The role of Ureaplasma urealyticum in adverse pregnancy outcome

We investigated Ureaplasma urealyticum genital tract colonisation rates in an Australian population to determine whether colonisation was associated with adverse pregnancy outcome. Women attending an antenatal clinic were evaluated for lower genital tract colonisation at their first antenatal visit (162 women) and at 28 weeks gestation (120 women). Placentas from 92 women were cultured. U. urealyticum was the predominant isolate from the lower (57.4%) and upper (17.4%) genital tract in this population of pregnant women. U. urealyticum was a persistent coloniser during mid-trimester of pregnancy (in 88% of women colonised) whereas M. hominis, G. vaginalis, and Group B streptococcus were present as transient flora of the lower genital tract. Lower genital tract colonisation during pregnancy was not directly associated with adverse pregnancy outcome. However preterm delivery in afebrile, asymptomatic women, could possibly be associated with chorioamnionitis (four of 16 preterm births). Screening of women with a history of preterm birth may prevent upper genital tract infections and preterm delivery.

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Intra-amniotic ureaplasma infection : adverse outcomes vary depending on gestation

Background: Ureaplasmas are the most prevalent bacteria isolated from preterm deliveries and the prognosis for neonates varies depending on the gestation at delivery. Ureaplasmas vary their surface-exposed antigen (MBA, a virulence mechanism) during chronic intra-amniotic infections, but it is not known when changes first occur during gestation. Method: U. parvum serovar 3 (2x10e7CFU) was injected intra-amniotically (IA) into six experimental cohorts of pregnant ewes (of n=7), 3 days (d) or 7d before delivery at either: 100d, 124d or 140d gestation (term=145d). Control ewes received IA 10B broth. Fetuses were delivered surgically and ureaplasmas cultured from amniotic fluid (AF), chorioamnion, fetal lung (FL) and umbilical cord. Ureaplasmas were tested by western blot to demonstrate MBA variation. Results: The highest number of ureaplasmas were recovered from FL at 100d gestation after 3 days of infection (p<0.03). Six of 7(86%) 100d–3d FL demonstrated an ureaplasma MBA variant, but only 17% and 15% of FL showed an MBA variant after 3d infection at 124d and 140d gestation respectively. Greatest variation of the MBA occurred in AF and FL at 124d gestation after 7d infection. The least MBA variation was observed at 140d; however, at this time the most severe histological chorioamnionitis was observed. Conclusions: After intra-amniotic ureaplasma injections, higher numbers of ureaplasmas gained access to the FL at 100d gestation than observed at later gestations. This may exacerbate the adverse outcomes for neonates delivered early in gestation. In late gestation, ureaplasma MBA variation was minimal, but chorioamnionitis was the most severe. Adverse pregnancy outcomes associated with IA ureaplasma infection may vary depending on the duration of gestation, the number of ureaplasmas isolated from the fetal tissues and the degree of MBA variation.

Ureaplasma speioces are associated with chorioamnionitiis in late preterm placentas

Background: Preterm birth is a major cause of neonatal morbidity and mortality, with 75% of preterm births occurring late preterm. Previous studies have investigated the microbial diversity within placentas delivered early preterm but there has been no investigation of the prevalence of bacteria, particularly Ureaplasma spp. in late preterm placentas. Method: Women giving birth late preterm (320 – 366 weeks of gestation) in Cincinnati were recruited for this study. Samples of chorioamnion were collected aseptically at the time of delivery, shipped to QUT and tested for Ureaplasma spp. and other bacteria by culture and/or PCR assays. The presence of bacteria was correlated with adverse pregnancy outcomes, including histological chorioamnionitis (tissue sections read by US pathologists). Results: To date, Ureaplasma spp. have been detected in 15/270 (5.5%) of placentas by culture and 19/270 (7%) by PCR. Ureaplasma presence correlated with histological chorioamnionitis (12/19%; 63%). However, the presence of other bacteria was not associated with chorioamnionitis (5%). Chorioamnionitis was unevenly distributed in ethnic groups, with a higher incidence in African-Americans’ (6/7; 86%), compared to Caucasians’ (6/12; 50%) who were colonised with ureaplasmas. Conclusion: This study is the first to report the prevalence of ureaplasmas in women (7%) who deliver late preterm. Ureaplasma spp. were associated with a higher incidence of chorioamnionitis (63% compared to 15% for non-infected women). This data strongly suggests that ureaplasmas are a cause of late preterm deliveries and African-American women are at greater risk of chorioamnionitis.

