Abstract
Dientamoeba fragilis is a commonly encountered trichomonad which has been implicated as a cause of gastrointestinal disease in humans. Despite the frequency of reports recording infections with this parasite, little research has been undertaken in terms of antimicrobial susceptibility. The aim of this study was to evaluate the susceptibility of D. fragilis to several commonly used antiparasitic agents: diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, secnidazole, ronidazole, tetracycline, and tinidazole. Antibiotic susceptibility testing was performed on four clinical strains of D. fragilis, designated A, E, M, and V, respectively. Molecular testing followed, and all strains were determined to be genotype 1. The activities of antiprotozoal compounds at concentrations ranging from 2 μg/ml to 500 μg/ml were determined via cell counts of D. fragilis trophozoites grown in dixenic culture. Minimum lethal concentrations (MLCs) were as follows: ornidazole, 8 to 16 μg/ml; ronidazole, 8 to 16 μg/ml; tinidazole, 31 μg/ml; metronidazole, 31 μg/ml; secnidazole, 31 to 63 μg/ml; nitazoxanide, 63 μg/ml; tetracycline, 250 μg/ml; furazolidone, 250 to 500 μg/ml; iodoquinol, 500 μg/ml; paromomycin, 500 μg/ml; and diloxanide furoate, >500 μg/ml. This is the first study to report the profiles of susceptibility to a wide range of commonly used treatments for clinical isolates of D. fragilis. Our study indicated 5-nitroimidazole derivatives to be the most active compounds in vitro against D. fragilis.
INTRODUCTION
Initially described by Jepps and Dobell (18), Dientamoeba fragilis is a protozoan parasite implicated as a cause of gastrointestinal diseases in both developed and developing regions of the world. Infection rates typically range from 0.5% to 16%, with higher rates seen in outbreaks or where personal hygiene is suboptimal (3, 17, 21, 26). The protozoan is recognized to cause chronic infections. In a prospective study, 6,750 patients were screened for D. fragilis, and 32% of patients presented with chronic symptoms (31), including abdominal pain, diarrhea, and nausea. Some authors have linked D. fragilis to irritable bowel syndrome (IBS)-like symptoms (4, 32).
Currently, a majority of evidence supports the pathogenic potential of D. fragilis (5). As such, it suggests the need for not only the correct diagnosis but also appropriate treatment (29, 30). The parasite responds to a number of antimicrobial compounds, with studies reporting the complete resolution and elimination of parasites following therapy with iodoquinol (8) metronidazole (24), paromomycin (13), and secnidazole (16). In an Australian study, complete resolution and eradication of the organism were observed for most patients following treatments with iodoquinol, paromomycin, or combination therapy, while treatment relapses/failures were recorded only with the use of metronidazole (29). It is of note that there are no current treatment guidelines for D. fragilis infections in place.
While D. fragilis can be readily cultured from clinical samples (4), long-term cultures are notoriously difficult to maintain (12), and this has hampered the in vitro study of this organism, including susceptibility testing. The objective of this study was to determine the in vitro susceptibility of a number of clinical isolates of D. fragilis to diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, ronidazole, secnidazole, tetracycline, and tinidazole. The results obtained will assist in the development of recommendations for the treatment of dientamoebiasis.
MATERIALS AND METHODS
Parasite culture.
Four strains of D. fragilis, A, E, M, and V, were isolated and propagated in vitro using Loeffler slopes, as described by Barratt et al. (4).
Antimicrobial agents/susceptibility testing.
Susceptibility testing was performed for the following agents: diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, ronidazole, secnidazole, tetracycline, and tinidazole. Metronidazole (Pfizer, NSW, Australia) in liquid form at 5 mg/ml was used as a stock solution and diluted with phosphate-buffered saline (PBS) buffer to cover a concentration range of 2 μg/ml to 500 μg/ml by doubling dilution. Tetracycline (Bioline, Alexandria, NSW, Australia) suspended in 90% ethanol at 12.5 mg/ml was diluted to 5 mg/ml and diluted in the same manner described for metronidazole. Ornidazole (Queensland Institute of Medical Research) in powder form was dissolved in 50% ethanol to 5 mg/ml and diluted thereafter. Paromomycin sulfate (Sigma-Aldrich, Sydney, NSW, Australia) and diloxanide furoate, furazolidone, iodoquinol, nitazoxanide, ronidazole, secnidazole, and tinidazole (all from West Lindfield Compounding Chemist, NSW, Australia) in powder form were suspended in 10% ethanol to make stock solutions at 5 mg/ml. Further dilutions were prepared by doubling dilution to cover from 2 μg/ml to 500 μg/ml. One milliliter of the respective dilutions was added to Loeffler slopes supplemented with rice starch and PBS overlay to contain a total liquid volume of 8 ml. A control consisting of 1 ml of 10% ethanol diluted into the same volume was performed in duplicate for all drugs in powder form to rule out any inhibitory effects on D. fragilis. As PBS buffer was used as a diluent for metronidazole and tetracycline, the same volume of PBS buffer was used as a control.
The cell concentration was determined using Kova slides viewed under a phase-contrast microscope at a magnification of ×400. As a decline in numbers of D. fragilis trophozoites occurs in negative controls after 92 h postexperiment, susceptibility testing with each compound was performed for only 4 days. Minimal lethal concentrations (MLCs) were determined to be the concentration of the drug at which no trophozoites were observed over the treatment period.
Characterization of bacterial flora and susceptibility testing from xenic culture.
