Caracterização de beta-lactamases de espectro ampliado (ESBLs), genes de resistência aos antimicrobianos e conteúdo plasmidial em cepas de Escherichia coli e Salmonella spp. não tifóides isoladas do ambiente hospitalar e da comunidade

Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2). A cepa de S. Typhimurium produtora de SHV-5 pertenceu a um único clone (A-ST19) e o gene blaSHV-5 foi identificado em plasmídeo com o replicon IncL/M com aproximadamente 55Kb. Foi identificado pela primeira vez no Brasil o ST313 em um clone de S. Typhimurium (D-ST313), comumente associado com doenças invasivas severas, particulamente no continente africano. Genes que codificam para a resistência aos antimicrobianos não-beta-lactâmicos e integrons classe 1 foram identificados entre as cepas de E. coli e de Salmonella spp. multirresistentes produtoras ou não de ESBLs. Em conclusão: i) nossos resultados referentes à E. coli confirmaram a disseminação de enzimas CTX-M (principalmente variantes do grupo CTX-M-2) desde, pelo menos, o ano de 2000, em hospitais no Rio de Janeiro; demonstraram a implicação dos plasmídeos IncA/C na disseminação de genes blaCTX-M; indicaram a possível evolução intra-plasmídeo de blaCTX-M-59 a partir de blaCTX-M-2; a observação da diversidade e multiplicidade de plasmídeos poderiam fornecer plataformas genéticas para a dispersão de diferentes genes e/ou elementos de resistência aos antimicrobianos; ii) em relação à Salmonella spp. este estudo descreveu, pela primeira vez, o isolamento, a partir de fórmula infantil, de uma cepa de S. Typhimurium produtora de ESBL; foi demonstrada a associação do gene blaSHV-5 com plasmídeo do tipo IncL/M, que é considerado epidêmico; foi identificado o clone D-ST313 de S. Typhimurium, que está associado a doenças invasivas severas no continente africano, que reuniu cepas isoladas exclusivamente do ambiente hospitalar.

Emergence of CTX-M Group 1-ESBL producing Klebsiella pneumonia from a tertiary care centre in Karachi, Pakistan.

http://www.jidc.org/index.php/journal/article/view/20818098/422 Background: Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previously from Pakistan but the genotypic characteristics of these enzymes is not known. Hence the aim of the study was first to characterise the genotypic content of these beta-lactamases and secondly to assess the clonal relationship of these isolates. Methodology: We analysed 65 non-duplicate ESBL positive, K. pneumoniae isolates prospectively collected based on phenotype as detected using the two-disc method. Isolates were collected from different sources: blood cultures (46.15%; n = 30); tracheal aspirates (24.6%; n = 16); urine (10.7%; n = 7); wound swabs, pus and tissue (18.4%; n = 12). ESBL production was confirmed by the ESBL E-test method and the presence of the blaCTX-M encoding genes was confirmed by polymerase chain reaction. The clonal relationship of clinical isolates was studied by Pulsed Field Gel Electrophoresis. Results: The results showed that 93.84% (n = 61) isolates of K. pneumoniae were positive for the blaCTX-M-1 group. One isolate showed PCR signals for blaCTX-M-25 group. None of our isolates were positive for CTX-M groups 2, 8 and 9. The majority of blaCTX-M positive isolates were genetically unrelated and no epidemic clones were identified. Conclusion: This study reports the emergence of CTX-M groups 1 and 25 producing isolates of K. pneumoniae with genetic diversity in Karachi, Pakistan.

Clinico-epidemiological features of infections caused by CTX-M type extended spectrum beta lactamase-producing Escherichia coli in hospitalised patients

A review of medical records of 45 of 53 hospitalised patients with positive cultures for CTX-M type ESBL-producing Escherichia coli between 01 January and 31 May 2004 was conducted. The mean age of the population studied was 73.1 (+/-14.6) years and the majority (55.6%) had been under the care of the internal medicine or elderly care service. In the majority (77.8%) of instances the isolate was attributed to a clinical infection rather than colonisation and the commonest clinical specimen to yield the organism was urine, which was positive in 57.8% of patients. Acquisition of the organism was categorised as nosocomial in 68.9% of patients; in this subgroup, the median duration of inpatient stay prior to recovery of the organism was 24 (range 3-240) days. Haemodialysis-dependence was the most common of the comorbidities evaluated. The mean number of antibiotics prescribed per patient in the 30 days prior to first isolation of the organism was 1.7 (range 0-4). Furthermore, the mean number of antibiotic-days exposure per patient during this period was 13.9 (range 0-48). The most frequently received class of antibiotic was beta-lactam/beta-lactamase inhibitor combinations. Of 35 infections, 26 (74.2%) were successfully treated. Overall 12 patients with infection died (34.3%); attributable mortality was presumed in seven (20%).

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Dimerization on the Structure and Biological Activity of Antimicrobial Peptide Ctx-Ha

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mechanism of Action and Relationship Between Structure and Biological Activity of Ctx-Ha: A New Ceratotoxin-like Peptide from Hypsiboas albopunctatus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Vaughan, Suzi and Armstrong, Wendy (2009) Threads of tension. In: Vaughan, Suzi and Schmidt, Christine Margaret, (eds.) Five Fashion Musings. Post Pressed, Brisbane, Qld, pp. 9-15.

five:fashion musings is an upcoming 2009 publicaton to celebrate the fashion discipline's five-year milestone at the Queensland Unviesity of Technology. it represents a body of work by fashion practitioners, aceademic and educators commissioned to explore their research in fashion theory, practice and pedogogy through five key themes.

