Susceptibility to rifaximin of Vibrio cholerae strains from different geographical areas

Maria Scrascia1, Maria Forcillo1, Francesco Maimone1,2 and Carlo Pazzani1,2,*

1 Dipartimento di Anatomia Patologica e di Genetica, Sezione di Genetica, Università di Bari, Via G. Amendola 165/A, 70126 Bari; 2 Interuniversity Research Centre for Sustainable Development, Università di Roma ‘La Sapienza’, Rome, Italy

Received 18 February 2003; returned 21 March 2003; revised 29 April 2003; accepted 6 May 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Four hundred and eight clinical strains of Vibrio cholerae isolated from different geographical areas and with different antimicrobial resistance patterns were tested for susceptibility to rifaximin, a non-absorbable antibiotic active in vitro against Gram-negative bacteria. The MICs ranged from 0.5 to 4 mg/L for all strains. These values and the pharmacokinetic properties suggest rifaximin as an attractive antimicrobial agent for cholera.

Keywords: rifamycin derivative, antimicrobial activity, cholera


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Vibrio cholerae O1 El Tor biotype is the causative organism of the current seventh cholera pandemic. In addition to O1 strains of the El Tor biotype, strains of V. cholerae O139 and specific strains of V. cholerae O1 classical biotype are also responsible for cholera outbreaks in Asian countries.1

Antimicrobial agents in the treatment of cholera cases are often recommended for reduction of symptomatology and vibrio excretion in the environment. The antimicrobials traditionally used have been tetracycline, trimethoprim–sulfamethoxazole, erythromycin and furazolidone.

Rifaximin, a rifamycin derivative, acts by binding irreversibly to the ß-subunit of the bacterial DNA-dependent RNA polymerase and inhibits RNA synthesis. Susceptibility testing of enterobacterial pathogens associated with traveller’s diarrhoea has suggested that rifaximin could be considered as a potential agent in the treatment of intestinal bacterial infections.2 In this study, we investigated the in vitro activity of rifaximin against V. cholerae strains isolated from different outbreaks in different regions and in different years.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sources and characterization of V. cholerae strains

A total of 408 V. cholerae strains were analysed in this study (Table 1). Of these, 359 V. cholerae O1 El Tor strains were isolated from clinical cases representative of outbreaks which occurred in Africa from 1985 to 1999, 12 El Tor strains were from outbreaks in Central and South America, and 32 strains from outbreaks in South Europe. Three clinical strains of V. cholerae O139 isolated in India and two reference O1 strains of the classical biotype were also included.


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Table 1.  Clonal relatedness and antimicrobial resistance pattern of 408 V. cholerae strains of different geographical origins
 
Rectal swabs, stool samples, or both (in Cary-Blair transport medium) were plated on thiosulphate-citrate-bile salts-sucrose (TCBS) (Oxoid, Milan, Italy) agar and incubated at 37°C for 18–24 h. Part of each specimen was enriched in alkaline peptone water pH 8.5 (Oxoid, Milan, Italy) and then plated on TCBS agar. Well-isolated suspect colonies were picked to Kligler iron agar slants and tested for urease and oxidase production (Oxoid, Milan, Italy). Isolates giving typical reactions were biochemically characterized by the API 20E system with additional tests for Vibrio spp. O-antigen groups were serologically identified by using commercially available antisera. V. cholerae isolates were also tested by agglutination with chicken erythrocytes and polymyxin B susceptibility. Original stock cultures of isolates were kept in 20% glycerol Luria-Bertani broth at –70°C.

Biotypes and genetic relationships of strains were identified by DNA-based typing methods. Fingerprints of genomic DNA were obtained using PCR to generate amplified polymorphic DNA (APD). The primers were six oligonucleotides selected from enterobacterial repetitive consensus sequences,3 from V. cholerae repetitive DNA sequences,4 and from the CTX{phi} phage genome of V. cholerae.5 Each strain was characterized by the combination of the six APD patterns. Combinations were compared and different APD clusters were identified to establish a reference framework to assess clonal relatedness.

Antibiotic susceptibility testing

The antibacterial susceptibility pattern of each isolate was determined by the disc diffusion method.6 The antimicrobial discs were used at the following concentrations: ampicillin (10 µg), chloramphenicol (30 µg), doxycycline (30 µg), kanamycin (30 µg), streptomycin (10 µg), spectinomycin (10 µg), tetracycline (30 µg), sulfamethoxazole (25 µg) and trimethoprim (5 µg) (Oxoid, Milan, Italy). MICs of rifaximin and tetracycline (Sigma, Milan, Italy), were tested for each isolate according to NCCLS guidelines7 with final concentrations ranging from 0.0125 to 256 mg/L. Rifaximin was a gift from Alfa Wassermann SpA, Bologna, Italy (Batch i/2096c). Escherichia coli ATCC 25922 was used as a quality control strain.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The clonal relatedness and the antimicrobial resistance patterns of the 408 V. cholerae O1 strains tested are shown in Table 1.