The bacterial flora present in dixenic cultures was characterized and identified. Supernatants from D. fragilis cultures were inoculated onto the following media: Columbia horse blood agar, Brilliance UTI agar, MacConkey agar, Sabouraud's agar, and anaerobic medium (Thermofisher Scientific Australia Pty Ltd., Scoresby, Victoria, Australia). The aerobic medium plates were incubated at 35°C for 24 to 48 h under aerobic conditions, while the anaerobic medium plates were incubated for 48 to 72 h under anaerobic conditions using an Anoxomat Mark II system (Mart Microbiology) with the following gas composition: 0.16% O2, 5% H2, 10% CO2, and 85% N2. All bacterial isolates were identified to species level using routine bacteriological procedures, including traditional phenotypic testing, biochemical testing, and RapID API strips (Biomeriéux, Baulkham Hills, Australia), according to the manufacturer's instructions.
Bacterial 16S rRNA gene sequencing.
Cultured clinical isolates were used for DNA extraction using a QIAamp DNA minikit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. PCR amplifications were performed using pure-Taq Ready-To-Go (Amersham Biosciences, Rydalmere, Australia) PCR beads (each containing ∼1.5 units Taq DNA polymerase, 10 mM Tris-HCl at pH 9, 50 mM KCl, 1.5 mM MgCl2, 200 mM each deoxynucleoside triphosphate, and stabilizers, including bovine serum albumin), 1.0 μl of genomic DNA, and 0.5 μM universal primers, forward primer with the sequence of 5′-AGAGTTTGATCMTGGCTCAG, and reverse primer with the sequence of 5′-AAGGAGGTGWTCCARCC. The following thermocycling profile was used: 3 min denaturation at 94°C and 30 cycles of 1 min at 94°C, 1.5 min at 57°C, and 2 min at 72°C. Purification of PCR products was performed using QIAquick PCR purification kits (Qiagen), according to the manufacturer's instructions. Sequencing of purified bacterial DNA was then performed by the Australian Genome Research Facility (AGRF). The sequence data were then compared to other bacterial sequences using BLASTN (34) and the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).
After the completion of susceptibility testing, the bacterial flora was again fully characterized and checked for the presence of bacterial species unaffected by the treatments.
Genotyping of D. fragilis isolates.
Genotyping of the D. fragilis isolates were performed as previously described by Stark et al. (31).
RESULTS
D. fragilis genotyping.
All D. fragilis isolates were identified to be genotype 1 by 18S ribosomal DNA analysis (GenBank accession number AY730405).
Bacterial flora identified from four dixenic cultures.
The bacterial flora identified from the dixenic cultures is shown in Table 1.
Table 1.
Bacterial species isolated from cultures of D. fragilis
| Isolate | O2 requirement | Organism identified |
|---|---|---|
| Clinical isolate V | Strict aerobe | Pseudomonas aeruginosa |
| Facultative anaerobe | Escherichia coli | |
| Obligate anaerobes | Clostridium tertium | |
| Clostridium hathewayi | ||
| Eubacterium limosum | ||
| Collinsella aerofaciens | ||
| Peptostreptococcus stomatis | ||
| Peptostreptococcus micros | ||
| Veillonella spp. | ||
| Bacteroides ovatus | ||
| Bacteroides fragilis | ||
| Parabacteroides distasonis | ||
| Prevotella oralis | ||
| Arcobacter butzleri | ||
| Eggerthella lenta | ||
| Clinical isolate E | Strict aerobe | P. aeruginosa |
| Facultative anaerobe | E. coli | |
| Strict anaerobes | P. micros | |
| P. stomatis | ||
| Veillonella spp. | ||
| E. limosum | ||
| Anaerococcus prevotii | ||
| Bacteroides capillosus | ||
| B. fragilis | ||
| C. aerofaciens | ||
| P. oralis | ||
| Clinical isolate M | Strict aerobe | P. aeruginosa |
| Facultative anaerobe | E. coli | |
| Obligate anaerobes | C. tertium | |
| P. micros | ||
| P. stomatis | ||
| B. fragilis | ||
| P. oralis | ||
| Clinical isolate A | Strict aerobe | P. aeruginosa |
| Facultative anaerobe | E. coli | |
| Obligate anaerobes | C. tertium | |
| P. micros | ||
| P. stomatis | ||
| Veillonella spp. | ||
| B. fragilis | ||
| P. oralis |
MLCs.
The results are shown in Tables 2 to 9. Briefly, mean MLC values for the compounds were as follows: ornidazole, 8 to 16 μg/ml; ronidazole, 8 to 16 μg/ml; tinidazole, 31 μg/ml; metronidazole, 31 μg/ml; secnidazole, 31 to 63 μg/ml; nitazoxanide, 63 μg/ml; tetracycline, 250 μg/ml; furazolidone, 250 to 500 μg/ml; iodoquinol, 500 μg/ml; paromomycin, 500 μg/ml; and diloxanide furoate, >500 μg/ml (as minimal or no effects were found with diloxanide furoate, iodoquinol, and paromomycin, tables for these compounds have been excluded). Minor differences in the MLCs between the clinical isolates were observed for a number of drugs, including furazolidone, nitazoxanide, ornidazole, ronidazole, and secnidazole, with isolate V showing slightly higher MLCs than those of the other isolates in general (Tables 2 and 4 to 7).
Table 2.
