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Health Protection Agency, Centre for Infections, Respiratory and Systemic Infection Laboratory, London NW9 5HT, United Kingdom
Health Protection Agency, Heartlands Hospital, Birmingham B9 5SS, United Kingdom, and Division of Immunity and Infection, Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom
Health Protection Agency, Regional Epidemiology Unit, Health Protection Agency, Leeds, United Kingdom
Scottish Centre for Infection and Environmental Health, Glasgow G3 7LN, United Kingdom
Department of Veterinary Bacteriology, University of Glasgow Veterinary School, Glasgow, United Kingdom
ABSTRACT
In the United Kingdom there has been a marked increase in the number of human infections caused by toxigenic Corynebacterium ulcerans. During 2002 and 2003 the organism was also isolated from several domestic cats with bilateral nasal discharge. As C. ulcerans has never previously been isolated from cats, the 16S rRNA gene from three cat isolates was sequenced to confirm their species identities. Fifty clinical isolates from the United Kingdom isolated from 1986 to 2003 and seven cat isolates were characterized by ribotyping to determine whether the ribotypes of the cat isolates were genotypically related to those found for human clinical isolates. For comparison, the genotypes of 11 overseas isolates and 13 isolates from H. R. Carne's collection isolated between 1933 and 1979 were also determined. Strains isolated from domestic cats were found to exhibit the predominant ribotypes observed among human clinical isolates, suggesting that C. ulcerans isolated from cats could be a potential reservoir for human infection.
INTRODUCTION
Corynebacterium ulcerans is a veterinary pathogen and causes mastitis in cattle and other domestic and wild animals (8, 10). The bacterium can carry the same bacteriophage that codes for the diphtheria toxin; and toxigenic strains of C. ulcerans have been associated with classical diphtheria, cutaneous diphtheria (1, 3, 15), as well as milder symptoms (3, 12). At least one death in the United Kingdom has been attributed to infection with this organism (2). Infections in humans are relatively rare and are usually associated with the ingestion of unpasteurized dairy products or a close association with farm animals (3). In the United Kingdom, between 1993 and 2003 six cases were reported to have classical diphtheria, and four of them had no identifiable risk factor. Most cases had no association with a farming community or the consumption of raw milk products, which suggested that there might be other routes of infection (2, 3).
The frequency and severity of infection with C. ulcerans appear to be increasing in the United Kingdom, and infection with toxigenic C. ulcerans strains appears to be more common than infection with toxigenic C. diphtheriae strains. Between 1986 and 2003 a total of 59 clinical isolates of C. ulcerans were submitted to the Streptococcus and Diphtheria Reference Unit (SDRU) for identification (Fig. 1). Among the 59 isolates submitted, 85% were toxigenic.
Between 2002 and 2003, seven isolates of C. ulcerans isolated from domestic cats in the United Kingdom were submitted to SDRU for confirmation and determination of toxin production. As C. ulcerans had never previously been identified in cats, 16S rRNA sequence identification and molecular typing for determination of whether the cat isolates were genotypically related to clinical isolates was undertaken.
MATERIALS AND METHODS
Bacterial strains. Fifty clinical isolates of C. ulcerans isolated between 1986 and 2003 from hospital laboratories in the United Kingdom and seven isolates from domestic cats in Glasgow, Leeds, and London submitted to SDRU during 2002 and 2003 were analyzed. Also, 11 clinical isolates referred from overseas laboratories (Germany, France, Ukraine, Italy, Canada, and The Netherlands) and 13 historical isolates of C. ulcerans from H. R. Carne's collection originally isolated between 1933 and 1979 were also analyzed for comparison.
Biotyping and toxigenicity testing. Biotyping was performed with all isolates by using the API CORYNE system, as described previously (6). All isolates were tested for toxin production by the Elek immunoprecipitation test (6, 7).
Sequencing of 16S rRNA genes. The 16S rRNA genes of three cat isolates (isolates CD02/59, CD02/62, and CD02/92) were sequenced to confirm the species identities. The gene was amplified as described elsewhere (11) by using a pair of universal primers, primers 27F and 1525R (MWG Biotech Ltd.), previously published by Lane (13). Template DNA for the PCRs was extracted as described previously by De Zoysa et al. (4). The reaction mixtures were cycled through the following temperature profile in a DNA engine (MJ Research): 94°C for 3 min, followed by 30 cycles at 94°C for 1 min, 67°C for 1 min, and 72°C for 1.5 min, with a final extension step at 72°C for 10 min.
The amplicons were purified and sequenced as described previously by De Zoysa et al. (5) by using previously published primers 27F, 1525R, 519R, 530F, 907R, and 926F (13). Raw chromatographic data were edited by using the Genebuilder sequence analysis software program (Applied Maths, Kortrijk, Belgium), and multiple alignments were made by using the Kodon sequence analysis software program (Applied Maths). The sequences obtained were compared with previously published 16S rRNA sequences of C. ulcerans (GenBank accession numbers X81912, X84256, and X81911), C. diphtheriae (GenBank accession numbers X82059 and X84248), and C. pseudotuberculosis (GenBank accession numbers X81907, X81916, D38579, and X84255).
Ribotyping. All 81 isolates of C. ulcerans (50 clinical isolates from the United Kingdom, 7 cat isolates, 11 overseas isolates, and 13 isolates from Carne's collection) were analyzed by ribotyping. Chromosomal DNA was extracted as described by De Zoysa et al. (4), and ribotyping was performed as described previously by Regnault et al. (14). Ribotype profiles were analyzed by using the computer software program Bionumerics (Applied Maths).
Nucleotide sequence accession number. The sequences from isolates 02/59 and 02/62 have been deposited in the GenBank sequence database under accession number AY987817.
