Protein markers for Alzheimer disease in the frontal cortex and cerebellum

Objective: To compare proteins related to Alzheimer disease ( AD) in the frontal cortex and cerebellum of subjects with early-onset AD (EOAD) with or without presenilin 1 (PS1) mutations with sporadic late-onset AD ( LOAD) and nondemented control subjects. Methods: Immunohistochemistry, immunoblot analysis, and ELISA were used to detect and assess protein levels in brain. Results: In EOAD and to a lesser extent in LOAD, there was increased amyloid beta (Abeta) deposition (by immunohistochemistry), increased soluble Abeta (by immunoblot analysis), and specific increases in Abeta(40) and Abeta(42) ( by ELISA) in the frontal cortex and, in some cases, in the cerebellum. Surprisingly, immunoblot analysis revealed reduced levels of PS1 in many of the subjects with EOAD with or without PS1 mutations. In those PS1 mutation-bearing subjects with the highest Abeta, PS1 was barely, if at all, detectable. This decrease in PS1 was specific and not attributable solely to neuronal loss because amyloid precursor protein (APP) and the PS1-interacting protein beta-catenin levels were unchanged. Conclusions: This study shows that in the frontal cortex and cerebellum from Alzheimer disease patients harboring certain presenilin 1 mutations, high levels of amyloid beta are associated with low levels of presenilin 1. The study provides the premise for further investigation of mechanisms underlying the downregulation of presenilin 1, which may have considerable pathogenic and therapeutic relevance.

Effect of moisture and temperature distribution on dried food microstructure and porosity

Dehydration of food materials requires water removal from it. This removal of moisture prevents the growth and reproduction of microorganisms that cause decay and minimizes many of the moisture-driven deterioration reactions (Brennan, 1994). However, during food drying, many other changes occur simultaneously resulting in a modified overall quality (Kompany et al., 1993). Among the physical attributes of dried food material porosity and microstructure are the important ones that can dominant other quality of dried foods (Aguilera et al., 2000). In addition, this two concerned quality attributes affected by process conditions, material components and raw structure of food stuff. In this work, temperature moisture distribution within food materials during microwave drying will be taken into consideration to observe its participation on the microstructure and porosity of the finished product. Apple is the selective materials for this work. Generally, most of the food materials are found in non-uniformed moisture contained condition. To develop non uniform temperature distribution, food materials have been dried in a microwave oven with different power levels (Chua et al., 2000). First of all, temperature and moisture model is simulated by COMSOL Multiphysics. Later on, digital imaging camera and Image Pro Premier software have been deployed to observation moisture distribution and thermal imaging camera for temperature distribution. Finally, Microstructure and porosity of the food materials are obtained from scanning electron microscope and porosity measuring devices respectively . Moisture distribution and temperature during drying influence the microstructure and porosity significantly. Specially, High temperature and moisture contained regions show less porosity and more rupture. These findings support other literatures of Halder et al. (2011) and Rahman et al (1990). On the other hand, low temperature and moisture regions depict uniform microstructure and high porosity. This work therefore assists in better understanding of the role of moisture and temperature distribution to a prediction of micro structure and porosity of dried food materials.

Strategies for improved disease resistance in micro-propagated bananas

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

Resequencing and fine-mapping of the chromosome 12q13-14 locus associated with multiple sclerosis refines the number of implicated genes