In vitro cell growth of the four isolates of D. fragilis during treatment with furazolidone
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 0.79 (2.65) | 1.5 (4.07) | 3.7 (10.43) | 0.014 (0.18) | 1 (0) | 1.4 (0) | 3.7 (9.02) | 2.6 (38.54) | 0.67 (1.59) | 1 (0) | 1.7 (2.3) | 4.4 (2.3) | 6.6 (4.95) | 3.2 (9.37) | 1 (0) | 1.3 (1.06) | 2 (2.83) | 6.7 (5.13) | 2.2 (1.94) |
| 2 | 1 (0) | 0.99 (1.59) | 1.9 (1.59) | 5 (3.01) | 0.6 (5.3) | 1 (0) | 0.72 (3.01) | 3.5 (6.01) | 2 (16.09) | 0.59 (7.07) | 1 (0) | 1.9 (1.06) | 4.6 (8.13) | 7 (4.77) | 2.8 (3.36) | 1 (0) | 1.3 (1.94) | 2 (1.06) | 6.3 (7.07) | 1.8 (3.89) |
| 4 | 1 (0) | 1.2 (2.3) | 1.4 (3.89) | 4 (0.88) | 0.12 (1.06) | 1 (0) | 1 (4.24) | 5.2 (4.07) | 4.3 (26.16) | 0.39 (0) | 1 (0) | 2 (0.88) | 4.3 (3.01) | 6.5 (3.89) | 2 (2.65) | 1 (0) | 1.3 (0.71) | 1.9 (2.12) | 6.2 (2.83) | 1.6 (3.18) |
| 8 | 1 (0) | 0.5 (0.53) | 0.68 (1.59) | 0.98 (1.77) | 0.058 (0.71) | 1 (0) | 0.68 (1.59) | 1.9 (7.25) | 1.1 (6.19) | 0.23 (0.35) | 1 (0) | 1.6 (1.59) | 3.9 (2.65) | 5.9 (4.24) | 2.7 (5.13) | 1 (0) | 1.5 (1.41) | 2.1 (1.41) | 5.6 (7.78) | 1.7 (0.88) |
| 16 | 1 (0) | 0.53 (1.24) | 0.65 (1.94) | 2.3 (3.01) | 0.029 (0.35) | 1 (0) | 0.75 (7.07) | 1.3 (5.66) | 2.3 (15.2) | 0.78 (8.84) | 1 (0) | 1.5 (2.83) | 2.8 (7.25) | 4.3 (6.36) | 1.3 (4.77) | 1 (0) | 1.2 (1.41) | 2 (3.36) | 3.3 (1.59) | 1.5 (1.41) |
| 31 | 1 (0) | 0.32 (0.71) | 0.2 (1.41) | 1.3 (5.48) | 0.56 (5.48) | 1 (0) | 0.62 (3.01) | 1.2 (13.26) | 1.9 (12.9) | 0.43 (1.06) | 1 (0) | 0.41 (0.53) | 2.1 (2.83) | 3 (1.59) | 0.93 (1.41) | 1 (0) | 0.63 (2.12) | 1.8 (1.41) | 2.7 (4.95) | 1.1 (2.12) |
| 63 | 1 (0) | 0.35 (0) | 0.1 (0.18) | 0.29 (1.06) | 0.56 (1.59) | 1 (0) | 0.27 (0.35) | 0.71 (0.71) | 2.7 (9.02) | 0.74 (9.19) | 1 (0) | 0.11 (0) | 0.18 (0.35) | 0.04 (0.35) | 0 (0) | 1 (0) | 0.06 (0.18) | 1.1 (2.12) | 0.2 (0.71) | 0.06 (0.18) |
| 125 | 1 (0) | 0.17 (1.1) | 0.18 (0.71) | 0.13 (0.2) | 0.23 (1.1) | 1 (0) | 0.2 (0.18) | 0.1 (0.71) | 0.42 (1.24) | 0.62 (1.77) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviations are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where 10% ethanol, used for dilution, was added into the medium.
Table 9.
In vitro cell growth of the four isolates of D. fragilis during treatment with tinidazole
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 3.25 (0.64) | 6.4 (1.84) | 1.8 (0.07) | 0 (0) | 1 (0) | 5.1 (0.66) | 3.2 (0.59) | 0.36 (0.52) | 0 (0) | 1 (0) | 3.8 (1.06) | 8 (4.95) | 20.5 (9.19) | 2.3 (1.41) | 1 (0) | 2.5 (0.7) | 5.5 (6.19) | 3.3 (0.7) | 2.1 (1.77) |
| 2 | 1 (0) | 1.95 (0.64) | 7.3 (0.99) | 1.4 (0.14) | 0 (0) | 1 (0) | 0.8 (0.29) | 2 (1.03) | 0.5 (0.15) | 0 (0) | 1 (0) | 2.9 (1.94) | 7.7 (1.59) | 17.8 (2.65) | 2 (1.06) | 1 (0) | 2.4 (3.71) | 4.9 (8.49) | 3.3 (1.94) | 1.9 (4.24) |
| 4 | 1 (0) | 1.6 (0.14) | 5.7 (0.49) | 1.3 (0.28) | 0 (0) | 1 (0) | 1.1 (0) | 1 (0.15) | 0 (0) | 0 (0) | 1 (0) | 2.6 (1.41) | 7.1 (5.48) | 16.8 (13.79) | 2.2 (2.12) | 1 (0) | 1.4 (0.35) | 3 (2.12) | 3.1 (4.6) | 2.6 (1.24) |
| 8 | 1 (0) | 2.3 (0.85) | 4.5 (0.64) | 2.8 (1.06) | 0 (0) | 1 (0) | 1.1 (0.66) | 1.1 (1.33) | 0 (0) | 0 (0) | 1 (0) | 2 (1.06) | 6.1 (2.65) | 9.4 (6.36) | 1.2 (1.41) | 1 (0) | 1.4 (1.77) | 2.7 (4.24) | 3.2 (5.48) | 2.7 (2.83) |
| 16 | 1 (0) | 0.95 (0.35) | 3.55 (1.2) | 1.2 (0.57) | 0 (0) | 1 (0) | 1.8 (1.55) | 1.5 (0.44) | 0 (0) | 0 (0) | 1 (0) | 0.7 (0.18) | 2.4 (2.65) | 4.7 (3.54) | 1 (0) | 1 (0) | 0.3 (0.18) | 1.2 (2.47) | 2.3 (3.18) | 2.6 (9.02) |
| 31 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where phosphate-buffered saline, used for dilution, was added into the medium.
