JAC Advance Access originally published online on June 9, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):288-296; doi:10.1093/jac/dkl228
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri
1 Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention Hangzhou 310006, People's Republic of China 2 Institute for Infectious Disease, Zhejiang University Hangzhou 310003, People's Republic of China
*Correspondence address: Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention, Hangzhou, 310006, People's Republic of China. Tel: +86-571-85177696; Fax: +86-571-85165007; E-mail: jingcaopan{at}sina.com
Received 9 February 2006; returned 21 March 2006; revised 29 April 2006; accepted 5 May 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
Objectives: To analyse the gene cassettes and determine the roles of class 1 and class 2 integrons in antibiotic-resistant strains of Shigella sonnei (n = 31) and Shigella flexneri (n = 33).
Methods: Various molecular techniques, including PCR and Southern-blotting analysis, were used to analyse various markers of class 1 and class 2 integrons in these 64 S. sonnei and S. flexneri isolates collected in Hangzhou, China. The gene cassette arrays in integrons were identified by DNA sequencing and/or restriction fragment length polymorphism. Two genomic DNA fragments, one containing intI1 from a S. flexneri isolate that contains intI1 but lacks 3'-conserved region and another containing intI2 from a S. sonnei isolate, were cloned into pUC19 vectors and sequenced. The links between integron gene cassette arrays and antibiotic resistance were analysed.
Results: Class 2 integrons were present in 80.6% (25/31) of the S. sonnei isolates and 87.9% (29/33) of the S. flexneri isolates. All of these integron 2-positive isolates contained constant gene cassette arrays of dfrA1 + sat1 + aadA1 which confer resistance to trimethoprim and streptomycin. It was demonstrated that the class 2 integron was located in the Tn7 region inside the attTn7 locus downstream of glmS in Shigella. Class 1 integrons were found in 9.4% (6/64) of Shigella spp. isolates. An atypical class 1 integron without a 3'-conserved segment on the Shigella chromosome, termed Shigella atypical class 1 integron (SAI), was present in 84.9% (28/33) of S. flexneri isolates. The SAI contained two gene cassettes, blaOXA30 and aadA1; however, the SAI conferred resistance to ampicillin, but not to streptomycin, in Escherichia coli host. The blaOXA30 and aadA1 cassettes of the SAI seemed to be always coordinately excised or integrated.
Conclusions: Multiple and complex mechanisms involving mobile genetic elements in class 1 and class 2 integrons and antibiotic resistance have been developed in the evolution of Shigella strains.
Keywords: Tn7 , blaOXA30 , dfrA1 , aadA1
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
Shigellosis is a common diarrhoeal disease in developing as well as industrialized countries.1 In Asia, the incidence and deaths were estimated to be 91 million and 414 000 annually, respectively.2 Among Shigella species, Shigella flexneri is the most common serotype, followed by Shigella sonnei. Treatment with antibiotics has been effective in alleviating the dysenteric syndrome of shigellosis, reducing the duration of pathogen excretion to prevent disease transmission and lowering the risk of potential complications for the past several decades. However, at the same time, Shigella isolates have progressively acquired resistance to antibiotics, including ampicillin, streptomycin, trimethoprim/sulfamethoxazole and tetracycline.3–6 Determinants of antibiotic resistance in Shigella isolates are frequently borne within mobile genetic elements, including the R plasmids, transposons, integrons and genomic islands, on the bacterial genome.7–11 Mobile genetic elements may facilitate the dissemination of resistance determinants among species, even genera.
