoblastoma/genética , Lentivirus/genética , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Aflatoxina B1/toxicidade , Linhagem Celular Tumoral , Citocromo P-450 CYP1A2/biossíntese , Impressões Digitais de DNA/métodos , Humanos , Fígado/metabolismo , Mycoplasma/química , Fenacetina/farmacocinética , Plasmídeos/genética
10.
Arch Microbiol ; 202(2): 411-420, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31828363

RESUMO

We describe two novel species of Acholeplasma sp. strain N93 and Mycoplasma sp. strain LR5794 which were isolated from the nasopharynx of a horse from the United Kingdom and from the oral cavity of a North American raccoon from Canada, respectively. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma and Acholeplasma species. Both strains are facultative anaerobes, resistant to penicillin, and produce acid from glucose but do not hydrolyze arginine and urea. Both strains grew well in microaerophilic and anaerobic atmospheric conditions at 35-37 °C using PPLO (pleuropneumonia-like organisms) medium. Acholeplasma sp. N93 does not require serum for growth. Colonies of both strains showed a typical fried-egg appearance and transmission electron microscopy of bacterial cells revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of several genetic loci. The genetic analysis indicated that Acholeplasma sp. N93 and Mycoplasma sp. LR5794 were most closely related to A. hippikon and A. equifetale, and M. molare and M. lagogenitalium, respectively. However, both novel strains were genetically unique in comparison to other well-known Mycoplasma and Acholeplasma species. Based on the isolation source history, phenotypic, genotypic, and phylogenetic characteristics of these novel strains, we propose the name Acholeplasma equirhinis sp. nov. for Acholeplasma sp. isolated from the nasopharynx of a horse [the type strain is N93T (= DSM 106692T = ATCC TSD-139T = NCTC 14351T)], and the name Mycoplasma procyoni sp. nov. for the Mycoplasma sp. isolated from the oral cavity of a North American raccoon [the type strain is LR5794T (= DSM 106703T = ATCC TSD-141T = NCTC 14309T)].


Assuntos
Acholeplasma/isolamento & purificação , Cavalos/microbiologia , Boca/microbiologia , Mycoplasma/isolamento & purificação , Nasofaringe/microbiologia , Guaxinins/microbiologia , Acholeplasma/classificação , Acholeplasma/genética , Animais , Canadá , DNA Bacteriano/genética , Mycoplasma/classificação , Mycoplasma/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Reino Unido
11.
Syst Appl Microbiol ; 43(1): 126047, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31859015

RESUMO

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1AT (= ATCC BAA-1893T = DSM 22456T), and Mycoplasma struthionis sp. nov., with type strain 237IAT (= ATCC BAA-1890T = DSM 22453T), are proposed.


Assuntos
Doenças das Aves/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma/classificação , Infecções Respiratórias/veterinária , Struthioniformes/microbiologia , Animais , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Genes Bacterianos/genética , Genoma Bacteriano/genética , Mycoplasma/química , Mycoplasma/citologia , Mycoplasma/fisiologia , Infecções por Mycoplasma/microbiologia , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S/genética , Infecções Respiratórias/microbiologia , Análise de Sequência de DNA , Especificidade da Espécie
12.
mBio ; 10(6)2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31874918

RESUMO

Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal "jellyfish"-like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal "tentacle"-like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the α- and ß-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and showed that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.IMPORTANCE The genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human "walking pneumonia," is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms, always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.


Assuntos
Adenosina Trifosfatases/metabolismo , Mycoplasma/citologia , Mycoplasma/enzimologia , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Bactérias/metabolismo , Transporte de Íons , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia de Contraste de Fase , Movimento , Propriedades de Superfície
13.
Rev Bras Parasitol Vet ; 28(4): 728-734, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31721928

RESUMO

Free-ranging and feral dogs represent a group of unattended companion animals. They impact wild animal populations by predating native species, displacing predators and introducing exotic pathogens. The aim of this work was to describe the molecular occurrence of Rickettsia, Ehrlichia, Anaplasma, Mycoplasma and Bartonella in feral dogs. The study was carried out in the last relict of a protected area in Mexico City. Blood clots samples from 19 dogs were obtained and analyzed for detection of specific fragments of the 16S-rRNA gene for Anaplasma, Ehrlichia and Mycoplasma and citrate synthase (gltA) for Bartonella and Rickettsia. Our results showed that DNA from three bacteria species (Bartonella vinsonii subsp. berkhoffii, Ehrlichia canis and Mycoplasma haemocanis) was present with frequencies ranging from 5.3 to 15.8%. This is the first record of B. vinsonii subsp. berkhoffii and M. haemocanis in dogs from México, and also the first finding of Ehrlichia canis in Mexico City. It is important to perform surveillance of feral dog populations in order to identify the impact of these pathogens on wild animal populations and Public Health in order to establish prevention and protection programs.