Table 4.
In vitro cell growth of the four isolates of D. fragilis during treatment with nitazoxanide
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 0.93 (0.88) | 4.3 (11.31) | 1.5 (1.41) | 0.16 (0) | 1 (0) | 1 (1.59) | 2.1 (15.2) | 3.1 (36.06) | 0.55 (3.89) | 1 (0) | 1.8 (1.06) | 4.6 (4.95) | 6.3 (9.19) | 2.6 (1.41) | 1 (0) | 1.9 (1.24) | 6.1 (5.3) | 8.5 (6.54) | 3.8 (2.12) |
| 2 | 1 (0) | 0.87 (0.18) | 3 (10.25) | 0.55 (3.01) | 0.22 (1.06) | 1 (0) | 0.47 (1.77) | 2.1 (13.97) | 1.5 (18.21) | 0.1 (3.01) | 1 (0) | 2.1 (1.24) | 5.5 (1.77) | 6.5 (4.42) | 2.4 (2.12) | 1 (0) | 1.3 (1.77) | 5 (9.19) | 8.1 (5.3) | 3 (2.12) |
| 4 | 1 (0) | 0.73 (1.06) | 2.6 (4.6) | 1.1 (8.31) | 0.21 (0.18) | 1 (0) | 0.63 (4.42) | 1.9 (7.25) | 1.9 (7.6) | 0.07 (0.35) | 1 (0) | 1.9 (1.41) | 5.3 (2.3) | 6.7 (2.12) | 2.2 (3.89) | 1 (0) | 1.2 (0.35) | 2.8 (3.36) | 7.4 (7.25) | 3.2 (2.65) |
| 8 | 1 (0) | 0.46 (0.18) | 0.62 (0.46) | 0.58 (1.59) | 0.08 (0.18) | 1 (0) | 0.18 (1.77) | 0.89 (2.47) | 1.3 (3.89) | 0.12 (0.88) | 1 (0) | 1.4 (2.83) | 2.9 (5.66) | 4.7 (8.31) | 2 (2.83) | 1 (0) | 1.1 (3.54) | 1.8 (1.41) | 5.6 (5.13) | 2.4 (3.89) |
| 16 | 1 (0) | 0.063 (0) | 0.047 (0.18) | 0.17 (0.53) | 0.063 (0.35) | 1 (0) | 0.065 (0.53) | 0.095 (0.35) | 1.1 (1.59) | 1.2 (0) | 1 (0) | 0.41 (1.56) | 1.8 (0.88) | 0.38 (0.21) | 0 (0) | 1 (0) | 0.21 (1.59) | 1.1 (2.69) | 5.5 (9.05) | 1.9 (0.14) |
| 31 | 1 (0) | 0.13 (0) | 0.047 (0.53) | 0.22 (2.12) | 0.032 (0.35) | 1 (0) | 0.017 (0.53) | 0.017 (0.53) | 0.25 (0.18) | 0.31 (9.19) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where 10% ethanol was added into the medium.
Table 7.
In vitro cell growth of the four isolates of D. fragilis during treatment with secnidazole
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 1.1 (1.77) | 4 (3.89) | 5.8 (16.79) | 3.5 (0.71) | 1 (0) | 0.82 (11.84) | 3.8 (52.5) | 0.34 (1.77) | 0.52(5.48) | 1 (0) | 2.2 (2.83) | 5 (6.19) | 6.1 (7.42) | 2.2 (0.53) | 1 (0) | 2.1 (1.23) | 6.2 (3.89) | 8.3 (3.36) | 3 (1.06) |
| 2 | 1 (0) | 1.4 (3.01) | 5.7 (0.88) | 4.7 (7.78) | 4.6 (10.08) | 1 (0) | 0.9 (3.71) | 1.4 (0.53) | 0.01 (0) | 0.15 (1.06) | 1 (0) | 1.8 (1.24) | 4.6 (8.49) | 6 (3.89) | 2.6 (3.36) | 1 (0) | 1.5 (1.41) | 5.8 (7.78) | 7.8 (3.54) | 3.2 (11.31) |
| 4 | 1 (0) | 1.3 (4.07) | 5.7 (4.07) | 5.1 (1.24) | 2.3 (4.07) | 1 (0) | 0.51 (0.71) | 1.5 (17.5) | 0.07 (1.41) | 0.35 (1.41) | 1 (0) | 1.5 (2.83) | 4.3 (3.18) | 5.9 (2.65) | 2.1 (2.83) | 1 (0) | 1.1 (0.71) | 4 (4.42) | 6.9 (6.36) | 2.8 (2.3) |
| 8 | 1 (0) | 0.84 (0.18) | 5.2 (5.83) | 5 (0.88) | 2.2 (4.42) | 1 (0) | 0.14 (1.94) | 0.5 (3.36) | 0 (14.85) | 0.78 (7.95) | 1 (0) | 0.54 (2.47) | 2.2 (4.07) | 3 (1.94) | 1.3 (4.95) | 1 (0) | 0.57 (1.59) | 2.3 (2.83) | 4.1 (5.3) | 2 (3.54) |
| 16 | 1 (0) | 0.7 (2.3) | 7.4 (6.01) | 6.1 (0.88) | 3.2 (17.15) | 1 (0) | 0.075 (1.06) | 0.14 (1.24) | 0.085 (3.18) | 0.97 (29.79) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 31 | 1 (0) | 0.45 (2.47) | 5.9 (1.06) | 7.1 (10.43) | 3.1 (4.42) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where 10% ethanol, used for dilution, was added into the medium.