Integrons are gene-capture systems that harbour antibiotic resistance genes and may provide a flexible approach for bacteria to adapt to the pressure caused by antibiotics. The resistance to some antibiotics in Shigella species is associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes. The gene cassettes within class 1 integrons found on chromosome or plasmid in Shigella spp. often encode resistance to ampicillin (oxa-1), streptomycin (aadA) and/or trimethoprim (dfrA).12–14 Class 2 integrons borne on Tn7 are often present in S. sonnei isolates and their gene cassette arrays are usually constant, consisting of dfrA1, sat1 and aadA1. These genes confer resistance to trimethoprim, streptothricin and streptomycin, respectively.4,13,15–17
The objectives of the present study were to analyse the molecular characteristics of class 1 and class 2 integrons, including their distribution and locations in the genome, and the link between gene cassettes and antibiotic resistance in S. flexneri and S. sonnei isolates collected from Hangzhou, China, during a period of 4 years (1998–2002). In addition, the characteristics of an atypical class 1 integron without 3'-conserved segment (3'-CS) and its link to antibiotic resistance in S. flexneri isolates were identified.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
Bacterial isolates
A total of 64 Shigella isolates (31 S. sonnei and 33 S. flexneri including 13 of serotype 2a, 1 of 1a, 9 of 2b, 2 of 4a, 2 of 6, 3 of x variant and 3 of y variant) were collected from Hangzhou First People's Hospital, Yuhang Center for Disease Control and Prevention, and Lingai Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, People's Republic of China, from 1998 to 2002 (Table 1). Among the isolates, 28 S. sonnei and 31 S. flexneri isolates were collected from patients with sporadic diarrhoea, 3 S. sonnei isolates were collected from three patients in one outbreak, and 2 S. flexneri 2a isolates were collected from a patient and a well-water sample involved in another outbreak. The susceptibility of the isolates to ampicillin, streptomycin and trimethoprim was tested by the disc diffusion method on Mueller–Hinton agar, and the MICs of those antibiotics were determined by the agar dilution method, according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS; now the Clinical Laboratories Standards Institute).18,19 Escherichia coli ATCC 25922 was used as a quality control strain for both the disc diffusion method and the agar dilution method. The susceptibility data of Shigella isolates tested were accepted only when the zone diameter or MIC for E. coli ATCC 25922 tested in parallel was within the acceptable ranges given in the NCCLS guidelines.
|
PCR and Southern-blotting analysis
Class 1 integrons were analysed by PCR using three sets of primers as described previously: intI1L and intI1R,20 inF and inB21 and qacE
1-F and sul1-B.21 The primers cover the 5'-conserved segment (5'-CS) (intI1), the cassette region and the 3'-CS of class 1 integrons, respectively (Table 2). The PCR products with primers inF and inB were sequenced to determine the gene cassettes of class 1 integrons. Class 2 integrons were examined by PCR with primers intI2L and intI2R specific for the intI2 gene (Table 2).20 Class 1 integron-containing S. sonnei isolate C103-1 and intI2-positive S. flexineri 2a isolate C101, both confirmed by sequencing their PCR products, were used as positive controls for PCR tests.
|
Genomic DNA and plasmid were isolated from four representative isolates that were positive for intI1 and negative for other gene cassette regions (inF–inB) as determined by PCR. The genomic DNA was then digested with BamHI and PstI. The restricted genomic DNA and the plasmid DNA were then electrophoresed in 1.0% agarose gels, transferred and fixed to nylon membrane, and hybridized to digoxigenin-labelled DNA probes specific for intI1 using the DIG high prime DNA labelling and detection kit (Roche) according to the manufacturer's instructions. A 3.3 kb BamHI–PstI DNA fragment containing intI1 from genomic DNA of S. flexneri 2a isolate C101-1 was cloned into vector pUC19 (termed pUC19-C101) and sequenced (Table 3). An atypical class 1 integron without 3'-CS was identified in this cloned DNA fragment (Figure 1). A pair of primers, intI1ca-F and IS1ca-R (Table 2) that are specific for intI1 and IS1, were designed to amplify the variable region in all Shigella isolates, and the PCR products from one S. sonnei isolate and four S. flexneri isolates with serotypes 2a, 2b, x variant and y variant were sequenced directly. Another pair of primers, inv-oxaR and inv-aadL (Table 2) specific for blaOXA30 and aadA1, was used to identify the gene cassette array (Figure 1).
