Pii: s0378-1135(02)00257-2

Veterinary Microbiology 89 (2002) 303–309 Distribution and characterization of class 1 integrons in Salmonella enterica serotype Gallinarum Hyuk Joon KwonTae Eun Kim, Sun Hee ChoJae Goo Seol aInstitute of iNtRON Biotechnology, Seoul, 138-200, South Korea bDepartment of Microbiology, College of Medicine, Cheju National University, cDepartment of Biochemistry, College of Medicine, Cheju National University, dClinical Pathology Laboratory, Bayer Korea Ltd., Seoul 157-200, South Korea eDepartment of Avian Diseases, College of Veterinary Medicine, Seoul National University, 103, Seodoon-dong, Suwon, 441-744 Kyounggi, South Korea fDepartment of Infectious Diseases, College of Veterinary Medicine, School of Agricultural Biotechnology, Seoul National University, 103, Seodoon-dong, Suwon, 441-744 Kyounggi, South Korea Received 10 January 2002; received in revised form 16 July 2002; accepted 31 July 2002 Fowl typhoid caused by Salmonella enterica subsp. enterica serotype Gallinarum biotype Gallinarum is the most important chicken disease in Korea. Due to appearance of new or multipleantibiotics resistances in the recently isolated strains, it was difficult to control the disease usingantibiotics in our country. Therefore, the prevalence and genetic contents of class 1 integrons inbiotype Gallinarum isolated between 1992 and 2001 were investigated by PCR and direct sequen-cing, respectively. Out of 90 strains, 35 (39%) carried class 1 integrons. The 1.0, 1.6 and 2.0 kbpamplicons were amplified in 32 strains (36%), 2 strains (2%) and 1 strain (1%), respectively. The 1.0,1.6 and 2.0 kbp amplicons contained one (aadA1a), two (aadB-aadA1b) and three cassettes (dhfrXII-orfF-aadA2), respectively, providing resistances against aminoglycosides (aadA1a, aadA1b, aadB,and aadA2) and trimethoprim (dhfrXII). The integron-carrying strains of biotype Gallinarumappeared in 1996 and acquired additional cassettes in 2000. Although the resistances to ampicillin,tetracycline and chloramphenicol are unrelated to class 1 integrons, relatively high prevalence of * Corresponding author. Tel.: þ82-31-290-2737; fax: þ82-31-290-2737.
E-mail address: [email protected] (H.S. Yoo).
0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 5 7 - 2 H.J. Kwon et al. / Veterinary Microbiology 89 (2002) 303–309 integron in biotype Gallinarum may be a dormant threat to the chemotherapy of the disease in the nearfuture because of potency to acquire additional antibiotics resistances.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Integron; Class I; Salmonella enterica subsp. enterica serotype Gallinarum; Biotype Gallinarum Salmonella enterica subsp. enterica serotype Gallinarum biotype Gallinarum is a non- motile avian pathogen ) causing fowl typhoid, an acutesepticemic disease-featuring anemia, leukocytosis, and hemorrhages in adult chickens(Since the first outbreak of fowl typhoid reported in 1992 in Korea, ithas spread nationwide. The early isolates of biotype Gallinarum showed the same plasmidprofiles and antibiograms (However, recent isolates have acquiredadditional antibiotic resistances.