Table 5.
In vitro cell growth of the four isolates of D. fragilis during treatment with ornidazole
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 1.6 (0.53) | 6.1 (5.13) | 0.28 (0.18) | 0 (0) | 1 (0) | 1.3 (0.53) | 11 (14.5) | 4.6 (1.24) | 0.58 (0.35) | 1 (0) | 3.8 (1.06) | 8 (4.95) | 20.5 (9.19) | 2.3 (1.41) | 1 (0) | 2.5 (0.7) | 5.5 (6.19) | 3.3 (0.7) | 2.1 (1.77) |
| 2 | 1 (0) | 1.1 (1.94) | 4.6 (7.25) | 0 (0) | 0 (0) | 1 (0) | 1.1 (1.06) | 11 (6.36) | 6.7 (5.83) | 1.3 (0.71) | 1 (0) | 3.5 (0.53) | 7.9 (10.08) | 18.5 (3.89) | 2 (0.53) | 1 (0) | 1.5 (1.59) | 4.2 (1.06) | 2.8 (2.3) | 1.9 (2.47) |
| 4 | 1 (0) | 0.36 (0.88) | 3.7 (2.65) | 0.24 (0.35) | 0 (0) | 1 (0) | 0.33 (0.71) | 10 (0.88) | 5.9 (2.47) | 0.62 (0.88) | 1 (0) | 2.3 (1.41) | 6.7 (9.55) | 14.9 (5.83) | 2.1 (0.35) | 1 (0) | 0.4 (0.35) | 1.1 (0.53) | 2.9 (1.41) | 1.8 (3.01) |
| 8 | 1 (0) | 0 (0) | 2.5 (3.89) | 2.9 (9.37) | 0.2 (0.53) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 16 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 31 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where 10% ethanol was added into the medium.
Table 6.
In vitro cell growth of the four isolates of D. fragilis during treatment with ronidazole
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 1.2 (0.2) | 3.2 (0.22) | 0.84 (0.18) | 0 (0) | 1 (0) | 1.6 (0.23) | 3.1 (0.08) | 1.1 (0.18) | 0 (0) | 1 (0) | 3.8 (1.06) | 8 (4.95) | 20.5 (9.19) | 2.3 (1.41) | 1 (0) | 2.5 (0.7) | 5.5 (6.19) | 3.3 (0.7) | 2.1 (1.77) |
| 2 | 1 (0) | 1 (0.14) | 2.2 (0.77) | 1.1 (0.2) | 0 (0) | 1 (0) | 1.5 (0.51) | 2.7 (0.61) | 0.96 (0.18) | 0 (0) | 1 (0) | 2.7 (3.01) | 7.3 (7.42) | 17 (14.14) | 2.6 (2.12) | 1 (0) | 2.3 (2.65) | 5.3 (10.08) | 2.8 (3.01) | 1.9 (1.94) |
| 4 | 1 (0) | 0.3 (0.07) | 1.3 (0.28) | 0.7 (0.37) | 0 (0) | 1 (0) | 1 (0) | 0.9 (0.02) | 0.2 (0.05) | 0 (0) | 1 (0) | 0.7 (0.88) | 6.7 (4.6) | 10.5 (3.01) | 1.7 (1.24) | 1 (0) | 1.2 (1.94) | 3.9 (12.37) | 2.7 (3.36) | 1.8 (1.77) |
| 8 | 1 (0) | 0.1 (0.06) | 0.2 (0.02) | 0.4 (0.13) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0.8 (1.06) | 3.3 (4.07) | 3 (9.37) | 1.5 (1.59) |
| 16 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 31 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where phosphate-buffered saline, used for dilution, was added into the medium.
Table 8.
