Lucas Miranda Marques

Invasion of Ureaplasma diversum in Hep-2 cells

BackgroundUnderstanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay.ResultsThe isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma.ConclusionsThe results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Prevalence of Mycoplasma genitalium and Mycoplasma hominis in urogenital tract of Brazilian women

BackgroundThe role of Mycoplasma hominis and M. genitalium in urogenital tract infections remains unknown. Furthermore these mollicutes present a complex relationship with the host immune response. The role of inflammatory cytokines in infections also makes them good candidates to investigate bacterial vaginosis and mycoplasma genital infections. Therefore, the aim of this study was to detect the above-mentioned mollicutes by quantitative Polymerase Chain Reaction (qPCR) methodologies in vaginal swabs and dosage of cytokines.MethodsVaginal swabs and peripheral blood were collected from 302 women, including healthy individuals. The molecular findings were correlated with some individual behavioral variables, clinical and demographic characteristics, presence of other important microorganisms in vaginal swabs, and levels of interleukin (IL)-1β and IL-6.ResultsM. hominis and M. genitalium were detected in 31.8% and 28.1% of samples, respectively. The qPCR results were associated with clinical signs and symptoms of the infections studied. The frequency of Trichomonas vaginalis, Gardnerella vaginalis, Neisseria gonorrhoeae and Chlamydia trachomatis was 3.0%, 21.5%, 42.4%, and 1.7% respectively. Increased levels of IL-1β were associated with the presence of M. hominis and signs and/or symptoms of the genital infection of women studied.ConclusionIL-1β production was associated with the detection of M. hominis by qPCR. The sexual behavior of women studied was associated with the detection of mycoplasma and other agents of genital infections.

Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil

MOST Mycoplasma species found in calf respiratory tracts are considered to be opportunistic pathogens; however, several species have been described as the primary agents for respiratory diseases (Simecka and others 1992). Mycoplasma bovis and Mycoplasma dispar have been considered the most important agents of pneumonia in cattle herds in which Mycoplasma mycoides subspecies mycoides SC is absent (Rebhun and others 2000). This short communication described a study to determine the prevalence of Mycoplasma species in the respiratory tracts of calves in Brazil. A total of 301 nasal swabs were collected from calves of both sexes, up to one year of age, from 10 different farms: seven in the State of Sao Paulo, two in Minas Gerais and one in Bahia. The samples were collected from 155 animals with respiratory disease and 146 clinically healthy animals by vigorously rubbing a swab over the surface of the nasal cavity. The nasal mucus samples were transported in 3 ml of modified Friis medium (Friis 1971) at 4°C and then incubated at 37°C for four days. The DNA was extracted from the samples by the method described by Fan and others (1995). The primers GPO-3 and MGSO (Van Kuppeveld and others 1992) were used in a PCR for the detection of Mollicutes. Samples that were positive for Mollicutes were submitted to other PCR assays using primers to detect M bovis (Gonzalez and others 1995), M mycoides subspecies mycoides SC (Dedieu and others 1994), M dispar (Marques and others 2007), and Ureaplasma diversum (Cardoso and others 2000). The PCR products were analysed on a 1·5 per cent agarose gel containing 0·5 μM/ml ethidium bromide in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8·0). Table 1 shows the prevalence of Mollicutes and the four species investigated. Mollicutes were detected in 76 (52·06 per cent) of the nasal mucus samples from healthy animals and in 141 (90·96 per cent) of the samples from animals with respiratory disease. Although M dispar and U diversum were detected in both sick and healthy animals, M bovis was detected only in calves with respiratory disease. M mycoides subspecies mycoides SC was not detected in any of the samples. M dispar and U diversum were detected more frequently in samples from animals with respiratory disease than healthy animals. Of the 301 nasal mucus samples, 102 (33·88 per cent) were positive for at least one of the species tested for. The frequency of Mollicute co-infections in healthy and sick animals is shown in Table 2. M mycoides subspecies mycoides SC is the classical primary agent of contagious bovine pleuropneumonia. According to the World Organisation for Animal Health (OIE), healthy cattle herds must be free of this microorganism. This species was not detected in the present study; however, the authors recommended that a wide-ranging investigation using PCR should be carried out in Brazil. M bovis has previously been isolated in healthy animals, and was originally described as a natural microorganism of the bovine respiratory system (Knudtson and others 1986, Ter Laak and others 1992b). Currently, M bovis is the second most important mycoplasma in bovine respiratory diseases (Gevaert 2006). In the present study, M bovis was detected only in sick animals, either as a single agent or in association with M dispar or U diversum. The results obtained are in agreement with other studies that have mentioned M bovis as a species associated with respiratory disease outbreaks in cattle herds (Bashiruddin and others 2001, Byrne and others 2001). M dispar was detected more frequently in sick calves than in healthy animals. Ter Laak and others (1992a) reported M dispar to be the main species isolated from lung samples from sick calves (49 per cent of the samples). In contrast, Ter Laak and others (1992b) showed a frequency of 40 per cent of M dispar detection in healthy animals. In the present study, U diversum was detected by PCR in 12 (8·22 per cent) nasal mucus samples from healthy calves, and in 49 (31·6 per cent) samples from sick animals. Ter Laak and others (1992b) also detected U diversum in healthy animals as well; however, the frequency of detection was higher in sick animals. In the present study, healthy animals that were PCRpositive for M dispar and U diversum were in close contact with sick animals infected with these species, suggesting that M dispar and U diversum may be important pathogens in the development of respiratory disease. The detection of Mollicutes in nasal mucus from both sick and healthy calves may indicate the opportunistic role of these bacteria. The host-parasite relationship of mycoplasmas is a complex subject, especially in calves, in which the disease process in the respiratory tract is not well understood. Co-infections with mycoplasmas in the respiratory tracts of calves are common (Thomas and others 2002, Levisohn and others 2004). In the present study, three mycoplasma species were detected together in four samples, but an association between U diversum and M dispar was most common. Brazil is one of the most important cattle meat producers in the world. Consequently, it is important to monitor disease occurrence among Brazilian herds, including mycoplasma infections. The PCR results for identifying mycoplasmas in TABLE 2: Frequency of co-infections with more than one Mycoplasma species in the respiratory tracts of healthy calves and calves with respiratory disease