Changing learning to improve practice : hand hygiene education in Queensland medical schools

The aim of Queensland Health’s ‘Clean hands are life savers’ program is to change the culture and behaviour of healthcare workers related to hand hygiene. Hand hygiene is considered to be the most effective means of preventing pathogen cross-transmission and healthcare-associated infections. Most hospitals throughout Queensland as well as Australia now manage a hand hygiene program to increase the hand hygiene compliance of all healthcare workers. Reports taken from routine hand hygiene observations reveal that doctors are usually less compliant in their hand-washing practices than other healthcare worker groups. The Centre for Healthcare Related Infection Surveillance and Prevention (CHRISP) has attempted to have an impact on this challenging group through their Medical Leadership Initiative. With education as a core component of the program, efforts were made to ensure our future doctors were receiving information that aligned with Queensland Health standards during their formative years at medical school. CHRISP met with university instructors to understand what infection prevention education was currently included in the curriculum and support the introduction of new learning activities that specifically focused on hand hygiene. This prompted change to the existing curriculum and a range of interventions were employed with mixed success. Although met with challenges, methods to integrate more infection prevention teaching were found.

Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)(1). Assembly of an infectious virion proceeds in two stages(2). In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation(3). However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-angstrom resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

Cryo-electron tomography of Marburg Virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

The hypothermic patient

Concepts used in this chapter include: Thermoregulation:- Thermoregulation refers to the body’s sophisticated, multi-system regulation of core body temperature. This hierarchical system extends from highly thermo-sensitive neurons in the preoptic region of the brain proximate to the rostral hypothalamus, down to the brain stem and spinal cord. Coupled with receptors in the skin and spine, both central and peripheral information on body temperature is integrated to inform and activate the homeostatic mechanisms which maintain our core temperature at 37oC.1 Body heat is lost through the skin, via respiration and excretions. The skin is perhaps the most important organ in regulating heat loss. Hyporthermia:- Hypothermia is defined as core body temperature less than 350C and is the result of imbalance between the body’s heat production and heat loss mechanisms. Hypothermia may be accidental, or induced for clinical benefit i.e: neurological protection (therapeutic hypothermia). External environmental conditions are the most common cause of accidental hypothermia, but not the only causes of hypothermia in humans. Other causes include metabolic imbalance; trauma; neurological and infectious disease; and exposure to toxins such as organophosphates. Therapeutic Hypothermia:- In some circumstances, hypothermia can be induced to protect neurological functioning as a result of the associated decrease in cerebral metabolism and energy consumption. Reduction in the extent of degenerative processes associated with periods of ischaemia such as excitotoxic cascade; apoptotic and necrotic cell death; microglial activation; oxidative stress and inflammation associated with ischaemia are averted or minimised.2 Mild hypothermia is the only effective treatment confirmed clinically for improving the neurological outcomes of patient’s comatose following cardiac arrest.3

Characterization of in vitro Chlamydia muridarum persistence and utilization in an in vivo mouse model of Chlamydia vaccine

Problem: Chlamydia trachomatis genital tract infections are easily treated with antibiotics, however the majority of infections are asymptomatic and therefore untreated, highlighting the need for a vaccine. Because most infections are asymptomatic, vaccination could potentially be administered to individuals who may have an acute infection at that time. In such individuals the effect of vaccination on the existing infection is unknown; however one potential outcome could be the development of a persistent infection. In vitro chlamydial persistence has been well characterized in various strains, however there have been no reported studies in C. muridarum. Method of Study: We performed ultrastructural characterization, and transcriptome analysis of selected genes. We then used the transcriptional profiles of the selected genes to examine whether intranasal immunization of mice during an active genital infection would induce persistence in the upper reproductive tract of female mice. Results and Conclusions: We found that persistence developed in the oviducts of mice as a result of immunization. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. This highlights the importance of the timing of vaccination in individuals.

Proteomic and bioinformatics tools to understand virulence mechanisms in Staphylococcus aureus

Staphylococcus aureus, one of the major pathogenic bacteria, is associated with substantial morbidity and mortality. The disease burden of staphylococcal infections is significant, which is primarily attributed to its adaptability and resistance to environmental stresses. S. aureus has the ability to develop multiple resistances to antimicrobial agents. These high resistances make pathogenicity of S. aureus one of the most complex mechanisms to understand and manage. Proteomic and bioinformatics approaches show great potential in exploring microbial adaptation strategies, ability to cause disease by pathogenic bacteria and the development of diagnostic tools. A summary of the latest developments in the application of ‘omics’ technologies to understand resistance mechanisms in S. aureus and their future role in antistaphylococcal vaccine and/or drug discovery is given here.

