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. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.

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 beta-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n = 14) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/92/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n = 10) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies. (Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN204442-JN204461.)

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.Methods: PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTRr) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance.Results: We demonstrated that several lines of highly MTRr G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTRr Trichomonas vaginalis.Conclusions: These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.

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 ID90 values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID90 values around 100 μM (MTR-susceptible isolates typically have an ID90 of 5–12.8 μM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells.

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 3.2 µM Mz while the clinically, highly Mz-resistant isolate has an MIC of 50–100 µM. This isolate was cross-resistant to other members of the 5-NI family of compounds including tinidazole and other experimental compounds and maintained resistance under anaerobic conditions. In addition, this isolate was cross-resistant to the 5-nitrothiazole compound nitazoxanide and the 5-nitrofuran derivative, furazolidone. Adenosine analogues toyocamycin and 2-fluoro-2′-deoxyadenosine with no nitro group were also less effective against the clinically Mz-resistant isolate than a Mz-susceptible one. Three other isolates which were determined to be Mz-resistant soon after isolation lost resistance in the long term. One other isolate has maintained some level of permanent Mz resistance (MIC of 25 µM). A multi-drug resistance mechanism may be involved in these clinically Mz-resistant isolates.

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. Initial mapping studies identified and ordered Not I chromosome segments (summating to 1.8 Mb) of the estimated 2 Mb chromosome 3. The resulting map was confirmed with the release of the Giardia genome sequence and this revitalized mapping. The result is that 93% of the WB isolate genome sequence has now been assigned to one of five major chromosomes, and community access to these data has been made available through GiardiaDB, the database for Giardia genomes.

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. METHODS Consenting patients presenting at the Goroka Base Hospital Sexually Transmitted Diseases (STD) Clinic and local women were asked to provide two vaginal swabs: one for culturing of the parasite; and one for polymerase chain reaction detection of T. vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae. T. vaginalis isolates were assayed for Mz susceptibility and a selection was genotyped. RESULTS The prevalence of T. vaginalis was determined to be 32.9% by culture and polymerase chain reaction of swabs among 82 local women and patients from the STD clinic. An unexpectedly high level of in vitro Mz resistance was determined with 17.4% of isolates displaying unexpectedly high resistance to Mz. The ability to identify isolates of T. vaginalis by genotyping was confirmed and the results revealed a more homogeneous T. vaginalis population in Papua New Guinea compared with isolates from elsewhere. CONCLUSION T. vaginalis is highly prevalent in the Goroka region and in vitro Mz resistance data suggest that clinical resistance may become an issue.

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. The probes were hybridised to NotI-cleaved whole chromosomes, and the combined size of different NotI segments identified by the probes was 2,225 kb indicating the probes were well distributed along the 3-Mb chromosome. Six scaffolds (CH991814, CH991779, CH991793, CH991763, CH991764, and CH991761) were identified as belonging to the 3-Mb chromosome, and these scaffolds were ordered and oriented according to scaffold features including I-PpoI sites and hybridisation pattern. However, the combined size of scaffolds was more than 4 Mb. Approximately, 1 Mb of scaffold CH991763 carrying previously identified sequences specific for the 1.5-Mb chromosome(s) including subtelomeric sequence was reassigned, and several other anomalies were addressed such that the final size of the apparently 3-Mb chromosome is estimated to be 2,885 kb. This work addresses erroneous computer-based assignment of a number of contigs and emphasises the need for alternative and confirmatory methods of scaffold construction.

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. Determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the potential of combining systematic synthetic approaches with biological and electrochemical evaluations in developing improved 5-NI drugs.

ABSTRACT. 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. Ultrathin sections of this parasite were examined using transmission electron microscopy and the size and area of the cell and hydrogenosomes were compared between drug‐resistant laboratory lines and clinically resistant isolates. Clinical metronidazole‐resistant T. vaginalis had similar‐sized hydrogenosomes as a metronidazole‐sensitive isolate. Inducing metronidazole resistance in both of these isolates caused down sizing of hydrogenosomes. Inducing toyocamycin resistance did not cause any ultrastructural changes to the cell or to the hydrogenosome. No correlation between hydrogenosome number and the drug‐resistant status of T. vaginalis isolates and lines was observed. This report demonstrates that clinical metronidazole resistance is not associated with down‐sized hydrogenosomes, thus indicating that an alternative resistance mechanism is used by T. vaginalis.

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. GOAL:The goal of this study was to develop a method that increases the time of viability of T. vaginalis in urine. STUDY DESIGN:Urine samples were inoculated with trichomonads, held at either room temperature or 37 degrees C, and processed through a column and frit, which was then placed in either a tube of culture medium containing antibiotics or a TV InPouch. RESULTS:The column and polyethylene frit system was found to increase the duration of viability for T. vaginalis from urine specimens at least 6-fold. CONCLUSION:This novel method, which uses a column and frit system to increase the duration of viability of the organism, has the potential to increase the sensitivity of diagnostic tests.

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 ∼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 genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

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.

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. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

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 (EC50) of 1.1–2.7μM (ritonavir concentration) against G. duodenalis and 6.8–8μM against metronidazole-sensitive and clinically metronidazole-resistant T. vaginalis. Minimal inhibitory concentrations were 2–2.5μM and 10–50μM for G. duodenalis and T. vaginalis, respectively. Within the range of human plasma concentrations for ritonavir, only G. duodenalis was inhibited. Lopinavir alone was less inhibitory than ritonavir but was associated with a blockage in cytokinesis of G. duodenalis trophozoites. Saquinavir was not effective. These findings are significant considering the association between human immunodeficiency virus and T. vaginalis, and between G. duodenalis and homosexual behaviour.

ABSTRACT 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.

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. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of ∼50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and α-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.

ABSTRACT 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.