Children's Fiction About 9/11: Ethnic, Heroic and National Identities

In this study, Lampert examines how cultural identities are constructed within fictional texts for young people written about the attacks on the Twin Towers. It identifi es three significant identity categories encoded in 9/11 books for children:ethnic identities, national identities, and heroic identities,arguing that the identities formed within the selected children’s texts are in flux, privileging performances of identities that are contingent on post-9/11 politics. Looking at texts including picture books, young adult fiction, and a selection of DC Comics, Lampert finds in post-9/11 children’s literature a co-mingling of xenophobia and tolerance; a binaried competition between good and evil and global harmony and national insularity; and a lauding of both the commonplace hero and the super-human. The shifting identities evident in texts that are being produced for children about 9/11 offer implicit and explicit accounts of what constitutes good citizenship, loyalty to nation and community, and desirable attributes in a Western post-9/11 context. This book makes an original contribution to the field of children’s literature by providing a focused and sustained analysis of how texts for children about 9/11 contribute to formations of identity in these complex times of cultural unease and global unrest.

Year 8, 9 and 10 students’ understanding and access of percent knowledge

This paper reports on Years 8, 9 and 10 students’ knowledge of percent problem types, use of diagrams, and type of solution strategy. Non- and semi-proficient students displayed the expected inflexible formula approach to solution but proficient students used a flexible mixture of estimation, number sense and trial and error instead of expected schema based methods.

The National Aboriginal and Torres Strait Islander Women's Health Strategy on Talkin with Tiga Bayles, 98.9 AM National Indigenous Radio Service (NIRS), Wednesday 21 July 2010. (9.00 - 10.00am).

Radio Program. Talkin with Tiga Bayles, 98.9 AM National Indigenous Radio Service (NIRS), 9.00-10.00am, Wednesday 21 July 2010. (1 hour program).----- Bronwyn Fredericks discssed the National Aboriginal and Torres Strait Islander Women’s Health Strategy was launched at the Australian Women’s Health Network (AWHN) National Conference in Hobart on the 19 May 2010. Within this radio interview the background of the Strategy is discussed, funding, who did the consultations and the writing. In the interview Bronwyn Fredericks outlines the process of the Strategy’s development and its uses for the future.----- It is important to note that this Strategy does not replace other national or State and Territory documents which identify priorities and needs. The aim is to supplement existing work.

Merging fact with fiction : a study of year 9 students' developing scientific literacy through the writing of hybridised scientific narratives on a socioscientific issue

International assessments of student science achievement, and growing evidence of students' waning interest in school science, have ensured that the development of scientific literacy continues to remain an important educational priority. Furthermore, researchers have called for teaching and learning strategies to engage students in the learning of science, particularly in the middle years of schooling. This study extends previous national and international research that has established a link between writing and learning science. Specifically, it investigates the learning experiences of eight intact Year 9 science classes as they engage in the writing of short stories that merge scientific and narrative genres (i.e., hybridised scientific narratives) about the socioscientific issue of biosecurity. This study employed a triangulation mixed methods research design, generating both quantitative and qualitative data, in order to investigate three research questions that examined the extent to which the students' participation in the study enhanced their scientific literacy; the extent to which the students demonstrated conceptual understanding of related scientific concepts through their written artefacts and in interviews about the artefacts; and the extent to which the students' participation in the project influenced their attitudes toward science and science learning. Three aspects of scientific literacy were investigated in this study: conceptual science understandings (a derived sense of scientific literacy), the students' transformation of scientific information in written stories about biosecurity (simple and expanded fundamental senses of scientific literacy), and attitudes toward science and science learning. The stories written by students in a selected case study class (N=26) were analysed quantitatively using a series of specifically-designed matrices that produce numerical scores that reflect students' developing fundamental and derived senses of scientific literacy. All students (N=152) also completed a Likert-style instrument (i.e., BioQuiz), pretest and posttest, that examined their interest in learning science, science self-efficacy, their perceived personal and general value of science, their familiarity with biosecurity issues, and their attitudes toward biosecurity. Socioscientific issues (SSI) education served as a theoretical framework for this study. It sought to investigate an alternative discourse with which students can engage in the context of SSI education, and the role of positive attitudes in engaging students in the negotiation of socioscientific issues. Results of the study have revealed that writing BioStories enhanced selected aspects of the participants' attitudes toward science and science learning, and their awareness and conceptual understanding of issues relating to biosecurity. Furthermore, the students' written artefacts alone did not provide an accurate representation of the level of their conceptual science understandings. An examination of these artefacts in combination with interviews about the students' written work provided a more comprehensive assessment of their developing scientific literacy. These findings support extensive calls for the utilisation of diversified writing-to-learn strategies in the science classroom, and therefore make a significant contribution to the writing-to-learn science literature, particularly in relation to the use of hybridised scientific genres. At the same time, this study presents the argument that the writing of hybridised scientific narratives such as BioStories can be used to complement the types of written discourse with which students engage in the negotiation of socioscientific issues, namely, argumentation, as the development of positive attitudes toward science and science learning can encourage students' participation in the discourse of science. The implications of this study for curricular design and implementation, and for further research, are also discussed.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Proceedings of the 21st Australian Software Engineering Conference (ASWEC 2010) : 6-9 April 2010, Auckland, New Zealand.

ASWEC is a joint conference of Engineers Australia and the Australian Computer Society reporting through the Engineers Australia/ACS Joint Board on Software Engineering.