Assuntos
Anaplasma/isolamento & purificação , Bartonella/isolamento & purificação , Doenças do Cão/microbiologia , Ehrlichia/isolamento & purificação , Infecções por Bactérias Gram-Negativas/veterinária , Mycoplasma/isolamento & purificação , Rickettsia/isolamento & purificação , Anaplasma/genética , Animais , Animais Selvagens , Bartonella/genética , Doenças do Cão/epidemiologia , Cães , Ehrlichia/genética , Feminino , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Masculino , México/epidemiologia , Mycoplasma/genética , Filogenia , Rickettsia/genética
14.
J Med Microbiol ; 68(12): 1747-1758, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31671056

RESUMO

Introduction. The Mollicutes class unites cell wall lacking bacteria many of which are membrane parasites and opportunistic bacteria.Aim. This study describes a novel morphological form found in the five species belonging to the bacterial class Mollicutes, and referred to as microcolonies (MCs).Methodology. MCs were obtained as described below and characterized with bacteriological and immunological methods, and microscopy.Results. In contrast to typical colonies (TCs), MCs are characterized by tiny propeller-shaped colonies formed by rod-like cells tightly packed in parallel rows. These colonies were observed within routinely cultivated cultures of type strains 7-12 days post-plating. Rod-like cells were visualized using a scanning electron microscope within TCs with a 'fried-egg-like' appearance. MCs were not observed to revert to TCs. MCs were resistant to antibiotics and other treatments effective against TCs. Pure MC cultures were generated in vitro by treatment of Mycoplasma cultures with hyperimmune serum, antibiotics or argon non-thermal plasma. MCs of Mycoplasma hominis strain H-34 were characterized in detail to confirm that they belonged to that species. MCs tested positive via PCR with M. hominis-specific primers, direct fluorescence and epifluorescence tests, and Western blotting with the camel-derived nanobody aMh-FcG2a, which is specific to the MH3620 transporter protein. Meanwhile, MCs behaved differently in standard bacteriological tests. Pure MC cultures were also isolated directly from clinical samples of the serum, synovial liquid and urine of patients within flammatory urogenital tract diseases, asthma or arthritis. In total, 79 independent MC cultures were isolated from clinical samples including M. hominis (n=70), Mycoplasma pneumoniae (n=2), Mycoplasma fermentans (n=2) and Mycoplasma spp. (n=5).Conclusion. MCs play an unknown role in infection pathology and display prominent antibiotic resistance, making them a challenge for the future studies on Mollicutes.


Assuntos
Mycoplasma/citologia , Tenericutes/isolamento & purificação , Farmacorresistência Bacteriana , Humanos , Tenericutes/citologia , Tenericutes/efeitos dos fármacos , Tenericutes/crescimento & desenvolvimento
15.
BMC Res Notes ; 12(1): 720, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675990

RESUMO

OBJECTIVE: We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. RESULTS: Our findings indicate that qPCR worked optimally with a 6 µl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 µl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 µl cell culture sample (23.42 vs 23.49 average Ct levels, respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from U937 cell cultures.


Assuntos
DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Mycoplasma/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Reprodutibilidade dos Testes , Células U937
16.
Nucleic Acids Res ; 47(21): e137, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31750522

RESUMO

Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Protease La/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Temperatura Baixa , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Bacterianos/genética , Mycoplasma/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética
17.
Rev Bras Parasitol Vet ; 28(4): 632-643, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31596318

RESUMO

This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.