In vitro cell growth of the four isolates of D. fragilis during treatment with tetracycline
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 2.3 (0.53) | 15 (0.71) | 22 (3.71) | 12 (13.61) | 1 (0) | 2.3 (5.48) | 8.8 (3.54) | 2.3 (7.25) | 0.07 (1.24) | 1 (0) | 1.8 (1.06) | 4.6 (4.95) | 6.3 (9.19) | 2.6 (1.41) | 1 (0) | 1.9 () | 6.1 (5.3) | 8.5 (6.54) | 3.8 (2.12) |
| 2 | 1 (0) | 1.4 (3.36) | 11 (1.59) | 22 (1.59) | 24 (2.83) | 1 (0) | 0.95 (0.71) | 7.4 (9.55) | 2.4 (12.2) | 0.1 (1.06) | 1 (0) | 1.4 (3.36) | 11 (1.59) | 22 (1.59) | 24 (2.83) | 1 (0) | 0.95 (0.71) | 7.4 (9.55) | 2.4 (12.2) | 0.1 (1.06) |
| 4 | 1 (0) | 1.5 (0.35) | 5.4 (4.95) | 11 (10.78) | 12 (5.48) | 1 (0) | 1.1 (0.18) | 6.9 (9.54) | 0.89 (1.94) | 0.14 (0.18) | 1 (0) | 1.5 (0.35) | 5.4 (4.95) | 11 (10.78) | 12 (5.48) | 1 (0) | 1.1 (0.18) | 6.9 (9.54) | 0.89 (1.94) | 0.14 (0.18) |
| 8 | 1 (0) | 0.66 (0) | 3.8 (2.3) | 4.4 (6.19) | 8.2 (9.37) | 1 (0) | 0.78 (0.18) | 2.5 (4.95) | 2.9 (16.79) | 0.09 (1.59) | 1 (0) | 0.66 (0) | 3.8 (2.3) | 4.4 (6.19) | 8.2 (9.37) | 1 (0) | 0.78 (0.18) | 2.5 (4.95) | 2.9 (16.79) | 0.09 (1.59) |
| 16 | 1 (0) | 0.73 (1.41) | 2.5 (7.25) | 6.2 (7.78) | 9.1 (11.67) | 1 (0) | 0.96 (0.88) | 2.7 (11.14) | 2.7 (21.57) | 0.21 (0.88) | 1 (0) | 0.73 (1.41) | 2.5 (7.25) | 6.2 (7.78) | 9.1 (11.67) | 1 (0) | 0.96 (0.88) | 2.7 (11.14) | 2.7 (21.57) | 0.21 (0.88) |
| 31 | 1 (0) | 0.73 (1.06) | 2.1 (5.83) | 4.5 (3.54) | 4.4 (2.12) | 1 (0) | 0.99 (1.06) | 0.7 (0.53) | 2.1 (10.08) | 0.04 (0.35) | 1 (0) | 0.73 (1.06) | 2.1 (5.83) | 4.5 (3.54) | 4.4 (2.12) | 1 (0) | 0.99 (1.06) | 0.7 (0.53) | 2.1 (10.08) | 0.04 (0.35) |
| 63 | 1 (0) | 0.36 (0.88) | 0.6 (1.06) | 1.1 (2.47) | 1.3 (5.3) | 1 (0) | 1 (2.83) | 1.4 (5.83) | 0.98 (0.18) | 0.57 (9.02) | 1 (0) | 0.34 (0.18) | 0.7 (0.18) | 1.1 (3.18) | 0.16 (1.23) | 1 (0) | 0.32 (0.71) | 0.74 (1.2) | 0.12 (0.02) | 0 (0) |
| 125 | 1 (0) | 0.13 (0.88) | 0.07 (0.18) | 0.03 (0) | 0.13 (0.18) | 1 (0) | 0.99 (3.18) | 0.62 (0.71) | 1 (5.66) | 0.8 (9.37) | 1 (0) | 0.18 (0.35) | 0.06 (0) | 0 (0) | 0 (0) | 1 (0) | 0.14 (1.06) | 0.12 (0.18) | 0.16 (0.88) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where phosphate-buffered saline, used for dilution, was added into the medium. Concentrations below the MLC have been omitted.
Bacterial flora identified posttreatment.
The bacterial flora identified posttreatment are presented in Table 1.
(i) Ronidazole.
At the concentrations above the MLC, ronidazole treatment led to the removal of the majority of the bacterial flora, while it left Escherichia coli, Pseudomonas aeruginosa, Clostridium spp., and Bacteroides fragilis unaffected.
(ii) Tinidazole.
Tinidazole treatment led to the removal of Eubacterium limosum, Collinsella aerofaciens, Veillonella spp., and Parabacteroides distasonis, while it left E. coli, P. aeruginosa, Clostridium spp., Bacteroides spp., Peptostreptococcus spp., and Eggerthella lenta unaffected.
(iii) Ornidazole.
At ornidazole concentrations above the MLC, E. coli, P. aeruginosa, and B. fragilis were unaffected by the treatment, while ornidazole led to the removal of Clostridium spp., E. limosum, C. aerofaciens, Veillonella spp., Bacteroides ovatus, Peptostreptococcus spp., P. distasonis, Prevotella oralis, Arcobacter butzleri, and E. lenta.
(iv) Metronidazole/secnidazole.
The following bacterial species were identified after treatment with either metronidazole or secnidazole: E. coli, P. aeruginosa, Bacteroides spp., Peptostreptococcus spp., P. oralis, and C. aerofaciens. Treatment at high concentrations resulted in the removal of the following bacterial species: Clostridium spp., Veillonella spp., P. distasonis, E. limosum, Anaerococcus prevotii, P. distasonis, A. butzleri, and E. lenta.
(v) Iodoquinol.
The following bacteria were isolated after treatment with iodoquinol: Clostridium spp., Bacteroides spp., C. aerofaciens, Peptostreptococcus spp., P. oralis, and Veillonella spp., as well as both E. coli and P. aeruginosa. A total of five species of bacteria were removed following iodoquinol treatment: E. limosum, P. distasonis, A. prevotii, A. butzleri, and E. lenta.
(vi) Paromomycin.