|
|
To find the location of the class 2 integron in the Shigella genome, HindIII-digested genomic DNA and the plasmid DNA that were isolated from an intI2-positive S. sonnei isolate and an intI2-positive S. flexneri isolate were hybridized to a digoxigenin-labelled probe specific for intI2. A 10.7 kb HindIII-restricted genomic DNA fragment from S. sonnei isolate C202 was cloned into vector pUC19 (termed pUC-C202) (Table 3) and sequenced. A primer set, intI2ca-F and intI2ca-R (Table 2) specific for intI2 and ybfA in Tn7 (Figure 2), was designed to amplify the gene cassettes of class 2 integrons in all Shigella isolates. The amplified products were digested by EcoRII to identify the gene cassette arrays using restriction fragment length polymorphism (RFLP) analysis. Three of these products from three S. flexneri isolates with serotypes 2a, 2b and x variant were directly sequenced.
|
Three primer sets were designed to identify the insert site of Tn7 in Shigella spp. isolates according to the sequences of the right end of Tn7, and glmS known to be upstream of the insertion site of Tn7 in E. coli (accession number V00620 [GenBank] ), and the HindIII-restricted genomic DNA fragment. The primer set of yi41-F/Tn7L-R (Table 2) was used to amplify the joint fragment between IS4 on the chromosomal backbone and the left end of Tn7, and the primer set of Tn7R-F/glmS-R (Table 2) was used to amplify the joint fragment between the right end of Tn7 and glmS on the backbone, whereas the primer set of yi41-F/glmS-R was used to amplify the joint fragment between the IS4 and glmS on the backbone that lacks Tn7 insertion (Figure 2).
The primers used in the present study were either derived from previous publications or designed by primer design software FastPCR (http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm) and Primer 3 online (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
Identification of recombination patterns of gene cassettes
The inverse PCR technique and the conduct assay22 were used to examine the recombination function of the gene cassettes on the atypical class 1 integron without 3'-CS carried in the BamHI–PstI fragment in pUC-C101.
For the inverse PCR analysis, two sets of divergent primers, inv-oxaL/inv-oxaR and inv-aadL/inv-aadR (Table 2) (Figure 1) specific for blaOXA30 and aadA1 cassettes of the atypical class 1 integron, were used to detect free circular cassettes in boiled overnight broth of E. coli strain DH5
containing pUC-C101. The amplified PCR products were sequenced to determine the recombination site.
E. coli strains and plasmids used in the conduct assay are listed in Table 3. The donor E. coli cells (strain UB3p) were constructed using the following steps: (i) plasmid R388 was introduced into E. coli strain UB1637 (Strr) by conjugation. The R388 was a natural conjugative IncW plasmid harbouring a class 1 integron with a gene cassette array of dfrB2 + orfA between 5'-CS and 3'-CS that confers resistance to trimethoprim through its dfrB2 cassette.22 (ii) The R388-containing UB1637 cells were then sequentially transformed with p112 (Ampr), a plasmid expressing the intI1 integrase gene, and pSU-C101 (Kanr) which was constructed by subcloning the BamHI–PstI fragment on pUC-C101 into pSU38 (Kanr).
Overnight cultures of 0.5 mL of donor cells (E. coli strain UB3p) and 0.5 mL of recipient cells [E. coli strain UB5201 (Nalr)] were mixed and pelleted. The mixture was resuspended in fresh Luria–Bertani (LB) broth and plated on LB agar, then incubated at 37°C for 3 h. Dilutions of the mixed cultures were screened on LB media containing nalidixic acid (60 mg/L) and ampicillin (100 mg/L) for transconjugants. Nine transconjugants were randomly selected for identification of gene cassette arrays in the class 1 integrons on the recombination R388s by the RFLP analyses, using XmnI and EcoRII-restricted PCR products that were amplified with primers inF and inB. In addition, the deduced gene cassette arrays from RFLP analysis were confirmed by PCR using primer sets inF/inv-oxaL, inv-oxaR/inv-aadL, inv-aadR/inB and inv-oxaR/inB that are specific for 5'-CS, different cassettes or 3'-CS.
