a Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612; USA; b Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA; c World Health Organization, Geneva, Switzerland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, there has been no consensus on the optimum treatment regimen for H. pylori infection. Only limited combinations of antimicrobials have been found to be successful for its eradication and many of these regimens contain metronidazole. Paromomycin, an oral non-absorbable aminoglycoside antibiotic, has activity against various gastrointestinal pathogens, including Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. Attainment of high intra-luminal concentrations in the gastrointestinal tract suggests it is a sensible alternative to study for activity against H. pylori.
The in-vitro activities of paromomycin alone and in combination with metronidazole or metronidazole plus hydroxymetronidazole (2:1 ratio) were examined to determine whether activity is enhanced when these antimicrobials are used concurrently.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antimicrobials were obtained as powders and prepared as solutions on the day of use according to NCCLS guidelines or manufacturers' recommendations. Paromomycin (aminosidine sulphate) was provided by Farmitalia Carlo Erba (Milan, Italy), metronidazole by Searle (Skokie, IL, USA) and hydroxymetronidazole by Rhône-Poulenc Rorer (Alfortville, France). Sterile water was used as a solvent and diluent for paromomycin powder. Metronidazole and hydroxymetronidazole were dissolved in dimethylsulphoxide (DMSO) and diluted in sterile water. Metronidazole and hydroxymetronidazole were prepared in serial dilutions as separate solutions and then combined in a 2:1 concentration ratio. Molten medium (50°C) was added to the antimicrobial solution, the tubes were inverted three times and the contents poured into sterile Petri plates.
The medium used for MIC determinations and chequerboard titrations was Mueller-Hinton (Difco, Detroit, MI, USA) supplemented with 10% sterile defibrinated horse blood (Remel) at a neutral pH. Control and antimicrobial-containing plates were prepared 1 day before testing and refrigerated.
The inocula were prepared by suspending organisms in sterile tryptic soy broth (Remel) and adjusting the turbidity to that of a 2.0 McFarland standard (10 6 cfu/mL by prior colony count of a representative strain). The organisms were inoculated on to the agar plates containing antimicrobial agents with a Steers replicator (Craft Machine Inc., Chester, PA, USA), delivering 8 µL/spot to give a final inoculum of 10 4 cfu/spot. Plates were incubated at 37°C in 10% CO 2 and examined for growth after 5 days.
The MIC was defined as the lowest concentration of antibiotic at which no visible growth or only
a faint haze occurred.
1 MICs were determined for paromomycin, metronidazole
and metronidazole/hydroxymetronidazole. All procedures were performed in duplicate. Isolates
were determined to be resistant to metronidazole at an MIC 16 mg/L.
In order to quantitate the degree of synergy, the fractional inhibitory concentration (FIC) indices
for both combinations were calculated according to Eliopoulos et al.
2 The FIC index was interpreted as follows: 0.5,
synergy; 0.5-0.75, partial synergy; 0.75-1.0, additive effects; 1.0-4.0,
indifference; and >4.0,
antagonism.
3
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of synergy testing of paromomycin and metronidazole or metronidazole/hydroxymetronidazole for H. pylori are shown in Tables I and II.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a single published report, the MIC of paromomycin against H. pylori was reported to be in the range 0.5-4.0 mg/L. 4 Oral paromomycin is not significantly absorbed and should achieve high concentrations within the gastrointestinal lumen, so exceeding the MIC for the organism at the site of infection. Its antibacterial activity in vitro has yet to be evaluated.
Metronidazole plays a key role in many available treatment regimens. The MIC of metronidazole against H. pylori has been reported to be in the range 0.25-64 mg/L. 4 ,5 Hydroxymetronidazole, the major metabolite of metronidazole in humans, also has antibacterial activity. It has been suggested that in-vitro susceptibility to metronidazole may be underestimated if the active metabolite of metronidazole is not taken into account. 6 For H. pylori the activity of hydroxymetronidazole is the same as, or within 1-2 dilutions of the parent compound in broth. 7 We have also found (unpublished observation, J. M. Meyer, S. Ryu, S. L. Pendland, T. P. Kanyok & L. H. Danziger) that the MICs of hydroxymetronidazole are approximately 1-2 dilutions below the parent compound when tested against our isolates of H. pylori on solid media.
