Dkg318.fm

Journal of Antimicrobial Chemotherapy (2003) 52, 303–305
DOI: 10.1093/jac/dkg318
Advance Access publication 1 July 2003
Susceptibility to rifaximin of Vibrio cholerae strains from different
geographical areas
Maria Scrascia1, Maria Forcillo1, Francesco Maimone1,2 and Carlo Pazzani1,2*
1Dipartimento di Anatomia Patologica e di Genetica, Sezione di Genetica, Università di Bari, Via G. Amendola 165/ A, 70126 Bari; 2Interuniversity Research Centre for Sustainable Development, Università di Roma ‘La Sapienza’, Received 18 February 2003; returned 21 March 2003; revised 29 April 2003; accepted 6 May 2003 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
V. cholerae O139 isolated in India and two reference O1 strains of theclassical biotype were also included.
Vibrio cholerae O1 El Tor biotype is the causative organism of the Rectal swabs, stool samples, or both (in Cary-Blair transport medium) current seventh cholera pandemic. In addition to O1 strains of the were plated on thiosulphate-citrate-bile salts-sucrose (TCBS) (Oxoid, El Tor biotype, strains of V. cholerae O139 and specific strains of Milan, Italy) agar and incubated at 37°C for 18–24 h. Part of each speci- V. cholerae O1 classical biotype are also responsible for cholera out- men was enriched in alkaline peptone water pH 8.5 (Oxoid, Milan, Italy) and then plated on TCBS agar. Well-isolated suspect colonies were picked Antimicrobial agents in the treatment of cholera cases are often to Kligler iron agar slants and tested for urease and oxidase production recommended for reduction of symptomatology and vibrio excretion (Oxoid, Milan, Italy). Isolates giving typical reactions were biochemic- in the environment. The antimicrobials traditionally used have ally characterized by the API 20E system with additional tests for Vibrio been tetracycline, trimethoprim–sulfamethoxazole, erythromycin spp. O-antigen groups were serologically identified by using commer- cially available antisera. V. cholerae isolates were also tested by agglu- Rifaximin, a rifamycin derivative, acts by binding irreversibly to tination with chicken erythrocytes and polymyxin B susceptibility.
the β-subunit of the bacterial DNA-dependent RNA polymerase and Original stock cultures of isolates were kept in 20% glycerol Luria- inhibits RNA synthesis. Susceptibility testing of enterobacterial Biotypes and genetic relationships of strains were identified by DNA- pathogens associated with traveller’s diarrhoea has suggested that based typing methods. Fingerprints of genomic DNA were obtained rifaximin could be considered as a potential agent in the treatment of using PCR to generate amplified polymorphic DNA (APD). The primers intestinal bacterial infections.2 In this study, we investigated the in were six oligonucleotides selected from enterobacterial repetitive con- vitro activity of rifaximin against V. cholerae strains isolated from sensus sequences,3 from V. cholerae repetitive DNA sequences,4 and different outbreaks in different regions and in different years.
from the CTXφ phage genome of V. cholerae.5 Each strain was charac-terized by the combination of the six APD patterns. Combinations were Materials and methods
compared and different APD clusters were identified to establish a refer-ence framework to assess clonal relatedness.
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 The antibacterial susceptibility pattern of each isolate was determined representative of outbreaks which occurred in Africa from 1985 to 1999, by the disc diffusion method.6 The antimicrobial discs were used at the 12 El Tor strains were from outbreaks in Central and South America, following concentrations: ampicillin (10 µg), chloramphenicol (30 µg), and 32 strains from outbreaks in South Europe. Three clinical strains of doxycycline (30 µg), kanamycin (30 µg), streptomycin (10 µg), spectino- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*Corresponding author. Fax: +39-080-544-3386; E-mail: [email protected].
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The British Society for Antimicrobial Chemotherapy 2003; all rights reserved.
M. Scrascia et al.
Table 1. Clonal relatedness and antimicrobial resistance pattern of 408 V. cholerae strains of different geographical origins
AMP DOX KAN SMX SPT STR TET (57)/eastern AfricaAMP KAN SMX SPT STR TMP (2)/southern AfricaDOX SMX SPT STR TET TMP (6)/eastern and southern AfricaSMX SPT STR TMP (74)/eastern and southern AfricaSPT (89)/northern, eastern and southern Africa AMP, ampicillin; DOX, doxycycline; KAN, kanamycin; SPT, spectinomycin; STR, streptomycin; TET, tetracycline; SMX, sulfamethoxazole; TMP,trimethoprim.
mycin (10 µg), tetracycline (30 µg), sulfamethoxazole (25 µg) and Table 2. MIC s and MIC s (mg/L) of rifaximin and
trimethoprim (5 µg) (Oxoid, Milan, Italy). MICs of rifaximin and tetra- tetracycline among V. cholerae O1 El Tor strainsa distributed cycline (Sigma, Milan, Italy), were tested for each isolate according to NCCLS guidelines7 with final concentrations ranging from 0.0125 to256 mg/L. Rifaximin was a gift from Alfa Wassermann SpA, Bologna,Italy (Batch i/2096c). Escherichia coli ATCC 25922 was used as a quality Results and discussion
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 aThe MIC range of RFX and TET for the three strains of V. cholerae relationships were determined by comparison and analysis of com- O139 and the two strains of V. cholerae O1 classical biotype was binations of the amplified DNA patterns. On this basis, all strains were clustered into 23 clones. The 359 strains isolated in Africa from1985 to 1999 consisted of 16 clones. The European and American The distribution of the MICs of rifaximin and tetracycline for the strains were classified in two clones, respectively, and the Indian V. cholerae El Tor isolates by geographic area is given in Table 2.
MICs of tetracycline were determined for comparison between Tests for antimicrobial susceptibility revealed six patterns within rifaximin and a traditionally used antimicrobial. Tetracycline showed the African strains. The large group of El Tor strains resistant to lower activity for the African isolates (MIC = 64 mg/L) than for ampicillin, sulfamethoxazole, spectinomycin, streptomycin, trimetho- the European (MIC = 16 mg/L) and American (MIC = 2 mg/L) prim (131 strains) and the group resistant to ampicillin, doxycycline, isolates. No differences in the MIC s (2–4 mg/L) and MIC s kanamycin, sulfamethoxazole, spectinomycin, streptomycin, tetra- (2–4 mg/L) of rifaximin were observed among the three groups of O1 cycline (57 strains) isolated from East African countries in 1985, 1998 and 1999 belonged to four and two clones, respectively. Two The MIC values of rifaximin for V. cholerae El Tor were far susceptibility patterns were found in the European and Americanstrains, respectively.
lower than those found among other enteric bacterial pathogens All strains were resistant to spectinomycin (100% of isolates) and isolated from cases of traveller’s diarrhoea (MIC = 16 mg/L and the vast majority were resistant to streptomycin (74%) and sulfa- methoxazole (74%). Resistances to trimethoprim and ampicillin were Rifaximin is a poorly absorbed antibiotic and in pharmacokinetic present in 60% and 47% of all strains, respectively, corresponding studies, high levels of rifaximin (up to 8000 µg/g) were detected in to 81% and 63% of the multidrug-resistant isolates. A significant faeces of patients and healthy volunteers after 3 days of a daily dosage percentage of isolates were also resistant to tetracycline (22%), doxy- of 800 mg.8 The MICs of rifaximin for all V. cholerae strains analysed are well below the levels of faecal concentrations which rifaximin Susceptibility of Vibrio cholerae to rifaximin
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