Nucleotide sequence accession numbers
The sequences of the 3.3 kb BamHI–PstI DNA fragment containing the atypical class 1 integron from genomic DNA of S. flexneri 2a isolate C101-1 and the 10.7 kb HindIII-restricted genomic DNA fragment containing a class 2 integron from S. sonnei isolate C202 were deposited in the GenBank database under accession numbers AY574195 and AY639870, respectively.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
Class 1 integrons and gene cassettes in Shigella spp
Among the 31 S. sonnei isolates, 4 (12.9%) were found to be positive for all three markers of class 1 integrons, in which three kinds of gene cassette arrays, aadA2, dfrA17 + aadA5 and dfrA1 + aadA1a, were identified (Tables 1 and 4). Of the 33 S. flexneri isolates, 2 isolates (6.1%), 1 S. flexneri 6 and 1 S. flexneri y variant, were positive for all three markers of class 1 integrons and harboured the gene cassette arrays of dfrV and dfrA17 + aadA5, respectively (Tables 1 and 4). No empty integron [i.e. positive by PCR analysis for gene cassette region (inF–inB) but negative for gene cassettes] was found. All of these four gene cassette arrays found among Shigella spp. isolates in this study are also often present in clinical and environmental isolates of the Enterobacteriaceae.23–27 These findings suggest that the transfer of antibiotic resistance genes can occur through gene cassettes on class 1 integrons among Shigella spp. and other bacteria.
|
Atypical class 1 integron in Shigella spp
For the three markers of class 1 integrons, we found that 29 (87.9%) S. flexneri isolates and 1 (3.2%) S. sonnei isolate were positive for intI1, but were negative for their gene cassette regions (inF–inB). Among the 29 intI1-positive isolates, only 3 S. flexneri isolates were positive for 3'-CS (Table 1), suggesting that these isolates might have an atypical class 1 integron without 3'-CS.
To find the location of the atypical integrons and their gene cassettes, four isolates that were positive for intI1 and negative for gene cassette regions (inF–inB) (2 isolates of S. flexneri 2a, 1 S. flexneri 2b and 1 S. sonnei) were selected, and genomic DNA and plasmid DNA isolated from these isolates were analysed by Southern-blotting analysis with a digoxigenin-labelled probe specific for intI1. A positive band of 3.3 kb was observed in BamHI and PstI-restricted genomic DNA isolated from all four isolates, but no signal was detected in plasmid DNA of these isolates, suggesting that the hypothesized atypical integron is only present in the chromosomes of these isolates. The 3.3 kb fragment from one of the four isolates, S. flexneri 2a isolate C101-1, was then cloned into vector pUC19 (termed pUC-C101) and sequenced. Analysis of the DNA sequence revealed that this atypical integron consists of intI1, attI, two gene cassettes of blaOXA30 and aadA1, and part of IS1 (Figure 1; see more sequence details in Figure S1 in Supplementary data available at JAC Online). Because the atypical integron is negative for class 1 integrons as determined by commonly used PCR, we termed it Shigella atypical class 1 integron (SAI). Due to the confusion in the nomenclature of the aadA family in the public nucleotide sequence database, we termed the aadA1 in the SAI aadA1SAI in this paper.
To confirm the presence of the atypical integron in S. flexneri and S. sonnei isolates, we used PCR with primer set intI1ca-F/IS1ca-R to amplify the variable region of the atypical integron. The gene cassette arrays were then identified by PCR analysis with another primer set inv-oxaR/inv-aadL. Among the 29 S. flexneri and 1 S. sonnei isolates that were positive for intI1 and negative for cassette regions (inF–inB), 28 S. flexneri isolates and 1 S. sonnei isolate were positive in both PCRs. The amplified PCR fragment sizes were 2453 bp when primer set intI1ca-F and IS1ca-R was used and 456 bp when primer set inv-oxaR and inv-aadL was used. Of five nucleotide sequences of the PCR products with primer set intI1ca-F/IS1ca-R from one S. sonnei and four S. flexneri isolates with serotypes 2a, 2b, x variant and y variant, four were 100% identical to that of the gene cassette region of SAI on pUC-C101, and one from S. flexneri 2a isolate had only a synonymous mutation at the 106th glutamic acid codon of aadA1SAI from GAA to GAG. One S. flexneri x variant isolate was negative in both PCRs. No positive PCRs were observed in the six class 1 integron-containing isolates (2 S. flexneri and 4 S. sonnei) and the other intI1 negative isolates (2 S. flexneri and 26 S. sonnei) when primer set intI1ca-F/IS1ca-R was used (Table 1). These results indicate that the atypical integron was present in 84.9% of S. flexneri isolates and 3.2% of S. sonnei isolates in this study. The atypical integron and class 1 integron did not coexist in the isolates examined.
