Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity.

Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential.

Development of metronidazole-resistant lines of Blastocystis sp.

Metronidazole (MTR) is frequently used for the treatment of Blastocystis infections, but with variable effectiveness, and often with treatment failures as a possible result of drug resistance. We have developed two Blastocystis MTR-resistant (MTR(R)) subtype 4 WR1 lines (WR1-M4 and WR1-M5), with variable susceptibility to a panel of anti-protozoal agents including various 5-nitroimidazoles, nitazoxanide and furazolidone. WR1-M4 and WR1-M5 were developed and assessed over an 18-month period and displayed persistent MTR resistance, being more than 2.

Barcoding of Giardia duodenalis isolates and derived lines from an established cryobank by a mutation scanning-based approach.

We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G.

Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia.

Objectives: The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.

Chromosome sequence maps of the Giardia lamblia assemblage A isolate WB.

Two genotypes, assemblages A and B, of the pathogenic gut protozoan parasite Giardia lamblia infect humans. Symptoms of infection range from asymptomatic to chronic diarrhea. Giardia chromosomes have long been characterized but not until the publication of the first Giardia genome sequence was chromosome mapping work, commenced nearly two decades ago, completed.

Sequence map of the 2 Mb Giardia lamblia assemblage A chromosome.

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2'-deoxyadenosine.

This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.

A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro.

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardialamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID(90) values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 microM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID(90) values around 100 microM (MTR-susceptible isolates typically have an ID(90) of 5-12.

Hydrogenosomes of laboratory-induced metronidazole-resistant Trichomonas vaginalis lines are downsized while those from clinically metronidazole-resistant isolates are not.

Trichomonas vaginalis is the most common sexually transmitted protozoan in the world and its resistance to metronidazole is increasing. The purpose of this study was to demonstrate that clinical metronidazole resistance in T. vaginalis does not occur via the same mechanism as laboratory-induced metronidazole resistance--that is, via hydrogenosome down sizing.

Metronidazole resistance in Trichomonas vaginalis from highland women in Papua New Guinea.

Background: The prevalence of the sexually transmissible protozoan parasite Trichomonas vaginalis in the highlands of Papua New Guinea (PNG) has been reported to be as high as 46% and although not previously studied in Papua New Guinea, clinical resistance against metronidazole (Mz), the drug most commonly used to treat trichomoniasis, is well documented worldwide. This study was primarily aimed at assessing resistance to Mz in T. vaginalis strains from the Goroka region.

Sequence map of the 3-Mb Giardia duodenalis assemblage A chromosome.

The genome of the gut protozoan parasite Giardia duodenalis (assemblage A) has been sequenced and compiled as contigs and scaffolds (GiardiaDB- http://GiardiaDB.org ), but specific chromosome location of all scaffolds is unknown. To determine which scaffolds belong to the 3-Mb chromosome, a library of probes specific for this chromosome was constructed.

Synthesis and electrochemistry of 2-ethenyl and 2-ethanyl derivatives of 5-nitroimidazole and antimicrobial activity against Giardia lamblia.

Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame Mz resistance and were more effective than Mz in a murine giardiasis model.

A novel method to increase the viability of Trichomonas vaginalis in urine.

Objectives: Two of the major diagnostic methods for Trichomonas vaginalis, wet mount and culture, rely on the continued viability of the organism. Methods to increase the viability of T. vaginalis in urine are needed.

Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment.

The activity of protease inhibitors against Giardia duodenalis and metronidazole-resistant Trichomonas vaginalis.

Antiretroviral protease inhibitors were assessed in vitro for their activity against Giardia duodenalis and Trichomonas vaginalis. Kaletra (a co-formulation of ritonavir and lopinavir) was the most effective overall, with 50% effective drug concentrations (EC(50)) of 1.1-2.

Drug resistance in Giardia: clinical versus laboratory isolates.

The advantages and limitations of determining mechanisms of drug resistance in Giardia duodenalis laboratory isolates, which have been generated in a number of ways, is weighed against the difficulty of analysing mechanisms in clinical isolates with a large diversity of genetic and expression capabilities. Using isogenic strains to follow changes in enzyme regulation involved in drug resistance, we have been able to assess the full capability of the parasite in developing drug resistance mechanisms. The complementarity of the two approaches, clinical versus laboratory induced drug resistance, and continuing comparison with other organisms, particularly the anaerobic bacteria with which Giardia has strong affiliations, is emphasized.

Virulent Avian Giardia duodenalis Pathogenic for Mice.

Early in 1995, a sulphur-crested cockatoo captured in the wild died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites isolated from the dead bird and established in traditional Giardia axenic medium were infective to mice and established chronic infections associated with weight gain impairment. Genetically and morphologically, the Giardia isolated from the bird belonged to the duodenalis group.

Genotyping Trichomonas vaginalis.

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T.

Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum.

Measurement of action spectra of light-activated processes.

We report on a new experimental technique suitable for measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength. We demonstrate the efficiency of this new technique in determination of the action spectrum of the light mediated transport of rhodamine 123 into the parasitic protozoan Giardia duodenalis.

5-Nitroimidazole drugs effective against metronidazole-resistant Trichomonas vaginalis and Giardia duodenalis.

Metronidazole (Mz)-resistant Giardia and Trichomonas were inhibited by 1 of 30 new 5-nitroimidazole drugs. Another five drugs were effective against some but not all of the Mz-resistant parasites. This study provides the incentive for the continued design of 5-nitroimidazole drugs to bypass cross-resistance among established 5-nitromidazole antiparasitic drugs.

Rearranged subtelomeric rRNA genes in Giardia duodenalis.

Giardia duodenalis has linear chromosomes capped with typical eukaryotic repeats [(TAGGG)n], subtelomeric rRNA genes, and telomere gene units. The absence of two closely associated NotI sites in the large-subunit rRNA gene was used as an indicator in hybridizations of one- and two-dimensional NotI-cleaved Giardia chromosome separations that some chromosomes carry only rearranged and, by deduction, nonfunctional rRNA genes.

Efficacy of antigiardial drugs.

The flagellated protozoa Giardia duodenalis is the most commonly detected parasite in the intestinal tract of humans. Infections with the parasite result in diarrhoeal disease in humans and animals, with infants at risk from failure-to-thrive syndrome. The incidence of giardiasis worldwide may be as high as 1000 million cases.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis.

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer.