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Mycoplasma hominis variable adherence-associated antigen: a major adhesin and highly variable surface membrane protein
Full text linkMycoplasma hominis is a member of the genus mycoplasma and has only been isolated from humans. It is most frequently isolated from the urogenital tract in the absence of symptoms, but has been isolated from wounds, brain abscess, inflamed joints, blood and placenta from pregnancy with adverse outcomes (especially preterm birth and occasionally term stillbirth). Controversy surrounds whether this organism is a commensal or a pathogen; however, Mycoplasma hominis has been shown to induce preterm birth and foetal lung injury in an experimental primate model as a sole pathogen. These bacteria are known to exist as a parasitic infection, due to a number of missing synthetic and metabolism pathway enzymes from their minimal genome; therefore, the ability to adhere to host cells is important. Here we provide a review that clarifies the different nomenclature (variable adherence-associated antigen and P50) that has been used to investigate the major surface adhesin for this organism, as well as reported mechanisms responsible for turning off its expression. Variation in the structure of this protein can be used to separate strains into six categories, a method that we were able to use to distinguish and characterise 12 UK strains isolated from between 1983 and 2012. We propose that the Vaa should be used in further investigations to determine if commensal populations and those that are associated with disease utilise different forms of this adhesin, as this is under-studied and identification of pathogenic determinants is overdue for this organism
Molecular typing of Mycoplasma pneumoniae: where do we stand?
No full textMycoplasma pneumoniae is a respiratory bacterial pathogen causing upper and lower respiratory disease in humans of all ages. It is considered a major cause of pneumonia, especially in children of school age and in some cases can result in serious extrapulmonary sequelae. A large increase in reported M. pneumoniae cases was documented in several European countries in 2011 [1]. In England and Wales, seasonal peaks of infection are detected from December to February each year with epidemics at approximately four yearly intervals, lasting 12–15 months [2]. Epidemics are not concurrent worldwide, however, differing countries also report cyclical patterns, as observed in England and Wales, such as Denmark, Sweden, Norway, Finland, Korea and Japan [3,4]. Additionally, in differing countries, seasonal peaks of infection have been observed in either summer or autumn and no definitive factor has been proven to account for seasonal variation or the formation of epidemic peaks.
Traditionally, molecular typing was used to characterize epidemic outbreaks of M. pneumoniae, however, it has been postulated that molecular typing of M. pneumoniae is hampered by the genetically homologous nature of the species [5]. Despite this, molecular typing methods have been developed for this organism including: PCR-restriction length polymorphism (RFLP) of the major surface adhesin P1 [5], multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) [6], multilocus sequence typing (MLST) [7] and the recent SNaPshot™ minisequencing assay [8]. The mechanisms driving fluctuations in incidence of M. pneumoniae infections have not been defined. It has been postulated that shifts in proportion of individual strains with specific P1 type or concurrent increased incidence of several strains may result in epidemics or immunity. Additionally, it is believed that the genotype of M. pneumoniae may be changing, generating diverse genetic material in each epidemic with a recent study reporting the detection of polyclonal strains in a single epidemic [9].
The initial molecular typing procedure targeted the gene encoding of the major surface adhesin, P1, of M. pneumoniae. RFLP analysis of the p1 gene was the most common genotyping method, enabling separation of M. pneumoniae isolates into two types, type 1 and 2 [5,10]. Studies utilizing the repetitive regions, RepMp2/3 and RepM4 in the p1 gene resulted in the identification of an additional six variants [11–13]. Speculation that a shift in P1 adhesin type may be the cause of epidemics has been disputed with evidence indicating the presence of multiple P1 adhesin types in observed increases of infection [6,9,10]. It was hypothesized that a decline in immunity or an increase of the immunologically naive population may result in the 4-year cycle of epidemic periods [14]. In other geographical locations, it has also been observed that multiple P1 types can be detected during outbreaks, and it has been suggested that although immunological pressure may favor shifts of P1 type, a co-circulation of P1 types appears to be common [15].
MLVA has been increasingly used internationally for strain characterization and is based on variation in the copy number of tandem repeated sequences, called VNTRs, found at different loci across the genome. The variation of the copy number of these tandem repeats depends on the isolate tested. Initially, 265 strains were grouped into 26 MLVA types, based on five VNTR loci (Mpn1, Mpn13–16) and additional novel types have since been reported [6,16]. MLVA was documented to be more discriminatory for M. pneumoniae strains than P1 typing, providing an additional level of classification for transmission studies. However, reports of observed instability in the Mpn1 locus has called into question the reliability of the marker. Additionally, inconsistency in nomenclature and identification of repeat regions has led to international standardization of the MLVA and the removal of Mpn1 as a locus [17]. Analysis of the 2010/2011 epidemic in the UK revealed a total of 11 distinct MLVA types present using the original typing method [14], however, reanalysis using international guidelines reduces the MLVA types detected to five distinct types [Unpublished Data]. The discriminatory power of the MLVA method for characterization of M. pneumoniae strains has reduced with the removal of the Mpn1 locus, necessitating either the identification of new loci or alternative typing methods.