Using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/), we found a DNA fragment on the chromosome of S. flexneri 2a strain YSH6000 with 99.4% sequence similarity to the SAI, which consists of the integrase gene intI1and two antibiotic resistance genes, oxa1 (100% identical to blaOXA30) and aadA1, followed by IS1 (accession number AF326777 [GenBank] ). In strain YSH6000, this DNA fragment and two contiguous determinants of resistance to chloramphenicol and tetracycline are located in a 16.7 kb IS1-flanked element, the Shigella resistance locus (SRL) which is located in a 66 kb pathogenicity island (PAI) on the bacterial chromosome. That region was designated the SRL PAI.11,28 Another similar fragment containing the recombinase gene and blaOXA30 (accession number AF255921 [GenBank] ) was also found in S. flexneri 2a isolates collected from Hong Kong and Shanghai.6 However, integrons have not been annotated to these sequences. Identification of these integrons will be helpful to understand more precisely the role of integrons in the mechanism of horizontal transfer of resistance genes.
Recombination patterns of SAI gene cassettes
In the inverse PCR analysis, two clear bands with lengths of 
1200 and 800 bp were amplified from boiled overnight broth of pUC-C101-containing E. coli strain DH5 by primer sets of inv-oxaL/inv-oxaR and inv-aadL/inv-aadR, respectively. The nucleotide sequence of the 1200 bp band indicated that the free circular molecule consisted of two cassettes of blaOXA30 and aadA1SAI, which was caused by the recombination between attI and attC of the aadA1SAI cassette (Figures 1 and 3). However, a predicted band with a length of 296 bp, suggesting recombination between attI and attC of blaOXA30, was not detected by the inverse PCR using primers inv-oxaL/inv-oxaR. The sequence of the 800 bp in the inverse PCR using primers inv-aadL/inv-aadR revealed the existence of a free circular cassette of aadA1SAI produced by recombination between attC of the blaOXA30 cassette and attC of the aadA1SAI cassette (Figures 1 and 3).
|
In the conduct assay, the gene cassette arrays in class 1 integrons on the recombination R388 were identified in 8 of 9 transconjugants. The array of blaOXA30 + aadA1SAI + dfrB2 + orfA was the most common (found in five transconjugants), followed by the array of blaOXA30 + aadA1SAI found in two transconjugants and the array of blaOXA30 + dfrB2 + dfrB2 + orfA found in one transconjugant. There was one failed identification of a cassette array in one transconjugant in spite of a positive result in the PCR analysis with primers inv-oxaR/inv-aadL. Because media containing nalidixic acid and ampicillin were used to screen for transconjugants, only these transconjugants who received the recombination R388, in the class 1 integron of which blaOXA30 cassette from the SAI on pSU-C101 was integrated, would survive. It was observed that the linked cassette of blaOXA30 and aadA1SAI, rather than a single blaOXA30 cassette, is present in almost all transconjugants, although there was not any known selective pressure favouring the presence of the aadA1SAI gene in the environment.
The above findings indicate that the blaOXA30 and aadA1SAI cassettes of the SAI are coordinately excised or integrated, although the attC site has been identified in each of the two cassettes based on sequence analysis. The reason for the recombination of the two linked cassettes remains unknown, but this pattern may partially explain the phenomenon of why almost all blaOXA30 cassettes found in integrons so far are linked with aadA1 cassettes.10,29–31 Although the SAIs containing ‘blaOXA30 + aadA1SAI’ in the Shigella spp. isolates lacked 3'-CS, class 1 integrons containing the gene cassette array of ‘blaOXA30 + aadA1’ with complete 3'-CS have been reported on plasmids in Salmonella enterica serovar Typhimurium and E. coli strains,29–31 suggesting that horizontal transfer of the cassette array may have existed in these isolates as well.
