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1 Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, H-6725 Szeged, Hungary
2 Anaerobe Reference Laboratory, NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff CF14 4XW, UK
Correspondence
Elisabeth Nagy
nagye{at}mlab.szote.u-szeged.hu
Received 27 April 2005
Accepted 30 October 2005
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Numerous molecular typing methods, such as arbitrarily primed-PCR, RFLP, PFGE and PCR ribotyping, have been used internationally to study C. difficile strains of different origins (Brazier, 2001). Most of these typing methods are suitable for following outbreaks, determining recurrences, characterizing endemic strains and tracing the spread of C. difficile. PCR ribotyping is a method that is widely used (Rotimi et al., 2003; Stubbs et al., 1999; Urbán et al., 2001) to determine the intraspecies genetic variation of C. difficile, and in which the 16S and 23S rRNA intergenic spacer region can be amplified by using specific primers under stringent amplification conditions. The sizes of the amplification products can range from 250 to 600 bp (Brazier, 2001).
In an earlier study in Hungary in collaboration with the Anaerobe Reference Laboratory (Cardiff), 65 C. difficile isolates were typed using PCR ribotyping (Urbán et al., 2001). The aim of the present study was to extend this investigation and to determine the distribution of PCR ribotypes in three Hungarian regions.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Culturing conditions and extraction of DNA. For PCR ribotyping, strains were grown on Fastidious Anaerobe Agar (LabM); the plates were incubated at 37 °C for 24 h under anaerobic conditions. After checking the colony morphology, fluorescence under UV light and the purity of the cultures, the cells were resuspended in a 5 % (w/v) solution of Chelex-100 (Bio-Rad). The solutions were incubated in boiling water for 12 min and then centrifuged at 15 000 g for 10 min. The supernatant was removed, placed in a fresh tube and stored at 4 °C for 12–24 h.
PCR ribotyping. Amplification reactions were performed in a 100 µl volume containing 50 pmol each primer (16S rRNA gene primer p3 5'-CTG GGG TGA AGT CGT AAC AAG G-3', position 1445–1466, and 23S rRNA gene primer p5 5'-GCG CCC TTT GTA GCT TGA CC-3', position 1–20), 200 µM dNTP (Pharmacia), 10 mM Tris/HCl (pH 8·8), 50 mM KCl, 1·5 mM MgCl2, 1 U Taq polymerase (Pharmacia), and 10 µl template DNA. PCR was carried out in a thermal cycler, for 1 cycle of 120 s at 95 °C, for starting denaturation, and 30 cycles of denaturation at 92 °C for 60 s, annealing at 55 °C for 60 s and extension at 72 °C for 90 s. After the last denaturation step, annealing was carried out at 55 °C for 45 s, and extension at 72 °C for 5 min (Stubbs et al., 1999).
Detection of the PCR products. Amplification products were concentrated to a final volume of 25–30 µl in a heat-block set at 75 °C for 1·5 h. PCR products were detected by electrophoresis in 3 % (w/v) metaphor agarose gels (BMA-BioWhittaker Molecular Applications) for 3 h, at 200 V and 60 mA in TAE buffer [40 mM Tris-acetate, 1 mM EDTA (pH 8·0)]. The DNA banding pattern was visualized under UV light after staining for 20 min in ethidium bromide (0·5 µg ml–1). A molecular mass ladder (Superladder-Low; ABgene) was included every six wells on the gels for normalization of the gel patterns. PCR ribotype profiles were analysed with GelCompar image analysis software (version 4.0; Applied Maths).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). During the period examined, the most prevalent types among the Hungarian isolates overall were PCR ribotypes 014 (24·8 %), 002 (13·3 %) and 085 (8·6 %). Most of the toxin-negative strains belonged to PCR ribotype 085 (9/22 isolates). The distribution of the ribotypes differed in the three geographical regions: in the Budapest region the dominant types were ribotypes 014, 002 and 018 (29, 19·4 and 12·9 %, respectively), whereas 014 (28·9 %), 049 (11·1 %), 085 (8·9 %) and 002 (8·9 %) were most frequent in West Hungary, and ribotypes 012 (20·7 %), 002 (13·8 %) and 014 (13·8 %) were characteristic of South Hungary. Type 049 (11·1 %) was present only in West Hungary, while ribotype 005 (9·7 %) was detected only in the Budapest region, and ribotype 035 (6·9 %) was observed only in South Hungary (Table 1). The presence of binary toxin-producing strains was demonstrated in West Hungary and the Budapest region, but not in South Hungary. The frequencies of the various ribotypes were compared between inpatients and outpatients. When the two-sided Fisher's exact test was used, the differences were not significant except in one case (ribotype 012), which was more prevalent among isolates obtained from inpatients.