Antibiotic resistance can be acquired through mutation or transfer of mobile genetic materials such as plasmids, transposons, and bacteriophages. In the mid-1980s, DNAsequencing of several seemingly unrelated antibiotic-resistant genes revealed commonupstream and downstream regions The upstream contained the attI site, a commonpromoter sequence, Pant, and the opposite strand, an integrase gene (int) (whereas the 30-conserved segment included an antiseptic-resistant gene (qacED1), a sulphonamide-resistant gene (sulI), and an open reading frame(orf5) of unknown function The central variable region includeddifferent combinations of inserted gene cassettes. To date about 60 different cassettesassociated with resistance genes have been characterized from integrons, which allow theirbacterial hosts to become resistant to broad spectra of antimicrobial agents (These genetic elements lack direct or indirect repeat sequences at their endsand many of the genes associated with transposition. For this reason, the new element istermed as integron in contrast to transposons. To date four types of integrons, with differentint genes, have been identified (with most fromclinical isolates belonging to class 1 The class 1 integrasecatalyzes site-specific recombination between attI of integron and a 59-base element of amobile gene cassette containing various antibiotic-resistant genes and unknown openreading frames (). Most of the gene cassettes lack their ownpromoters, and integrons act as natural expression vectors by supplying a commonpromoter, Pant, located in the conserved sequences upstream of the inserted genes ( Several studies have investigated the prevalence of integrons in multi-drug-resistant serotypes of Salmonella and have found them to be widespread ). However, the prevalence of integronsand the genetic structures of inserted cassettes of serotype Gallinarum biotype Gallinarum werenot identified. In Korea, resistance to various antibiotics is increasing in isolates from animalsHowever, the frequency, characteristics, and roles of integrons and gene H.J. Kwon et al. / Veterinary Microbiology 89 (2002) 303–309 cassettes have not yet been investigated. Therefore, in this study, S. enterica serotypeGallinarum biotype Gallinarum isolates from Korea were investigated to demonstrate theprevalence and characteristics of class 1 integrons and the gene cassettes in the organism.
The study included 90 apparently epidemiologically unrelated S. enterica serotype Gallinarum biotype Gallinarum isolated from chickens submitted to Clinical PathologyLaboratory, Bayer Korea or Avian Diseases Laboratory, College of Veterinary Medicine,Seoul National University, in Korea from 1992 to 2001 (Isolates were identified using a Vitek system after basic biochemical testsincluding oxidase, catalase tests. Once identified, the isolates were preserved at À70 8C inLB broth containing 20% glycerol (v/v).
2.2. Antimicrobial susceptibility testing The bacterial strains, biotype Gallinarum, were tested for their susceptibility to 12 antimicrobial agents () through the disk diffusion assay using a standard procedure 2.3. Detection of class 1 integrons by PCR All strains were tested more than once for the presence of class 1 integrons using primers 50CS and 30CS (). Total DNA of Salmonella was extracted usingG-spin genomic DNA extraction kit (for Gram-negative; iNtRON Biotechnology Co., Seoul, Table 1Distribution of integrons in S. enterica subsp. enterica serotype Gallinarum biotype Gallinarum isolated fromchickens in Korea H.J. Kwon et al. / Veterinary Microbiology 89 (2002) 303–309 Table 2Relationships between amplicon sizes, resistance genes, and resistance patterns found within S. entericaserotype Gallinarum biotype Gallinarum isolates Groups were divided by amplicon sizes, contents of cassettes, and antibiotics resistance patterns.
a Drugs and disk loads were as follows: streptomycin (S), 10 mg; trimethoprim-sulfamethoxazole (SXT), 1.25–23.75 mg; tetracycline (T), 5 mg; ampicillin (A), 10 mg; gentamicin (G), 10 mg; chloramphenicol (C), 30 mg.
Korea) following manufacturer’s protocol. The PCR solution was composed of 10Â buffer2 ml, dNTPs (2.5 mM) 0.4 ml, 50CS/30CS (10 pmol/ml) 0.5 ml each, Taq DNA polymerase(5 U/ml; iNtRON Biotechnology Co.) 0.2 ml, distilled water 15.4 ml and template DNA(50 ng/ml) 1 ml. The PCR was performed as follows: a cycle at 94 8C, 5 min; followed by35 cycles at 94 8C, 30 s; 55 8C, 30 s; and 72 8C, 2.5 min; and a final extension step at 72 8C,5 min. Amplicons were analyzed through electrophoresis on 1.0% agarose gels, and a 1 kbladder (iNtRON Biotechnology Co.) was used as the molecular size marker.