Genetic diversity was assessed by PCR fingerprinting with selected primers generating amplified polymorphic genomic DNA. Clonal relationships were determined by comparison and analysis of combinations of the amplified DNA patterns. On this basis, all strains were clustered into 23 clones. The 359 strains isolated in Africa from 1985 to 1999 consisted of 16 clones. The European and American strains were classified in two clones, respectively, and the Indian strains in three clones.

Tests for antimicrobial susceptibility revealed six patterns within the African strains. The large group of El Tor strains resistant to ampicillin, sulfamethoxazole, spectinomycin, streptomycin, trimethoprim (131 strains) and the group resistant to ampicillin, doxycycline, kanamycin, sulfamethoxazole, spectinomycin, streptomycin, tetracycline (57 strains) isolated from East African countries in 1985, 1998 and 1999 belonged to four and two clones, respectively. Two susceptibility patterns were found in the European and American strains, respectively.

All strains were resistant to spectinomycin (100% of isolates) and the vast majority were resistant to streptomycin (74%) and sulfamethoxazole (74%). Resistances to trimethoprim and ampicillin were present in 60% and 47% of all strains, respectively, corresponding to 81% and 63% of the multidrug-resistant isolates. A significant percentage of isolates were also resistant to tetracycline (22%), doxycycline (15%) and kanamycin (14%).

The distribution of the MICs of rifaximin and tetracycline for the V. cholerae El Tor isolates by geographic area is given in Table 2. MICs of tetracycline were determined for comparison between rifaximin and a traditionally used antimicrobial. Tetracycline showed lower activity for the African isolates (MIC90 = 64 mg/L) than for the European (MIC90 = 16 mg/L) and American (MIC90 = 2 mg/L) isolates. No differences in the MIC50s (2–4 mg/L) and MIC90s (2–4 mg/L) of rifaximin were observed among the three groups of O1 El Tor strains.


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Table 2.  MIC50s and MIC90s (mg/L) of rifaximin and tetracycline among V. cholerae O1 El Tor strainsa distributed by geographic area
 
The MIC values of rifaximin for V. cholerae El Tor were far lower than those found among other enteric bacterial pathogens isolated from cases of traveller’s diarrhoea (MIC50 = 16 mg/L and MIC90 = 32 mg/L).2

Rifaximin is a poorly absorbed antibiotic and in pharmacokinetic studies, high levels of rifaximin (up to 8000 µg/g) were detected in faeces of patients and healthy volunteers after 3 days of a daily dosage of 800 mg.8 The MICs of rifaximin for all V. cholerae strains analysed are well below the levels of faecal concentrations which rifaximin can reach in vivo. These findings indicate rifaximin as a candidate antimicrobial agent to be tested in cholera patients.


    Acknowledgements
 
We thank Ida Luzzi (Istituto Superiore di Sanità, Rome, Italy) for providing isolates from Italy, Albania and America. This study was supported by a grant from Alfa Wassermann SpA and by grant IC18-CT97-0231 (F.M.) from the European Commission under INCO-DC program.


    Footnotes
 
* Corresponding author. Fax: +39-080-544-3386; E-mail: pazzani{at}biologia.uniba.it. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Faruque, S. M., Albert, M. J. & Mekalanos, J. J. (1998). Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiology and Molecular Biology Reviews 62, 1301–14.[Abstract/Free Full Text]

2 . Gomi, H., Jiang, Z. D., Adachi, J. A. et al. (2001). In vitro antimicrobial susceptibility testing of bacterial enteropathogens causing traveler’s diarrhea in four geographic regions. Antimicrobial Agents and Chemotherapy 45, 212–6.[Abstract/Free Full Text]

3 . Versalovic, J., Koeuth, T. & Lupski, J. R. (1991). Distribution of repetitive DNA sequences in eubacteria and applications to fingerprinting of bacterial genomes. Nucleic Acids Research 19, 6823–31.[Abstract]

4 . Barker, A., Clark, C. A. & Manning, P. A. (1994). Identification of VCR, a repeat sequence associated with a locus encoding a hemagglutinin in Vibrio cholerae O1. Journal of Bacteriology 176, 5450–8.[Abstract]

5 . Pearson, G. D. N., Woods, A., Chiang, S. L. et al. (1993). CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proceedings of the National Academy of Sciences, USA 90, 3750–4.[Abstract]

6 . Bauer, A. W., Kirby, W. M., Sherris, J. C. et al. (1966). Antibiotic susceptibility testing by standardized single disc method. American Journal of Clinical Pathology 45, 493–6.[ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S8. NCCLS, Villanova, PA, USA.

8 . Jiang, Z. D., Ke, S., Palazzini, E. et al. (2000). In vitro activity and fecal concentration of rifaximin after oral administration. Antimicrobial Agents and Chemotherapy 44, 2205–6.[Abstract/Free Full Text]

9 . Coppo, A., Colombo, M., Pazzani, C. et al. (1995). Vibrio cholerae in the Horn of Africa: epidemiology, plasmids, tetracycline resistance gene amplification, and comparison between O1 and non-O1 strains. American Journal of Tropical Medicine and Hygiene 53, 351–9.[ISI][Medline]

10 . Di Pierro, M., Lu, R., Uzzau, S. et al. (2001). Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. Journal of Biological Chemistry 276, 19160–5.[Abstract/Free Full Text]





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