Class 2 integrons and cassettes in Shigella spp
Class 2 integrons were detected in 80.6% (25/31) of the S. sonnei isolates and 87.9% (29/33) of the S. flexneri isolates. However, class 2 integrons were present in all of the S. flexneri serotype 2a isolates (n = 13) and serotype 2b isolates (n = 9) examined (Table 1). Class 2 integrons were localized on chromosomes by Southern-blotting analysis of HindIII-restricted genomic DNA and plasmid DNA isolated from one S. sonnei isolate and one S. flexneri isolate that are both positive for intI2. A 10.7 kb HindIII-restricted genomic DNA fragment from S. sonnei isolate C202 was cloned into vector pUC19 (termed pUC-C202) (Table 2) and sequenced. The fragment contains partial fimbrial operon, IS4, and the left end of Tn7; it contained a complete class 2 integron, including intI2 and a gene cassette array of dfrA1 + sat + aadA1 + orfX that confer resistance to trimethoprim, streptothricin and streptomycin (Figure 2; see more sequence details in Figure S2 in Supplementary data available at JAC Online). To differentiate the aadA1SAI, the aadA1 in the class 2 integron was termed aadA1Tn7. The identity between nucleotide acid sequences of aadA1SAI and aadA1Tn7 was 99.4% (787/792). Compared with aadA1SAI, the valine codon (GTG) at the 4th amino acid residue was replaced by the alanine codon (GCG) and the glutamic acid codon (GAA) at the 235th amino acid residue was deleted in the sequence of aadA1Tn7.
The 3361 bp DNA fragment over the gene cassette region in the class 2 integron was amplified by PCR in all intI2-positive Shigella spp. isolates using primer set intI2ca-F/intI2ca-R (Table 1). All PCR products shared the same EcoRII-RFLP pattern. In addition, three nucleotide sequences of the PCR products from three S. flexneri isolates with serotypes 2a, 2b and x variant were 100% identical to that of the gene cassette region of the class 2 integron on pUC-C202. These results suggest that all gene cassette arrays in these class 2 integrons were dfrA1 + sat + aadA1 + orfX. In contrast to the various gene cassette arrays found in class 1 integrons, the cassette arrays in class 2 integrons are usually constant, as observed in this and several other studies, which is believed to be due to the mutation of an internal stop codon within intI2.4,13,15–17,23,32
It has been shown that Tn7 can be inserted in a single orientation into a specific target site, attTn7, downstream of the glmUS operon on the E. coli chromosome to minute 82.33 We found here that IS4 presents downstream of glmUS on genomes of S. flexneri 2a strain 2457T (GenBank accession number AE014073) and strain 301 (GenBank accession number AE005674, in which IS4 was annotated yi41), and adjacent to the left end of Tn7 in the HindIII fragment from S. sonnei isolate C202 (Figure 2), suggesting that the Tn7 in S. sonnei isolate C202 may insert into attTn7 downstream of glmUS. This hypothesis was supported by results from sequencing the right end of Tn7 and the 3' end of glmS amplified from isolate C202 using primers Tn7R-F and glmS-R (nucleotide sequence data are shown in Figure S2 in Supplementary data available at JAC Online) (Figure 2).
The Tn7 insertion site in the rest of the Shigella spp. isolates was identified by PCR analysis using three primer sets, yi41-F/Tn7L-R, Tn7R-F/glmS-R and yi41-F/glmS-R. Tn7s were inserted at the same site, attTn7, in all 54 intI2-positive Shigella spp. isolates, since the PCRs were positive for primer sets yi41-F/Tn7L-R (610 bp) and Tn7R-F/glmS-R (560 bp), but negative for primer set yi41-F/glmS-R (too far to be amplified). In contrast, the remaining 10 intI2-negative isolates were negative in both PCRs with primer sets yi41-F/Tn7L-R and Tn7R-F/glmS-R. Of these 10 isolates, 6 S. sonnei isolates were positive in PCRs using the primer set yi41-F/glmS-R (472 bp), indicating no Tn7 insertion; however, 4 S. flexneri isolates (2 of serotype 6 and 2 of serotype y variant) were negative in the same PCRs.