Mycobacterium abscessus infection and potable water

Mycobacterium abscessus is a rapidly growing mycobacteria responsible for progressive pulmonary disease, soft tissue and wound infections, and can contaminate clinical specimens. Nontuberculous mycobacteria (NTM) are generally considered environmental organisms though M. abscessus has not featured frequently in environmental studies, particularly those examining potable water. In a study of Brisbane potable water, M. abscessus was isolate from ten different locations. The incidence of disease due to M. abscessus has been increasing in Queensland. Aim: To compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods: From a study of Brisbane potable water between 2007 and 2009, ten isolates confirmed as M. abscessus were recovered. In addition, one strain was isolated from a rainwater tank of a patient with disease due to M. avium, and another from the swimming pool of a patient with M. intracellulare disease. A random sample of 74 clinical isolates referred to the QLD Mycobacterial reference laboratory during the same time period was available for comparison using repPCR strain typing (Diversilab). Results: The drinking water isolates formed two distinct strain patterns (A and B) that shared >90% similarity. The tankwater isolate (pattern C) shared >85% similarity with the potable water isolates, but the pool isolate (D) was distinctly different. Fifty-three clinical isolates clustered tightly (>95% similarity) with the Group A potable water isolates, 4 patients with Group B. Thirteen patient isolates clustered with the Rainwater tank isolate. One patient matched the pool isolate. There were a further 3 patient isolates that were unrelated to the water isolates. No differences were found between strain types in terms of geographic origin, gender, age, or site/type of infection. Conclusion: The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same area, strengthens the possibility that drinking water may be a source of infection in these patients.

Detection of banana bunchy top virus in virus-infected plants using polymerase chain reaction

Polymerase chain reaction (PCR) was developed for the detection of Banana bunchy top virus (BBTV) at maximum after 210 min and at minimum after 90 min using Pc-1 and Pc-2, respectively. PCR detection of BBTV in crude sap indicated that the freezing of banana tissue in liquid nitrogen (LN2) before extraction was more effective than using sand as the extraction technique. BBTV was also detected using PCR assay in 69 healthy and diseased plants using Na-PO4 buffer containing 1 % SDS. PCR detection of BBTV in nucleic acid extracts using seven different extraction buffers to adapt the use of PCR in routine detection in the field was studied. Results proved that BBTV was detected with high sensitivity in nucleic acid extracts more than in infectious sap. The results also suggested the common aetiology for the BBTV by the PCR reactions of BBTV in nucleic acid extracts from Australia, Burundi, Egypt, France, Gabon, Philippines and Taiwan. Results also proved a positive relation between the Egyptian-BBTV isolate and abaca bunchy top isolate from the Philippines, but there no relation was found with the Cucumber mosaic cucumovirus (CMV) isolates from Egypt and Philippines and Banana bract mosaic virus (BBMV) were found.

Wetlands, climate zones and Barmah Forest virus disease in Queensland, Australia

Barmah Forest virus (BFV) disease is the second most common mosquito-borne disease in Australia, but the linkages of the wetlands and climate zones with BFV transmission remain unclear. We aimed to examine the relationship between the wetlands, climate zones and BFV risk in Queensland, Australia. Data on the wetlands, climate zones, population and BFV cases for the period 1992 to 2008 were obtained from relevant government agencies. BFV risk was grouped as low-, medium- and high-level based on BFV incidence percentiles. The buffer zones around each BFV case were made using 1, 5, 10, 15, 20, 25 and 50 km distances. We performed a discriminant analysis to determine the differences between wetland classes and BFV risk within each climate zone. The discriminant analyses show that saline 1, riverine and saline tidal influence were the most significant contributors to BFV risk in all climate and buffer zones, while lacustrine, palustrine, estuarine and saline 2 and saline 3 wetlands were less important. These models had classification accuracies of 76%, 98% and 100% for BFV risk in subtropical, tropical and temperate climate zones, respectively. This study demonstrates that BFV risk varies with wetland class and climate zone. The discriminant analysis is a useful tool to quantify the links between wetlands, climate zones and BFV risk.

The role of HtrA as a chaperone and protease in bacterial pathogenesis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.