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These data suggest that the distribution of C. difficile may change over time and geographically. We have shown that there can be regional differences within a country. A number of ribotyping surveys in various countries have also demonstrated different distributions of ribotypes. For example, type 001 was detected in the majority of UK hospital infections (55 %), while only comprising 7·5 % of strains causing community-acquired infections in England and Wales (Stubbs et al., 1999). In Kuwait, PCR ribotypes 097 and 078 accounted for 
40 % of all isolates in intensive-therapy units (Rotimi et al., 2003).
In 2001, binary toxin-producing isolates were not observed among strains originating from Szeged University Hospital, whereas binary toxin genes were detected in three isolates between 2002 and 2004. The presence of toxin was also proven using Western blotting. Two of the three binary toxin-positive strains were isolated and identified from severe community-acquired diarrhoea after antibiotic therapy in West Hungary and the Budapest region (Terhes et al., 2004), and PaLoc analysis showed that the strains belonged to toxinotypes III and IV, which produce toxins A and B. These isolates caused severe diarrhoea, but the exact role of the binary toxin in pathogenesis has not yet been clarified. Some clinical studies suggest that the binary toxin can be an additional virulence factor that may enhance the severity of infection (Barbut et al., 2005).
During the past 10 years, numerous molecular typing methods have been developed to differentiate nosocomial C. difficile strains. Some of these methods can provide useful information, especially during hospital outbreaks. In each case, the most important expectations are that the methods should be discriminative, reproducible, simple and rapid. PFGE is accepted as the gold-standard technique for the typing of bacterial strains, but the majority of C. difficile isolates cannot be typed because of the degradation of the DNA. Moreover, this method is rather labour-intensive and time-consuming (Hyett et al., 1997; Kato et al., 1996). PCR ribotyping is one of the most frequently used methods for the epidemiological investigation of C. difficile. The present study compared the ribotypes of C. difficile strains from three parts of Hungary with the results of earlier international and national ribotyping studies. Both regional and international differences were observed in relation to the distribution of various ribotypes, and changes in the prevalence of the different ribotypes over time were also apparent. Because of the increasing numbers of hospital infections and outbreaks caused by C. difficile the introduction of typing methods in more countries is essential for establishing a database relating to the spread of the most frequent epidemic C. difficile strains. Such a database would be of great help in the future demonstration of outbreaks, and in the distinction between recurrences and reinfections.
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ACKNOWLEDGEMENTS |
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We are grateful to Frederic Barbut, Department of Microbiology and Infectious Control Unit, Hôpital Saint-Antoine, Assistance Publique-Hôpitaux de Paris, Paris, France for performing the binary toxin Western blotting and toxinotyping of binary toxin-producing isolates. We are grateful to Galina Riszkulova, Mária Tömöry, Anna Tusnádi, Mária Vollain and Kanjo Abdul Hamid for kindly providing C. difficile isolates.
This paper was presented in part as a poster (no. 1146) at the 15th European Congress of Clinical Microbiology and Infectious Diseases in Copenhagen, Denmark, on 2–5 April 2005.
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