Multiple sclerosis (MS) is a common chronic inflammatory disease of the central nervous system. Susceptibility to the disease is affected by both environmental and genetic factors. Genetic factors include haplotypes in the histocompatibility complex (MHC) and over 50 non-MHC loci reported by genome-wide association studies. Amongst these, we previously reported polymorphisms in chromosome 12q13-14 with a protective effect in individuals of European descent. This locus spans 288 kb and contains 17 genes, including several candidate genes which have potentially significant pathogenic and therapeutic implications. In this study, we aimed to fine-map this locus. We have implemented a two-phase study: a variant discovery phase where we have used next-generation sequencing and two target-enrichment strategies [long-range polymerase chain reaction (PCR) and Nimblegen's solution phase hybridization capture] in pools of 25 samples; and a genotyping phase where we genotyped 712 variants in 3577 healthy controls and 3269 MS patients. This study confirmed the association (rs2069502, P = 9.9 × 10−11, OR = 0.787) and narrowed down the locus of association to an 86.5 kb region. Although the study was unable to pinpoint the key-associated variant, we have identified a 42 (genotyped and imputed) single-nucleotide polymorphism haplotype block likely to harbour the causal variant. No evidence of association at previously reported low-frequency variants in CYP27B1 was observed. As part of the study we compared variant discovery performance using two target-enrichment strategies. We concluded that our pools enriched with Nimblegen's solution phase hybridization capture had better sensitivity to detect true variants than the pools enriched with long-range PCR, whilst specificity was better in the long-range PCR-enriched pools compared with solution phase hybridization capture enriched pools; this result has important implications for the design of future fine-mapping studies.

Integrative genomic profiling reveals conserved genetic mechanisms for tumorigenesis in common entities of non-Hodgkin's lymphoma

Recent developments in genomic technologies have resulted in increased understanding of pathogenic mechanisms and emphasized the importance of central survival pathways. Here, we use a novel bioinformatic based integrative genomic profiling approach to elucidate conserved mechanisms of lymphomagenesis in the three commonest non-Hodgkin's lymphoma (NHL) entities: diffuse large B-cell lymphoma, follicular lymphoma, and B-cell chronic lymphocytic leukemia. By integrating genome-wide DNA copy number analysis and transcriptome profiling of tumor cohorts, we identified genetic lesions present in each entity and highlighted their likely target genes. This revealed a significant enrichment of components of both the apoptosis pathway and the mitogen activated protein kinase pathway, including amplification of the MAP3K12 locus in all three entities, within the set of genes targeted by genetic alterations in these diseases. Furthermore, amplification of 12p13.33 was identified in all three entities and found to target the FOXM1 oncogene. Amplification of FOXM1 was subsequently found to be associated with an increased MYC oncogenic signaling signature, and siRNA-mediated knock-down of FOXM1 resulted in decreased MYC expression and induced G2 arrest. Together, these findings underscore genetic alteration of the MAPK and apoptosis pathways, and genetic amplification of FOXM1 as conserved mechanisms of lymphomagenesis in common NHL entities. Integrative genomic profiling identifies common central survival mechanisms and highlights them as attractive targets for directed therapy.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

Aqueous phase synthesis of copper nanoparticles : a link between heavy metal resistance and nanoparticle synthesis ability in bacterial systems

We demonstrate aqueous phase biosynthesis of phase-pure metallic copper nanoparticles (CuNPs) using a silver resistant bacterium Morganella morganii. This is particularly important considering that there has been no report that demonstrates biosynthesis and stabilization of pure copper nanoparticles in the aqueous phase. Electrochemical analysis of bacterial cells exposed to Cu2+ ions provides new insights into the mechanistic aspect of Cu2+ ion reduction within the bacterial cell and indicates a strong link between the silver and copper resistance machinery of bacteria in the context of metal ion reduction. The outcomes of this study take us a step closer towards designing rational strategies for biosynthesis of different metal nanoparticles using microorganisms.

Characteristics of nontuberculous mycobacteria from a municipal water distribution system and their relevance to human infections

This thesis documented pathogenic species of nontuberculous mycobacteria in the Brisbane water distribution system. When water and shower aerosol strains were compared with human strains of mycobacteria, the study found that the likelihood of acquiring infection from municipal water was specific for four main species. The method for isolation of mycobacteria from water was refined, followed by sampling from 220 sites across Brisbane. A variety of species (incl 15 pathogens) were identified and genotypically compared to human strains. For M. abscessus and M. lentiflavum, water strains clustered with human strains. Pathogenic strains of M. kansasii were found, though non-pathogenic strains dominated. Waterborne strains of M. fortuitum differed to human strains. Extensive home sampling of 20 patients with NTM disease, supported the theory that the risk of acquiring NTM from water or shower aerosols appears species specific for M. avium, M. kansasii, M. lentiflavum and M. abscessus.