To determine more accurately the contribution of the metabolite to the activity of the parent compound for H. pylori, we studied metronidazole independently and in combination with hydroxymetronidazole. To simulate in-vivo conditions, a fixed 2:1 concentration ratio of metronidazole to hydroxymetronidazole was used, which is similar to the ratio that exists in serum after a single 500 mg oral dose. 8
Synergic interactions between antimicrobials may contribute to the efficacy of selected treatment regimens. Synergy has been shown between metronidazole and bismuth subcitrate, amoxycillin and tetracycline. 5,7 Hydroxymetronidazole alone has demonstrated synergy with metronidazole, amoxycillin and tetracycline. 7 There have been no published reports of synergy studies using metronidazole and hydroxymetronidazole together with other compounds. We have previously studied clarithromycin in combination with its active metabolite, in an attempt to characterize the contribution of 14-hydroxyclarithromycin to synergy and the efficacy of treatment regimens containing clarithromycin. 9
In this investigation, partial synergy was demonstrated for the majority (11/19) of isolates when the combination of paromomycin and metronidazole alone or metronidazole plus hydroxymetronidazole was tested. When metronidazole was combined with paromomycin, three of the 19 isolates (16%) demonstrated synergy. Addition of hydroxymetronidazole to the parent compound increased the number to seven (37%). No antagonism was seen with either combination.
For metronidazole-sensitive strains, only one of 12 isolates demonstrated synergy to the combination of metronidazole and paromomycin. Addition of hydroxymetronidazole to the parent compound increased the number of isolates demonstrating synergy (Table I) to five. When metronidazole resistant strains were tested, there was a trend towards an increased number of isolates dem onstrating partial synergy when the metabolite was added. The small number of isolates studied limits any conclusions from this data.
Methods for performing susceptibility and synergy testing of H. pylori have not been standardized. A breakpoint of 16 mg/L for metronidazole was selected based on correlation with clinical failure. 10 The agar dilution method for synergy testing on Mueller-Hinton medium supplemented with 10% defibrinated horse blood is preferred in our laboratory, since it has consistently supported reliable growth and reproducible results. This choice of medium was recently corroborated by other investigators who found it superior to other media in its ability to support growth of H. pylori both semiquantitatively and qualitatively. 11
Paromomycin has been shown in this investigation to demonstrate substantial in-vitro activity against H. pylori. It appears to have synergic activity with metronidazole against both metronidazole sensitive and resistant isolates, enhanced by the addition of hydroxymetronidazole. Paromomycin warrants further investigation as a potential therapeutic agent for treatment of H. pyloriinfection.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . , Eliopoulos, G. M. & Moellering, R. C. (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 33096. Williams & Wilkins, Baltimore, MD.
3 . Neu, H. C.(1977)).Mecillinam - an amidino penicillin which acts synergistically with other ß-lactam compounds. Journal of Antimicrobial Chemotherapy 3, Suppl. B , 43-52.[ISI][Medline]
4 . Glupczynski, Y., Delmee, M., Bruck, C., Labbe, M., Avesani, V. & Burette, A.(1988)).Susceptibility of clinical isolates of Campylobacter pylori to 24 antimicrobial and anti-ulcer agents. European Journal of Epidemiology 4, 154-7.[ISI][Medline]
5 . Van Caekenberghe, D. L. & Breyssens, J.(1987)). In vitro synergistic activity between bismuth subcitrate and various antimicrobial agents against Campylobacter pyloridis (C. pylori). Antimicrobial Agents and Chemotherapy 31, 1429-30.[ISI][Medline]
6 . Pendland, S. L., Piscitelli, S. C., Schreckenberger, P. C. & Danziger, L. H.(1994)). In vitro activities of metronidazole and its hydroxy metabolite against Bacteroides spp. Antimicrobial Agents and Chemotherapy 38, 2106-10.[Abstract]
7 . Pavicic, M. J., Namavar, F., Verboom, T., Van Winkelhoff, A. J. & De Graaff, J.(1993)). In vitro susceptibility ofHelicobacter pylori to several antimicrobial combinations. Antimicrobial Agents and Chemotherapy 37, 1184-6.[Abstract]
8 . Houghton, G. W., Thorne, P. S., Smith, J., Templeton, R. & Collier, J.(1979) Comparison of the pharmacokinetics of metronidazole in healthy female volunteers following either a single oral or intravenous dose. British Journal of Clinical Pharmacology 8, 337-41.[ISI][Medline]
9 . Meyer, J. M., Ryu, S., Pendland, S. L. & Danziger, L. H.(1997). In vitro synergy testing of clarithromycin and 14-hydroxyclarithromycin with amoxicillin or bismuth subsalicylate against Helicobacter pylori. Antimicrobial Agents and Chemotherapy 41, 1607-8.[Abstract]
10 . Bouchard, S., Birac, C., Lamouliatte, H., Forestier, S. & Megraud, F.(1996)).Correlation between the MICs of metronidazole on Helicobacter pylori strains and the outcome of a lansoprazole-amoxicillin-metronidazole therapy. Gut 39, Suppl. 2,A6.
11 . Hartzen, S. H., Andersen, L. P., Bremmelgaard, A., Colding, H., Arpi, M., Kristiansen, J.et al. (1997)). Antimicrobial susceptibility testing of 230 Helicobacter pylori strains: importance of medium, inoculum and incubation time. Antimicrobial Agents and Chemotherapy 41, 2634-9.[Abstract]
Received 20 April 1998; returned 15 June 1998; revised 22 July 1998; accepted 13 October 1998