PCR products, representing different amplicons generated by the 33 strains of biotype Gallinarum were purified through simple ethanol precipitation and sequenced following aprevious method (DNA sequences obtained were compared to theGenBank database of the National Center for Biotechnology Information BLAST network( Results of the antimicrobial susceptibility test are summarized in . Strains isolated since 1996 acquired new resistance to antibiotics such as streptomycin, tetra-cycline, ampicillin, chloramphenicol, and gentamicin (Streptomycin- and tetra-cycline-resistant strains were isolated in 1996 and 1999, respectively. Additionalresistances to ampicillin, gentamicin, and chloramphenicol were observed in 2000 only1 year after the acquisition of tetracycline resistance. Multi-drug-resistant Salmonellastrains showing four or more different antibiotics resistances were already identified inserotypes Typhimurium DT104, Ohio, Panama, Brandenburg, Virchow, and Hadar (The appearance of multi-drug-resistant strains of biotype Gallinarum in ashort period of time to obtain additional resistant phenotypes urge further studies on themechanism of resistance spread and effective control methods.
H.J. Kwon et al. / Veterinary Microbiology 89 (2002) 303–309 Class 1 integrons were present in 35 strains (38%) of biotype Gallinarum (90 strains) isolated from chickens in Korea. To determine the association between class 1 integronsand the sul1 gene, strains of different integron structures were analyzed by PCR. When 35strains of biotype Gallinarum with 50CS-sulI primers ), amplicons of about 1.9, 2.5 and 2.9 kbp (representing variable regions ofabout 1.0, 1.6 and 2.0 kbp, respectively) were amplified, respectively (data not shown).
3.3. Relationships of class 1 integrons, resistance genes, and resistance patterns The class 1 integrons reported for Salmonella suggested that aadA, aadB, aacA4, and aacC1 (encoding aminoglycosides resistance), sat-1 (encoding streptothricin resistance),pse-1 and oxa1 (encoding ampicillin resistance), catB3 (encoding chloramphenicolresistance), and dhfr (encoding trimethoprim resistance) genes could be present in someof the variable regions (Thenucleotide sequences of both ends of the PCR products were determined. All 1.0 kbamplicons of biotype Gallinarum were revealed to contain aadA1a cassette related tostreptomycin/spectinomycin-resistance and the same variable region wasreported in various serotypes of Salmonella ) and other Enterobacter-iaceae Genetic structures of the 1.6and 2.0 kbp amplicons were determined by partial end sequencing. Two 1.6 and one2.0 kbp amplicons had (two different) gene cassettes, aadB-aadA1b and dhfrXII-orfF-aadA2, respectively (AadB and aadA2 encode for aminoglycosides adenyl-transferases related to gentamicin- and streptomycin/spectinomycin-resistances, respec-tively, and dhfrXII encodes for a trimethoprim-resistance-related protein (AadA genes are divided into sixgenetic subtypes, 1a, 1b, 2, 3, 4, 5 and 6 distinguished based on the nucleotide sequencesIntegrons bearing only one genetic variant of aadA (aadA1a) were more prevalent inbiotype Gallinarum (91%) compared to those carrying additional cassettes (9%). Theprevalence of streptomycin-resistant cassettes in biotype Gallinarum isolated from chick-ens may be explained by the fact that streptomycin has been used with vitamins as a stress-reducing drug in Korea. Gentamicin has been used for the treatment of fowl typhoid, andthe recent appearance of gentamicin-resistant isolates of biotype Gallinarum may berelated to the acquisition of the aadB cassette. Due to the presence of a single commonpromoter (Pant) in the upstream of gene cassettes, the near gene cassette is expressed morehighly than others ). A recently introduced gene cassette locatesbetween Pant and pre-existing cassettes, and it is expressed highly than downstream genecassettes ). The near localization of aadB and dhfrXII to Pant in eachintegron represents recent selection pressures by gentamicin and trimethoprim/sulfa-methoxazole (( The tetracycline-resistance was not related to the integron and associated cassettes, but may depend on other mobile genetic elements Ampicillin- and H.J. Kwon et al. / Veterinary Microbiology 89 (2002) 303–309 chloramphenicol-resistances can be transferred in the form of gene cassettes to the integron(), however, in the case of biotype Gallinarum, are not related to theintegron.
This work was partly supported by Brain Korea 21 Project and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University.
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