B. fragilis, B. ovatus, C. aerofaciens, E. coli, and P. aeruginosa were present in cultures for four isolates, while E. limosum, Peptostreptococcus spp., A. prevotii, Veillonella spp., Bacteroides merdae/B. caccae, P. distasonis, P. oralis, A. butzleri, and E. lenta were removed by treatment with paromomycin.
(vii) Tetracycline.
Following tetracycline treatment, the following bacterial species were present: E. coli, P. aeruginosa, Bacteroides spp., Clostridium spp., A. prevotii, Peptostreptococcus spp., and Veillonella spp. Treatment removed E. limosum, P. distasonis, P. oralis, A. butzleri, and E. lenta.
(viii) Furazolidone.
The bacterial species E. coli, P. aeruginosa, Clostridium spp., Bacteroides spp., Peptostreptococcus spp., P. oralis, and Veillonella spp. were present in cultures treated with furazolidone, and furazolidone treatment removed E. limosum, C. aerofaciens, P. distasonis, A. butzleri, and E. lenta for four isolates.
(ix) Nitazoxanide.
Following treatment with nitazoxanide, E. coli, P. aeruginosa, Bacteroides spp., C. aerofaciens, E. limosum, A. prevotii, Peptostreptococcus spp., and Veillonella spp. were present, and treatment removed E. limosum, P. distasonis, P. oralis, A. butzleri, and E. lenta.
(x) Diloxanide furoate.
Treatment with diloxanide furoate had no effect on the bacterial flora, as all bacterial species were identified in all isolates.
DISCUSSION
D. fragilis has been a frequently encountered pathogenic protozoon; however, very little research on the susceptibility of this organism has been conducted. This study suggests that the 5-nitromidazole derivatives (metronidazole, ornidazole, ronidazole, and tinidazole) are the most active components against D. fragilis trophozoites, with lethal concentrations ranging from 8 to 63 μg/ml.
Metronidazole, a common oral synthetic antimicrobial compound, was found to have an MLC of 31 μg/ml (Table 3), and this is in correlation with the minimal amoebicidal concentration of 32 μg/ml obtained by Chan et al. (10). The efficacy of metronidazole treatment for D. fragilis ranges from 70 to 80% (24).
Table 3.
In vitro cell growth of the four isolates of D. fragilis during treatment with metronidazole
| Drug concn (μg/ml) | Viable count (104 cells/ml) over 4 days (SD)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolate V |
Isolate E |
Isolate A |
Isolate M |
|||||||||||||||||
| Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | Day 0 | 1 day | 2 days | 3 days | 4 days | |
| 0b | 1 (0) | 1.3 (0.53) | 6.7 (4.42) | 12 (6.72) | 7.2 (4.07) | 1 (0) | 1.9 (3.01) | 4.7 (16.79) | 4.9 (26.52) | 0.75 (1.06) | 1 (0) | 4.3 (3.18) | 9.3 (1.94) | 9.3 (1.15) | 3.7 (2.12) | 1 (0) | 6 (2.47) | 30.3 (5.48) | 40.2 (12.73) | 17.9 (6.54) |
| 2 | 1 (0) | 1.1 (1.41) | 1.4 (3.01) | 5.9 (1.94) | 7.3 (4.6) | 1 (0) | 1.5 (3.54) | 3.8 (3.89) | 4.3 (22.63) | 0.42 (0.35) | 1 (0) | 3.1 (1.24) | 9.3 (3.18) | 10 (3.09) | 4.8 (4.07) | 1 (0) | 4.6 (2.47) | 46.6 (2.12) | 42.2 (1.77) | 15.9 (6.54) |
| 4 | 1 (0) | 0.68 (1.59) | 4 (5.13) | 8.5 (2.65) | 11 (0.35) | 1 (0) | 2.3 (7.78) | 4.5 (17.85) | 3.9 (17.85) | 0.57 (0) | 1 (0) | 3.2 (1.41) | 8.1 (5.3) | 12 (0.62) | 5.5 (1.77) | 1 (0) | 1.2 (0) | 26.9 (7.25) | 43.7 (3.01) | 12.9 (1.59) |
| 8 | 1 (0) | 0.18 (0.53) | 0.86 (1.06) | 2.6 (3.01) | 10 (3.36) | 1 (0) | 1.7 (2.65) | 1.3 (3.71) | 2.4 (3.01) | 1.1 (1.77) | 1 (0) | 1.2 (0.53) | 5.9 (3.71) | 9.9 (4.24) | 4.5 (2.92) | 1 (0) | 0.4 (0) | 15.5 (9.72) | 57.7 (16.44) | 20 (1.06) |
| 16 | 1 (0) | 0 (0) | 0.03 (0.18) | 0.25 (0.71) | 4.1 (2.3) | 1 (0) | 1.9 (6.89) | 1.3 (0.71) | 2.4 (1.59) | 3.3 (11.84) | 1 (0) | 1.25 (0.18) | 4.2 (1.77) | 5.4 (3.09) | 2.3 (0.35) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 31 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 63 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 125 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 250 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| 500 | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
The values for standard deviation are given in two decimal places. Two replicates were used for each concentration tested per isolate. All values have been divided by original values of day 0 (x) to produce the same values (x = 1) for initial concentration.
A drug concentration of 0 μg/ml refers to negative control, where phosphate-buffered saline, used for dilution, was added into the medium.