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.

Determination of bacterial aetiologic factor on tracheobronchial lavage in relation to clinical signs of bovine respiratory disease

This study aimed to determine the occurrence of Mannheimiahaemolytica, Pasteurella multocida and Mycoplasma spp., in relation to clinical signs of respiratory disease. Tracheobronchial lavage samples were collected from 96 (healthy and unhealthy) cattle in the State of São Paulo, Brazil. Mycoplasma spp. (12.5u2009%) and Pasteurellamultocida (15.50u2009%) were the most prevalent species. Bacillus sp., Staphylococcus sp., Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Klebsiella pneumoniae were also isolated. Mollicutes (70.83u2009%), Mycoplasmabovis (2.94u2009%) and Mycoplasma dispar (38.23u2009%) were identified using conventional PCR. Submassive sound on acoustic percussion of the thorax was associated with the absence of Mollicutes (P=0.025). Whistling (P=0.076) and coarse crackle (P=0.046) were associated with the absence of Mycoplasma dispar. Clear sound on acoustic percussion of the thorax was associated with the absence of Mycoplasmabovis (P=0.007). Coughing was associated with the presence of Pasteurellamultocida [P=0.035; confidence interval (CI), 1.12-26.89], but its absence was associated with mucopurulent (P=0.0215; CI, 1.55-34.5) and mucoid nasal discharge (P=0.068; CI, 19-28.5), submassive sound (P=0.031; CI, 1.23-75.5), fine crackle (P=0.058; CI, 1.23-20.1) and coarse crackle (P=0.046; CI, 2.38-70.8). The high prevalence of Pasteurella multocida and Mycoplasma spp. in unhealthy calves increases the importance of these micro-organisms in the pathogenesis of respiratory diseases. This study increases the information about the role of Mycoplasma dispar in respiratory diseases. Differences in some species in relation to clinical signs can be applied as a presumptive diagnosis.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

BackgroundBacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit.ResultsThe number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24xa0hours. However, after 72xa0hours a considerable decrease of apoptotic cells was observed.ConclusionsThe data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

Ureaplasma urealyticum and U. parvum in sexually active women attending public health clinics in Brazil

