Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan
Received 11 September 2004; returned 11 October 2004; revised 1 November 2004; accepted 10 November 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: A total of 105 Salmonella isolates were included in this study. The broth microdilution method was used to determine the MIC values of a range of antibiotics for these isolates. PCR and DNA sequencing were used for screening and characterization of class 1 and class 2 integrons.
Results: PCR sequencing analysis revealed the presence of seven profiles of class 1 integrons in addition to a new type of class 2 integron. The identified gene cassettes within class 1 integrons were as follows; aadA1, aadA2 and aadA5, which confer resistance to streptomycin and spectinomycin; aadB, which confers resistance to gentamicin, kanamycin and tobramycin; dfrA1 and dfrA17, which confer resistance to trimethoprim; blaPSE-1, which confers resistance to ampicillin; catB3, which confers resistance to chloramphenicol; and sat1, which confers resistance to streptothricin. Two strains of the multidrug-resistant S. Typhimurium DT104 were characterized in this study. DNA sequencing of class 2 integrons identified one with an unusual array of gene cassettes, sat, sat1 and aadA1.
Conclusions: In this study, we characterized the antibiotic resistance gene cassettes within class 1 integrons in different isolates of non-typhoid Salmonella serovars, and we also identified a new type of class 2 integron.
Keywords: multidrug resistance , Salmonella spp. , class 1 integrons , class 2 integrons
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the five classes of multi-resistance integrons that have been identified to date,4 integron classes 1 and 2 are the most frequent in Gram-negative bacteria.5 The structure of the class 1 integron includes 5' and 3' conserved segments and a variable region. The class 2 integron has an organization similar to that of class 1 but it is associated with transposon Tn7, and it is known to carry three classic gene cassettes, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively.6 An unusual class 2 integron array has been reported recently.7 Therefore, the aims of this study were (i) to investigate the prevalence and characterize class 1 and class 2 integrons among non-typhoid Salmonella serovars in Japan, and (ii) to monitor the spread of the multidrug-resistant S. enterica serovar Typhimurium DT104.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 105 Salmonella isolates were recovered from patients (13/105), retail chicken meats (12/105) and river water (80/105) in Hiroshima prefecture. The isolates were identified and serotyped by standard procedures.
Identification of S. enterica serovar Typhimurium DT104 by PCR
The PCR amplification of an internal segment of the 16S-to-23S spacer region of bacterial rRNA genes was used to identify S. enterica serovar Typhimurium DT104 as previously described.8
Antimicrobial susceptibility testing
The MIC values of the antibiotics were determined by the NCCLS broth microdilution method. The MIC values for Salmonella serovars were determined for the following antibiotics, supplied by different commercial companies: ampicillin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and neomycin. MIC breakpoints were evaluated according to NCCLS guidelines.9
Bacterial DNA preparation, PCR and DNA sequencing
Preparation of bacterial DNA templates and the PCR conditions used for detection of class 1 and class 2 integrons were carried out as previously described.10 Both DNA strands of the entire class 1 integron segments were sequenced using an ABI automatic DNA sequencer (Model 373; Perkin-Elmer). Two other primers were designed according to the preliminary DNA sequencing results of class 2 integrons. These primers were located within the PCR fragment and were used for complete sequencing of the whole class 2 integron segment.
Computer analysis of the sequenced data
A similarity search was carried out using the BLAST program available at the NCBI BLAST homepage (http://www.ncbi.nlm.nih.gov/BLAST/).
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, PCR screening results of 105 Salmonella isolates detected class 1 integrons in 12 (11.4%) isolates of different Salmonella serovars, which were mainly Typhimurium (3), Enteritidis (1), Infantis (4) and other serovars (4) (Table 1). These serovars showed seven profiles of class 1 integrons. DNA sequencing results of class 1 integron gene cassettes identified several classic antibiotic resistance genes, which are usually associated with Salmonella serovars.5 The resistance phenotypes were expressed for most of these genes (Table 1). The gene cassettes were as follows: aminoglycoside adenylyltransferase gene cassettes type A, aadA1, aadA2 and aadA5, which confer resistance to streptomycin and spectinomycin; aminoglycoside adenyltransferase type B, aadB, which confers resistance to gentamicin, kanamycin and tobramycin; dihydrofolate reductase gene cassettes, dfrA1 and dfrA17, which confer resistance to trimethoprim; ß-lactamase gene blaPSE-1, which confers resistance to ampicillin; chloramphenicol acetyltransferase, catB3, which confers resistance to chloramphenicol; and streptothricin acetyltransferase, sat1, which confers resistance to streptothricin. Thus our data indicate that the above mentioned gene cassettes are still predominant among Salmonella serovars.