Secnidazole, the first of 5-nitroimidazole derivatives known to offer a 3-day antiprotozoal activity from a single dosage due to its longer half-life, was shown to be effective in the treatment of dientamoebiasis by Girginkardesler et al. (16). The data presented here support such findings, as the minimum amoebicidal concentration was found to be 63 μg/ml (Table 7).
Tinidazole is another known antiprotozoal compound effective for a number of protozoal infections (14). This study suggests the use of tinidazole to be effective for inhibition of D. fragilis trophozoites in vitro, with MLCs being as low as 31 μg/ml.
Ronidazole has been show to be approximately five times more active than metronidazole in inhibition of Giardia intestinalis (7). Another study using a closely related enteric pathogen of felines, Tritrichomonas foetus, revealed that ronidazole had a higher lethal activity against T. foetus trophozoites than metronidazole (20). The data presented here agree with the high lethal activity of ronidazole, as the MLC was determined to be as low as 8 μg/ml.
Ornidazole was shown to be highly active against Dientamoeba, with MLCs from this study found to be as low as 8 μg/ml. Previously, a comparison of metronidazole and ornidazole was undertaken by Kurt et al. (23). Significant differences were found, with ornidazole showing higher parasite clearance and clinical improvement along with fewer side effects.
Furazolidone is a synthetic nitrofuran derivative used for the treatment of a broad range of bacterial and protozoal infections (26a). It is evident from our data that the treatment of D. fragilis with furazolidone displays only minimal inhibitory effects at best, and MLCs were as high as 250 μg/ml for clinical isolates. No studies to date have tested furazolidone for the purpose of treating D. fragilis infections.
Nitazoxanide was first introduced in 1984 as a cestocidal drug (27), and subsequent studies have demonstrated that it has inhibitory effects against Trichomonas vaginalis (2, 9) and other diarrhea-causing protozoa. Our study demonstrated that the use of nitazoxanide at a concentration of 63 μg/ml is lethal for D. fragilis and thus may be a possible treatment option; however, no clinical studies to date have reported on the use of nitazoxanide for the treatment of dientamoebiasis.
Tetracycline is a broad-spectrum antimicrobial which has been recommended as a possible treatment option for D. fragilis (26). In agreement with the findings of Chan et al. (10), the data presented here suggest that treatment of dientamoebiasis with tetracycline should be reconsidered, as relatively high MLC values of 250 μg/ml were obtained.
Iodoquinol is a halogenated hydroxyquinoline which has been used extensively to treat dientamoebiasis (22). This study suggests that it may be ineffective for the treatment of D. fragilis, as the MLC for all isolates was 500 μg/ml.
Paromomycin is a poorly absorbed aminoglycoside antibiotic that is also currently recommended by the Centers for Disease Control and Prevention (CDC) for the treatment of D. fragilis infections (1). It was shown here that the use of paromomycin as a therapeutic option may be questionable, as the MLCs were 500 μg/ml. The data presented here and in the previous study by Chan et al. (10), who reported a minimal amoebicidal concentration of 16 μg/ml for paromomycin, differ significantly.
Diloxanide furoate is a luminal amoebicide, and it would appear that diloxanide furoate has minimal to no inhibitory effects on D. fragilis infections in vitro, as the MLC for diloxanide furoate was found to be greater than 500 μg/ml. However, it should be noted that the hydrolysis reaction involved in the activation of diloxanide furoate appears to be temperature and [OH−] dependent, so it is possible that in vivo results may differ (15). No studies to date have tested diloxanide furoate for treatment of dientamoebiasis.
There is only one previously reported experiment of in vitro susceptibility testing of D. fragilis, and that study used an ATCC strain (ATCC 30948). In this study, the minimal amoebicidal concentrations for iodoquinol, paromomycin, tetracycline, and metronidazole were determined to be 128, 16, 32, and 32 μg/ml, respectively (10), in contrast to our data, which were 500, 500, 250, and 31 μg/ml, respectively. The previous study used the D. fragilis strain ATCC 30948 (genotype 2), which has not been found to be associated with gastrointestinal diseases to date and is rarely encountered in human samples (11, 25, 28). While the majority of studies have shown genotype 1 in nearly all cases (6, 31, 33, 36), testing of this genotype may be more appropriate in terms of clinical significance and may be a possible reason for the different MLC values between the two studies.
The true complexity of the bacterial flora contained within these clinical isolates of D. fragilis is summarized in Table 1. Elimination of certain species and/or the majority of the bacterial flora present in the cultures may indirectly result in detrimental effects to the D. fragilis trophozoites, as the parasite has long been known to utilize them as a food source. However, it was observed that the treatments did not affect the majority of the bacterial flora present. This supports the notion that elimination of D. fragilis in these is the result of the antiparasitic effects of the drug and not due to the antibacterial effects on the bacterial populations.
The results of this study show that the inhibitory effects of a number of antimicrobials currently used as recommended treatments, including iodoquinol, paromomycin, and tetracycline, make them inappropriate for treatment of D. fragilis infections. The use of newer antiprotozoal compounds with far fewer known side effects or combination therapies derived from current treatment options for D. fragilis infections appears to be a viable option for consideration. In summary, the data presented here indicated that the use of 5-nitroimidazoles is the most effective option for treating D. fragilis infections, with ornidazole and ronidazole being the most active in vitro compounds.
ACKNOWLEDGMENTS
We thank West Lindfield Compounding Chemist for providing diloxanide furoate, furazolidone, iodoquinol, nitazoxanide, and secnidazole and J. Upcroft of the Queensland Institute of Medical Research.
Footnotes
Published ahead of print 24 October 2011
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