Initial attempts at developing an MLST scheme for M. pneumoniae were unsuccessful due to low levels of polymorphisms found in the housekeeping genes examined, suggested to be because of the homogeneity of the M. pneumoniae species, and it was concluded that the use of an MLST scheme with housekeeping and structural genes was not useful for molecular typing. However, three housekeeping genes were examined for polymorphisms across 30 isolates of either P1 type 1, 2 or a variant strain and the other genes selected for analysis were examined against a single representative strain from each P1 type [18]. Recently, an MLST scheme was successfully developed to differentiate M. pneumoniae isolates based on sequence polymorphisms in eight housekeeping genes, which improved on existing typing methods for M. pneumoniae [7]. This MLST scheme discriminated between 57 M. pneumoniae isolates with a higher level than both MLVA (with the removal of Mpn1) and P1 typing and it may prove more optimal for epidemiological studies than other existing methods. Population modeling and phylogenetic analysis of concatenated MLST profiles revealed two distinct genetic clades of M. pneumoinae, showing similar topology to phylogenetic data and distinct genetic clustering obtained using genomic sequence analysis. The typing profiles obtained using the MLST method infers representation of the genetic phylogeny, reflecting that M. pneumoniae can be subdivided into two distinct genetic lineages [7]. Nevertheless, this MLST scheme has not yet been applied to localized outbreak or epidemic strain analysis or has not been demonstrated direct on clinical specimens. Recent development of a SNaPshot™ mini sequencing assay has resulted in identification of nine SNP types [8]. This method is rapid and appears to have greater discriminatory ability than MLVA and P1 typing. A direct comparison of MLST and SNaPshot™ minisequencing assay has not been undertaken and both methods may have similar discriminatory abilities. However, MLST resulted in a larger number of defined sequence types.
These methods are all PCR-based and do not necessarily require the growth of bacteria, which can be a lengthy process for M. pneumoniae. P1 typing, MLVA and MLST do not limit investigation through the requirement of specialist methodology. However, MLST can be laborious and expensive, with the cost of genomic sequencing reducing and becoming a more attractive option for genetic analysis of strains. Genomic sequencing may allow the concurrent identification of P1 type, MLVA profile and MLST sequence type directly from the genomic sequence as well as providing additional information, such as the presence of antibiotic resistance and toxin markers. Improvements in sequencing technology and the development of methodologies to produce longer sequence reads enables the reliable determination of repeated DNA sequences [19]. This is of importance for species such as Mycoplasma, in which large tracts of repeated sections within AT-rich genomes are common. For genetically homologous species such as M. pneumoniae, the use of genomic sequencing to analyze phylogeny inferred from single nucleotide polymorphism analysis will improve the ability to accurately segregate this species into distinct lineages allowing in-depth epidemiology studies. Due to the fastidious nature of M. pneumoniae and other human Mollicutes, such as Mycoplasma amphoriforme, the use of metagenomic approaches to identify pathogens in studies of human infections [20] will no doubt improve detection of infections caused by Mollicutes, while obviating the need for expensive and laborious culture and typing methods, simultaneously providing additional data such as the detection of mutations known to confer resistance
Detection of macrolide resistant Mycoplasma pneumoniae in England, September 2014 to September 2015
No full textIsolation of separate Ureaplasma species from endotracheal secretions of twin patients
Full text linkIsolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection
Genomic determination of minimum multi-locus sequence typing schemas to represent the genomic phylogeny of Mycoplasma hominis
Full text linkBackground: Mycoplasma hominis is an opportunistic human pathogen, associated with clinically diverse disease. Currently, there is no standardised method for typing M. hominis, which would aid in understanding pathogen epidemiology and transmission. Due to availability and costs of whole genome sequencing and the challenges in obtaining adequate M. hominis DNA, the use of whole genome sequence analysis to provide clinical guidance is unpractical for this bacterial species as well as other fastidious organisms.Results: This study identified pan-genome set of 700 genes found to be present in four published reference genomes. A subset of 417 genes was identified to be core genome for 18 isolates and 1 reference. Leave-one-out analysis of the core genes highlighted set of 48 genes that are required to recapture the original phylogenetic relationships observed using whole genome SNP analysis. Three 7-locus MLST schemas with high diversity index (97%) and low dN/dS ratios (0.1, 0.13, and 0.11) were derived that could be used to confer good discrimination between strains and could be of practical use in future studies direct on clinical specimens.Conclusions: The genes proposed in this study could be utilised to design a cost-effective and rapid PCR-based MLST assay that could be applied directly to clinical isolates, without prior isolation. This study includes additional genomic analysis revealing high levels of genetic heterogeneity among this species. This provides a novel and evidence based approach for the development of MLST schema that accurately represent genomic phylogeny for use in epidemiology and transmission studies.<br/
Development of a multilocus sequence typing scheme for the molecular typing of Mycoplasma pneumoniae
No full textThis work was funded by Public Health England. These studies were supported by funding initiatives by the National Institute for Social Care and Health Research (NISCHR; research support from the Welsh Government) via the registered research group Microbial and Infection Translational Research Group (MITReG) and Children and Young Persons Research Network (CYPRN).Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).Peer reviewe
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Mycoplasma pneumoniae Epidemiology in England and Wales: A National Perspective.
Full text linkInvestigations of patients with suspected Mycoplasma pneumoniae infection have been undertaken in England since the early 1970s. M. pneumoniae is a respiratory pathogen that is a common cause of pneumonia and may cause serious sequelae such as encephalitis and has been documented in children with persistent cough. The pathogen is found in all age groups, with higher prevalence in children aged 5-14 years. In England, recurrent epidemic periods have occurred at ~4-yearly intervals. In addition, low-level sporadic infection occurs with seasonal peaks from December to February. Voluntarily reports from regional laboratories and hospitals in England from 1975 to 2015 were collated by Public Health England for epidemiological analysis. Further data pertaining cases of note and specimens submitted to Public Health England from 2005 to 2015 for confirmation, molecular typing is included
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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