Ureaplasma urealyticum and U. parvum have been associated with genital infections. The purpose of this study was to detect the presence of ureaplasmas and other sexually transmitted infections in sexually active women from Brazil and relate these data to demographic and sexual health, and cytokines IL-6 and IL-1β. Samples of cervical swab of 302 women were examined at the Family Health Units in Vitória da Conquista. The frequency of detection by conventional PCR was 76·2% for Mollicutes. In qPCR, the frequency found was 16·6% for U. urealyticum and 60·6% U. parvum and the bacterial load of these microorganisms was not significantly associated with signs and symptoms of genital infection. The frequency found for Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis and Chlamydia trachomatis was 3·0%, 21·5%, 42·4% and 1·7%, respectively. Higher levels of IL-1β were associated with control women colonized by U. urealyticum and U. parvum. Increased levels of IL-6 were associated with women who exhibited U. parvum. Sexually active women, with more than one sexual partner in the last 3 months, living in a rural area were associated with increased odds of certain U. parvum serovar infection.

Potential of Maintaining a Healthy Vaginal Environment by Two Lactobacillus Strains Isolated from Cocoa Fermentation

Bacteria in the genera Mycoplasma and Ureaplasma do not have cell walls and therefore interact with host cells through lipid-associated membrane proteins (LAMP). These lipoproteins are important for both surface adhesion and modulation of host immune responses. Mycoplasma and Ureaplasma have been implicated in cases of bacterial vaginosis (BV), which can cause infertility, abortion, and premature delivery. In contrast, bacteria of the genus Lactobacillus, which are present in the vaginal microbiota of healthy women, are thought to inhibit local colonization by pathogenic microorganisms. The aim of the present study was to evaluate the in vitro interactions between lipoproteins of Mycoplasma and Ureaplasma species and vaginal lineage (HMVII) cells and to study the effect of Lactobacillus isolates from cocoa fermentation on these interactions. The tested Lactobacillus strains showed some important probiotic characteristics, with autoaggregation percentages of 28.55% and 31.82% for L. fermentum FA4 and L. plantarum PA3 strains, respectively, and percent adhesion values of 31.66 and 41.65%, respectively. The two strains were hydrophobic, with moderate to high hydrophobicity values, 65.33% and 71.12% for L. fermentum FA4 and L. plantarum PA3 in toluene. Both strains secreted acids into the culture medium with pH=4.32 and pH=4.33, respectively, and showed antibiotics susceptibility profiles similar to those of other lactobacilli. The strains were also able to inhibit the death of vaginal epithelial cells after incubation with U. parvum LAMP from 41.03% to 2.43% (L. fermentum FA4) and 0.43% (L. plantarum PA3) and also managed to significantly decrease the rate of cell death caused by the interaction with LAMP of M. hominis from 34.29% to 14.06% (L. fermentum FA4) and 14.61% (L. plantarum PA3), thus demonstrating their potential for maintaining a healthy vaginal environment.

Co-infection of sexually transmitted pathogens and Human Papillomavirus in cervical samples of women of Brazil

BackgroundSome sexually transmitted infectious agents, such as Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to Human Papillomavirus (HPV) and cervical lesion progression. Thus, the aim of this study was to determine any association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographical variables to the development and progress of cervical lesions.MethodsOne hundred and thirty-two women between 14 and 78xa0years and living at Vitória da Conquista, Bahia, Brazil, were included (62 individuals with cervical lesions and 70 without lesions). They answered a questionnaire to provide data for a socioeconomic and sexual activity profile. Samples of cervical swabs were collected and analyzed by PCR to detect genital microorganisms and HPV. Quantitative PCR was used to detect and quantify Ureaplasma urealyticum and Ureaplasma parvum. Univariate and multiple logistic regression were performed to measure the association with the cervical lesions, and an odds ratio (OR) with 95% confidence intervals (95%CI) were calculated. The Mann-Whitney U test was also used to compare the microorganism load in the case and control groups. The significance level was 5% in all hypotheses tested.ResultsCervical lesions were associated with: women in a stable sexual relationship (ORu2009=u200914.21, 95%CIu2009=u20093.67–55.018), positive PCR for HPV (ORu2009=u200916.81, 95%CIu2009=u20094.19–67.42), Trichomonas vaginalis (ORu2009=u20098.566, 95%CIu2009=u20092.04–35.94) and Gardnerella vaginalis (ORu2009=u20096.13, 95%CIu2009=u20091.53–24.61), adjusted by age and qPCR for U. parvum. U. parvum load showed a statistical difference between the case and control groups (p-valueu2009=u20090.002).ConclusionVariables such as stable relationship, HPV, T. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesions. Additional studies are needed to better understand the role of these factors in cervical lesion development.