RESULTS
Biotyping and toxigenicity testing. All isolates analyzed were identified as C. ulcerans (API number 0011326). Hydrolysis of hiss serum glycogen was used to distinguish C. ulcerans from C. pseudotuberculosis. Among the 81 isolates analyzed, 45 of 50 (90%) isolates from the United Kingdom, 8 of 11 overseas isolates, 9 of 13 Carne isolates, and all 7 cat isolates were toxigenic (Tables 1, 2, and 3).
Sequencing of 16S rRNA genes. A 1,500-bp amplicon was amplified by PCR, and nucleotide sequences of 1,398 bp in length were generated from the amplified fragments of the 16S rRNA genes of the three cat isolates. Upon alignment and comparison the 16S rRNA gene sequences, it was found that the sequences of two isolates (isolates 02/59 and 02/62) were identical. These two isolates were from two cats in the same household. Comparison of these sequences with those held in the GenBank database (National Center for Biotechnology Information, Bethesda, Md.) by using the BLAST program demonstrated that they the shared greatest identity (99.9%) with the 16S rRNA gene of C. ulcerans strain CCUG 2708 (GenBank accession number X81911). The third isolate, isolate 02/92 (from a Persian cat), demonstrated that it shared the greatest identity (99.9%) with the 16S rRNA gene of C. ulcerans type strain NCTC 7910 (GenBank accession number X84256).
Ribotyping. Analysis of the BstEII ribotype profiles of the 81 C. ulcerans isolates with the computer software program Bionumerics revealed nine distinct ribotypes. The ribotypes were provisionally designated U1 to U9. Figure 2 illustrates the nine ribotype profiles. Ribotype U1 was found in 20 isolates from the United Kingdom and four isolates from overseas (Germany, France, Ukraine, and Italy); ribotype U2 was found in 10 isolates from the United Kingdom, 1 isolate from overseas (Germany), and 8 isolates from Carne's collection (4 isolates were isolated in 1933; 3 others were isolated in 1956, 1965, and 1969, respectively; and information was not available for 1 strain). Ribotype U3 was found in 2 isolates from the United Kingdom and 1 isolate from overseas (Austria); U4 was found in 14 isolates from the United Kingdom and 1 isolate from overseas (The Netherlands); U5 was found in two isolates from the United Kingdom and five isolates from Carne's collection (one isolate was isolated in 1925, one isolate was isolated in 1968, and information was not available for three isolates); U6 was found in two isolates from the United Kingdom; U7, U8, and U9 are rare and were only found in four isolates from Canada (Tables 1 and 2).
The seven isolates from domestic cats also generated ribotypes documented among human clinical isolates. The following ribotypes were generated by the cat isolates: isolates 02/92 and 03/296 were of ribotype U1; isolates 02/59 and 02/62 (from cats in the same household) and isolate H03452 0239 were of ribotype U2; isolate H03489 0029 was of ribotype U4; and isolate 02/253 was of ribotype U6 (Table 3). Figure 3 represents the relationships between the ribotypes of the human and cat isolates, as determined with Bionumerics software.
DISCUSSION
The results obtained showed that 90% of the clinical isolates of C. ulcerans analyzed from the United Kingdom were toxigenic, and sequencing of the 16S rRNA genes from three cat isolates confirmed their species identities as C. ulcerans. Ribotyping revealed that all ribotypes were not equally common. Ribotype U1 was the predominant pattern seen among human clinical isolates in the United Kingdom, and the second and the third most predominant patterns seen in the United Kingdom were U4 and U2, respectively. All ribotypes generated by the cat isolates were documented among human clinical isolates, and six of seven cat strains belonged to one of the predominant ribotypes seen among the clinical strains. The two isolates from the cats in the same household were found to have indistinguishable ribotypes (ribotype U2). Ribotype U2 was found in eight of Carne's isolates (isolated mainly from the throat), and this ribotype was seen as far back as 1933. Also, ribotype U5 was found in five of Carne's isolates, and this ribotype was seen as far back as 1925 (in Norway). These findings show that these ribotypes have existed for over 70 years. This is an example of how stable ribotypes can remain over time. Ribotype patterns U7, U8, and U9 were found only in Canadian isolates; and these patterns were not seen among the European isolates.
Even though C. ulcerans has never previously been reported in cats, these results suggest that this organism in cats could be a potential reservoir for human infection. The source of human C. ulcerans infection in the United Kingdom is often unclear and is often not determined. The results obtained in this study also highlight the importance of determining the source of human infection.
Previously, it was thought that person-to-person spread of toxigenic C. ulcerans did not occur (3), and contact tracing was not recommended. However, this changed following isolation of the organism from siblings in 1996, and in 1998, the organism was isolated from a father and son (3). Therefore, it has been recommended that the public health response to human infection with C. ulcerans should be the same as that for C. diphtheriae (3).
Recently, a fatal case of necrotizing sinusitis due to toxigenic C. ulcerans was reported in a previously healthy farmer in Germany (16), and Hatanaka et al. (9) reported a case of diphtheria-like illness in a Japanese woman caused by toxigenic C. ulcerans; the patient had no direct contact with dairy livestock or unpasteurized dairy products; however, a week before the illness, the patient had been scratched by a cat with rhinorrhea (9). C. ulcerans has also been isolated from an asymptomatic carrier who had close contact with infected cattle (3). Therefore, it is not known whether more patients would have developed classical symptoms if they have not previously been immunized against diphtheria. As the main action of the diphtheria toxoid is to neutralize the diphtheria toxin, immunization is likely to protect individuals against an identical exotoxin produced by C. ulcerans.
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