Assuntos
Babesiose/diagnóstico , Doenças do Gato/microbiologia , Doenças do Gato/parasitologia , Infecções por Bactérias Gram-Negativas/veterinária , Anaplasma/genética , Anaplasma/imunologia , Anaplasma/isolamento & purificação , Animais , Babesia/genética , Babesia/imunologia , Babesia/isolamento & purificação , Babesiose/transmissão , Bartonella/genética , Bartonella/imunologia , Bartonella/isolamento & purificação , Brasil , Doenças do Gato/diagnóstico , Doenças do Gato/transmissão , Gatos , Ehrlichia/genética , Ehrlichia/imunologia , Ehrlichia/isolamento & purificação , Feminino , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/transmissão , Insetos Vetores , Masculino , Mycoplasma/genética , Mycoplasma/imunologia , Mycoplasma/isolamento & purificação
18.
Vet Microbiol ; 237: 108422, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585641

RESUMO

Mycoplasma flocculare is genetically closely related to M. hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, and is frequently isolated with this second species. In this article, we report on the development of the first multilocus sequence typing (MLST) scheme for M. flocculare, based on three genes (adk, rpoB and tpiA). In total, 5022 bp of sequence were analyzed. MLST was used to characterize seven M. flocculare isolates and the reference strain. Eight distinct sequence types were defined, showing the great intraspecies variability of M. flocculare, and the high discriminatory power of the new typing method. The relative contribution of recombinations to the genomic evolution of M. flocculare was revealed by calculating the index of association (IA: 0.0185). This MLST scheme is now available for the acquisition of new knowledge on M. flocculare epidemiology via an online database comprising the DNA sequences of each allele, available at http://pubmlst.org/mflocculare/.


Assuntos
Tipagem de Sequências Multilocus/métodos , Mycoplasma/genética , Polimorfismo Genético/genética , Filogenia
19.
Vet Res ; 50(1): 82, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615555

RESUMO

Effective vaccines against tuberculosis (TB) are needed in order to prevent TB transmission in human and animal populations. Evaluation of TB vaccines may be facilitated by using reliable animal models that mimic host pathophysiology and natural transmission of the disease as closely as possible. In this study, we evaluated the immunogenicity and efficacy of two attenuated vaccines, BCG and MTBVAC, after each was given to 17 goats (2 months old) and then exposed for 9 months to goats infected with M. caprae. In general, MTBVAC-vaccinated goats showed higher interferon-gamma release than BCG vaccinated goats in response to bovine protein purified derivative and ESAT-6/CFP-10 antigens and the response was significantly higher than that observed in the control group until challenge. All animals showed lesions consistent with TB at the end of the study. Goats that received either vaccine showed significantly lower scores for pulmonary lymph nodes and total lesions than unvaccinated controls. Both MTBVAC and BCG vaccines proved to be immunogenic and effective in reducing severity of TB pathology caused by M. caprae. Our model system of natural TB transmission may be useful for evaluating and optimizing vaccines.


Assuntos
Vacina BCG/imunologia , Doenças das Cabras/imunologia , Imunogenicidade da Vacina/imunologia , Mycoplasma/fisiologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/veterinária , Animais , Doenças das Cabras/transmissão , Cabras , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Tuberculose/imunologia , Tuberculose/transmissão , Vacinas Atenuadas/imunologia
20.
Vet Parasitol ; 274: 108929, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31568995

RESUMO

'Candidatus Mycoplasma haemobos' is an emerging pathogen in the genus Mycoplasma. The Rhipicephalus (Boophilus) microplus tick has been suspected to be the vector of 'C. M. haemobos'. To determine the role of R. (B.) microplus in transmission of 'C. M. haemobos', we tested the competence of R. (B.) microplus larvae to acquire 'C. M. haemobos' from positive female ticks and to serve as a 'C. M. haemobos' vector in mice. Using PCR and sequencing, we also analyzed the epidemic strains of 'C. M. haemobos' among R. (B.) microplus ticks collected from goats and sheep in southern Henan Province, central China. Our results identified three epidemic strains of 'C. M. haemobos', and the positive female ticks naturally infected could pass 'C. M. haemobos' at egg and larval stages. Furthermore, 'C. M. haemobos' infected larvae could transmit the pathogens to mice during feeding, and the negative larvae could acquire 'C. M. haemobos' from infected mice. Our study shows that R. (B.) microplus ticks could serve as a vector and reservoir of 'C. M. haemobos'.


Assuntos
Vetores Aracnídeos/microbiologia , Mycoplasma/isolamento & purificação , Rhipicephalus/fisiologia , Animais , China , Reservatórios de Doenças , Feminino , Larva , Camundongos , Camundongos Endogâmicos BALB C
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