Charlene Mason

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)–mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced β-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates β-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G2 phase of cell cycle. [Mol Cancer Ther 2008;7(3):548–58]

Population structure and characterization of viridans group streptococci (VGS) including Streptococcus pneumoniae isolated from adult patients with cystic fibrosis (CF)

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitis (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance.

Molecular characterization of macrolide resistance determinants [erm(B) and mef(A)] in Streptococcus pneumoniae and viridans group streptococci (VGS) isolated from adult patients with cystic fibrosis (CF)

OBJECTIVES Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci. METHODS Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months. RESULTS Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent. CONCLUSIONS Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.

Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis

OBJECTIVES Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). METHODS The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. RESULTS Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). CONCLUSIONS Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

Comparasion of five gene loci (rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ) to aid the molecular identification of viridans-group streptococci and pneumococci

Abstract Viridans-group streptococci (VGS) consist of several taxa which historically have been highly diverse. However, at times it may become necessary to have a reliable scheme for the identification of these organisms to the species level. The aim of this study is to compare the ability of five gene loci, namely rnpB, 16S rRNA, 16S–23S rRNA, sodA and dnaJ, to speciate such organisms through a sequence typing-based approach. Reference organisms consisting of six VGS species were compared based on sequence typing, followed by comparison of 31 wild-type respiratory isolates, and showed that employment of sequence typing using the rnpB gene locus was the most specific and reliable. Therefore, the use of rnpB sequencing for the identification of VGS to species level is a reliable and feasible option, based on a single gene target.

Chapter 11 Total synthesis and mechanism of action studies on the antitumor alkaloid, (-)-agelastatin a

Publisher Summary This chapter provides an overview of a very important chemical genomics tool that have been recently synthesized in laboratories—the antitumor alkaloid, (-)-agelastatin A. Its synthesis was undertaken solely for the purpose of getting answers to some important questions concerning the workings of the Wnt/GSK-3β/β-catenin/E-cadherin cell signaling pathways and the downstream genes and proteins that they control. With the synthetic (-)-agelastatin A that was prepared by route given in the chapter, it was possible to preliminarily investigate its effects on the Wnt/APC/β-catenin cell signaling pathway. In this regard, it was found that at 10 nM concentrations, (-)-agelastatin A dramatically decreases the levels of cellular (β-catenin within a metastatic Rama 37 C9-Met DNA mammary epithelial cell line). It has also been observed that it massively down-regulates the expression of the metastasis-inducing glycophosphoprotein, osteopontin (Opn), in such cells at this low concentration and that it additionally down-regulates Tcf-1. At 10 nM concentrations, (-)-agelastatin A also potently up-regulates Tcf-4 in such cells.

Comparison of antibiotic susceptibility in viridans group streptococci in low and high antibiotic-prescribing General Practices.

Antibiotic resistance has become a global public health issue. Most antibiotics are prescribed in the community, although there is less stewardship of such agents in the community compared to secondary and tertiary care. Few studies have attempted to examine the prescribing practices in General Practice and its impact on antibiotic resistance and, therefore, a study was performed in order to compare antibiotic susceptibilities of commensal viridans group streptococci (VGS) obtained from patient cohorts in General Practices (GP), who were high and low prescribers of oral antibiotics.

Optimisation of storage conditions for maintaining culturability of penicillin-susceptible and penicillin-resistant isolates of Streptococcus pneumoniae in transport medium

Abstract Methods employed by the World Health Organization (WHO) are used during this study to determine the optimum storage conditions for maintaining the culturability of Streptococcus pneumoniae in skimmed milk, tryptone, glucose and glycerin (STGG) transport medium. A comparison of S. pneumoniae strains sensitive and resistant to penicillin showed no significant difference in their survival ability in STGG medium. Furthermore, it is confirmed that storage at –70°C remains most effective for maintaining viability by culture of S. pneumoniae. Storage at –20°C would only be acceptable in the short-term, while storage at +4°C is not recommended. Of note, this study has shown STGG medium at room temperature to be an efficient growth medium for pneumococci in the short-term. This work will help to establish robust sampling protocols when performing community studies to ensure culturability of comparison between community and laboratory pneumococci survival.

Comparison of minimum inhibitory concentration by broth microdilution testing versus standard disc diffusion testing in the detection of penicillin, erythromycin and ciprofloxacin resistance in viridans group streptococci

The aim of this study was to investigate the reliability of disc diffusion testing with penicillin, erythromycin and ciprofloxacin within the viridans group streptococci (VGS). In total, the antibiotic susceptibilities of 167 VGS isolates were compared by standard disc diffusion and broth microdilution methods, and these phenotypic data were compared to the carriage of the respective gene resistance determinants [ermB and mefA/E (macrolides); QRDR, gyrA, gyrB, parC and parE (quinolones)]. Overall, there were 35 discrepancies [resistant by MIC and susceptible by zone diameter (21.0%)] between MIC and disc diameter when penicillin susceptibility was interpreted by Clinical and Laboratory Standards Institute criteria. Scattergrams showed a bimodal distribution between non-susceptible and susceptible strains when erythromycin susceptibility was tested by both methods. Thirty-four (20.4%) isolates were categorized as resistant by MIC breakpoints, while disc diameter defined these as having intermediate resistance. With ciprofloxacin, three isolates (1.8%) showed minor discrepancies between MIC breakpoints and disc diameter. Isolates non-susceptible to all three antimicrobial agents tested were reliably distinguished from susceptible isolates by disc diffusion testing, except for the detection of low-level resistance to penicillin, where broth microdilution or an alternative quantitative MIC method should be used. Otherwise, we conclude that disc diffusion testing is a reliable method to detect strains of VGS non-susceptible to penicillin, erythromycin and ciprofloxacin, as demonstrated with their concordance to their gene resistance characteristics.

The viridans group streptococci.

The viridans group streptococci (VGS) are an extremely diverse range of organisms within the genus Streptococcus, which are characterized by a greening of the blood in blood agar, on which they are normally grown. Oral streptococci largely comprise members of the VGS, which currently include 20 species, which are commensal inhabitants of the oropharyngeal cavity, and the gastrointestinal and genital tracts of mammals. On the basis of 16S rRNA sequence homology, these bacteria are categorised into four groups: the salivarius rRNA homology group, including Streptococcus thermophilus, Streptococcus vestibularis and Streptococcus salivarius; the mitis group, including Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sanguinis and Streptococcus parasanguinis; the anginosus group, including Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius; and the mutans group, including Streptococcus mutans, Streptococcus criceti, Streptococcus downei, Streptococcus ferus, Streptococcus macacae, Streptococcus ratti and Streptococcus sobrinus. This review aims at comparing conventional and molecular detection methods and the emergence of antibiotic resistance within the VGS.