|
One of the aims of this study was to monitor the spread of the multidrug-resistant S. Typhimurium DT104 in Japan. According to the resistance phenotypes and DNA sequencing results of class 1 integron gene cassettes, S. Typhimurium DT104 phenotypes were detected in only two out of 105 (1.9%) isolates examined. These two strains showed the typical multidrug-resistance phenotype of the previously identified DT104,3 as they were resistant to ampicillin, chloramphenicol, streptomycin, tetracycline (ACSSuT resistance type) and ciprofloxacin (Table 1). PCR and DNA sequencing results identified two different sizes of integrons: 1.2 kb and 1 kb (Table 1), each carrying a single resistance gene cassette, blaPSE-1 and aadA2, respectively. These two integrons are characteristic for the DT104 type.3 On the other hand, identification of these two strains of S. Typhimurium as a DT104 type was confirmed by PCR amplification of an internal segment of the 16S-to-23S spacer region of bacterial rRNA genes.8 These two strains showed the typical PCR amplicons and DNA sequences (100% identity) of the previously identified S. Typhimurium DT104 (data not shown).8 The incidence of S. Typhimurium DT104 in this study is somewhat similar to the ratio previously reported in 1999 in Japan by Izumiya et al.,11 who detected S. Typhimurium DT104 in 10 out of 447 (2.2%) non-typhoid strains. Therefore, our findings indicate that the prevalence of S. Typhimurium DT104 in Japan still remains about the same as that recorded 5 years ago.
New type of class 2 integron in S. Enteritidis strains: unusual array of class 2 gene cassettes
It is well known that the class 2 integron is associated with Tn7 and that it carries three conserved resistance gene cassettes; dfrA1, sat1 and aadA1 (Figure 1a).6 However, a new class 2 integron with unusual gene cassettes was recently characterized.7 In this integron, erythromycin esterase gene, ereA, replaced dfrA1 of the classic class 2 integron but it was inserted between the sat1 and aadA1 gene cassettes to form a large (approximately 2.9 kb in size) unusual array, sat1, ereA and aadA1 (Figure 1b). In this study, we also identified a new array of class 2 integrons in two isolates of S. Enteritidis (Table 1). This new integron was slightly larger (approximately 2.5 kb in size) than the classic type of class 2 integron (about 2.2 kb in size) and contained another sat gene, which replaced dfrA1 of the class 2 integron (Figure 1c) so that the new gene array, sat, sat1 and aadA1, contained two different types of sat gene, sat and sat1. Hence, there was a similarity between the S. Enteritidis class 2 integron and the recently detected integron, as both contained a new gene that replaced dfrA1, whereas sat1 and aadA1 of the class 2 integron were still conserved. These results support the explanation of Hansson et al.6 who suggested that the integron of Tn7 has acquired the trimethoprim gene cassette, dfrA1. In addition, the conservation of the internal stop codon in the integrase gene 2, intI2, suggests that the trimethoprim gene cassette in Tn7 may have been inserted by trans action of IntI1 in cells where both classes of integron were present.6 Our findings thus raise the intriguing question of what is the benefit of S. Enteritidis having two types of sat genes on an unusual array of class 2 integron, sat, sat1 and aadA1, which confer resistance against previously used feed additives.
|
The nucleotide sequences of the seven different profiles of class 1 integrons of non-typhoid Salmonella serovars have been assigned the DDBJ/GenBank/EMBL accession nos. AB126601, AB126602, AB126603, AB126604, AB186118, AB186119 and AB186122, whereas the new class 2 integron has been assigned the DDBJ/GenBank/EMBL accession no. AB161461.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . National Institute of Infectious Diseases. (2003). Salmonellosis in Japan as of June 2003. Infectious Agents Surveillance Report 24, 17980. [Online.] http://idsc.nih.go.jp/iasr/24/282/tpc282.html (24 January 2005, date last accessed).
3 . Threlfall, E. J., Frost, J. A., Ward, L. R. et al. (1994). Epidemic in cattle and humans of Salmonella typhimurium DT104 with chromosomally integrated drug resistance. Veterinary Record 134, 577.[ISI][Medline]
4 . Rowe-Magnus, D. A., Guerout, A. M. & Mazel, D. (2002). Bacterial resistance evolution by recruitment of super-integron gene cassettes. Molecular Microbiology 43, 165769.[CrossRef][ISI][Medline]
5
.
Goldstein, C., Lee, M. D., Sanchez, S. et al. (2001). Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrobial Agents and Chemotherapy 45, 7236.
6
.
Hansson, K., Sundstrom, L., Pelletier, A. et al. (2002). IntI2 integron integrase in Tn7. Journal of Bacteriology 184, 171221.
7
.
Biskri, L. & Mazel, D. (2003). Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron. Antimicrobial Agents and Chemotherapy 47, 332631.
8
.
Pritchett, L. C., Konkel, M. E., Gay, J. M. et al. (2000). Identification of DT104 and U302 phage types among Salmonella enterica serotype Typhimurium isolates by PCR. Journal of Clinical Microbiology 38, 34848.
9 . National Committee for Clinical Laboratory Standards (2002). Performance Standards for Antimicrobial Susceptibility TestingTwelfth Informational Supplement: Approved Standard M100-S12. NCCLS, Wayne, PA, USA.
10
.
DeLappe, N., O'Halloran, F., Fanning, S. et al. (2003). Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection. Journal of Clinical Microbiology 41, 191924.
11 . Izumiya, H., Tamura, K., Terajima, J. et al. (1999). Salmonella enterica serovar Typhimurium phage type DT104 and other multidrug resistant strains in Japan. Japanese Journal of Infectious Diseases 52, 133.[ISI][Medline]