Defense and counterdefense in the plant world

An important role of RNA interference (RNAi)-like pathways in plants is defense against viral infection. Viruses can overcome this defense by expressing proteins that suppress the pathway. A new study of Agrobacterium tumefaciens infection reveals that this plant pathogen, although a bacterium, also induces and then suppresses the host RNAi response. © 2006 Nature Publishing Group.

Exploring plant genomes by RNA-induced gene silencing

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms - for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken: sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intra-amniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

Factors associated with the isolation of Nontuberculous mycobacteria (NTM) from a large municipal water system in Brisbane, Australia

Background Nontuberculous mycobacteria (NTM) are normal inhabitants of a variety of environmental reservoirs including natural and municipal water. The aim of this study was to document the variety of species of NTM in potable water in Brisbane, QLD, with a specific interest in the main pathogens responsible for disease in this region and to explore factors associated with the isolation of NTM. One-litre water samples were collected from 189 routine collection sites in summer and 195 sites in winter. Samples were split, with half decontaminated with CPC 0.005%, then concentrated by filtration and cultured on 7H11 plates in MGIT tubes (winter only). Results Mycobacteria were grown from 40.21% sites in Summer (76/189) and 82.05% sites in winter (160/195). The winter samples yielded the greatest number and variety of mycobacteria as there was a high degree of subculture overgrowth and contamination in summer. Of those samples that did yield mycobacteria in summer, the variety of species differed from those isolated in winter. The inclusion of liquid media increased the yield for some species of NTM. Species that have been documented to cause disease in humans residing in Brisbane that were also found in water include M. gordonae, M. kansasii, M. abscessus, M. chelonae, M. fortuitum complex, M. intracellulare, M. avium complex, M. flavescens, M. interjectum, M. lentiflavum, M. mucogenicum, M. simiae, M. szulgai, M. terrae. M. kansasii was frequently isolated, but M. avium and M. intracellulare (the main pathogens responsible for disease is QLD) were isolated infrequently. Distance of sampling site from treatment plant in summer was associated with isolation of NTM. Pathogenic NTM (defined as those known to cause disease in QLD) were more likely to be identified from sites with narrower diameter pipes, predominantly distribution sample points, and from sites with asbestos cement or modified PVC pipes. Conclusions NTM responsible for human disease can be found in large urban water distribution systems in Australia. Based on our findings, additional point chlorination, maintenance of more constant pressure gradients in the system, and the utilisation of particular pipe materials should be considered.

Isolation of nontuberculous mycobacteria (NTM) from household water and shower aerosols in patients with pulmonary disease caused by NTM

It has been postulated that susceptible individuals may acquire infection with nontuberculous mycobacteria (NTM) from water and aerosol exposure. This study examined household water and shower aerosols of patients with NTM pulmonary disease. The mycobacteria isolated from clinical samples from 20 patients included M. avium (5 patients), M. intracellulare (12 patients), M. abscessus (7 patients), M. gordonae (1 patient), M. lentiflavum (1 patient), M. fortuitum (1 patient), M. peregrinum (1 patient), M. chelonae (1 patient), M. triplex (1 patient), and M. kansasii (1 patient). One-liter water samples and swabs were collected from all taps, and swimming pools or rainwater tanks. Shower aerosols were sampled using Andersen six-stage cascade impactors. For a subgroup of patients, real-time PCR was performed and high-resolution melt profiles were compared to those of ATCC control strains. Pathogenic mycobacteria were isolated from 19 homes. Species identified in the home matched that found in the patient in seven (35%) cases: M. abscessus (3 cases), M. avium (1 case), M. gordonae (1 case), M. lentiflavum (1 case), and M. kansasii (1 case). In an additional patient with M. abscessus infection, this species was isolated from potable water supplying her home. NTM grown from aerosols included M. abscessus (3 homes), M. gordonae (2 homes), M. kansasii (1 home), M. fortuitum complex (4 homes), M. mucogenicum (1 home), and M. wolinskyi (1 home). NTM causing human disease can be isolated from household water and aerosols. The evidence appears strongest for M. avium, M. kansasii, M. lentiflavum, and M. abscessus. Despite a predominance of disease due to M. intracellulare, we found no evidence for acquisition of infection from household water for this species.