To our knowledge, this is the first experimental identification of Tn7 insertion in Shigella spp. The location of Tn7 on Shigella chromosome can explain the phenomenon observed in an earlier study that co-transfer of streptomycin and trimethoprim resistance encoded by the class 2 integron in S. sonnei was not observed in conjugation experiments,13 since the class 2 integron was not located on a conjugative plasmid.
Resistance conferred by class 1 integrons, SAI and class 2 integrons
All 33 tested isolates (17 S. sonnei and 16 S. flexneri) harbouring class 2 integrons were resistant to both trimethoprim and streptomycin. However, of the six S. sonnei isolates without class 2 integrons, four isolates (two with class 1 integrons containing the aadA2 cassette and two without class 1 integrons) were susceptible to trimethoprim and two isolates with class 1 integrons containing cassettes dfrA17 + aadA5 or dfrA1 + aadA1a were resistant to trimethoprim, whereas five isolates were susceptible to streptomycin, though two, one and one of them contained class 1 integrons with cassettes of aadA2, dfrA17 + aadA5 and dfrA1 + aadA1a, respectively, but one other isolate without class 1 integron was resistant to streptomycin (Table 4). Moreover, of four S. flexneri isolates without class 2 integrons, two isolates (one serotype 6 and one serotype y variant) containing class 1 integrons with cassettes of dfrV or dfrA17 + aadA5 were resistant to both trimethoprim and streptomycin (Table 4), whereas other two isolates without class 1 integron (one of serotype 6 and one of serotype y variant) were susceptible to both.
The above findings suggest that (i) class 2 integrons confer resistance to trimethoprim and streptomycin and (ii) class 1 integrons that contain cassettes of dfrV, dfrA17 + aadA5 or dfrA1 + aadA1a confer resistance to trimethoprim, in Shigella spp. isolates. In addition, introduction of pUC-C202 into E. coli DH5 conferred host resistance to both trimethoprim and streptomycin, confirming that the class 2 integron is responsible for resistance to both trimethoprim and streptomycin.
All Shigella isolates carrying the SAI (found in 28 S. flexneri isolates and 1 S. sonnei isolate) were resistant to ampicillin, but some of the isolates that contain no SAI were also resistant to ampicillin (3 of 4 S. flexneri isolates tested and 9 of 30 S. sonnei isolates were resistant), suggesting that the SAI, as well as other determinants, are responsible for host resistance to ampicillin in Shigella isolates. Moreover, the SAI-mediated resistance to ampicillin was directly demonstrated by acquisition of resistance to ampicillin in susceptible host E. coli DH5 after transformation with pSU-C101 plasmid containing the SAI element.
Because all Shigella isolates carrying the SAI also contain class 2 integrons, the SAI-mediated streptomycin resistance could not be evaluated in these isolates. To our surprise, however, although the MIC for the transformant (E. coli DH5 harbouring pUC-C101) of streptomycin, as determined by the agar dilution method, was increased from 0.25 mg/L in the control transformant (E. coli DH5 harbouring pUC19) to 4.0 mg/L, introduction of pUC-C101 into E. coli DH5 did not confer resistance to streptomycin, as shown in a test using the disc diffusion method.
The gene cassettes of aadA2, dfrA17 + aadA5 or dfrA1 + aadA1a of class 1 integrons and blaOXA30 + aadA1 of the SAI element did not confer resistance to streptomycin in Shigella spp. in this study. Similar observations were made in several previous reports. Roe et al.34 reported that resistance to streptomycin was not observed in three E. coli isolates harbouring a class 1 integron that contains a single cassette of aadA1, but resistance to kanamycin in the same antibiotic class was observed. White et al.35 observed that, although an E. coli isolate harbouring a class 1 integron that contains cassettes of ‘dfrA17 + aadA5’ was resistant to both trimethoprim and streptomycin, dfrA17 alone conferred a high level of resistance to trimethoprim, whereas aadA5 conferred resistance to spectinomycin but not to streptomycin.
Conclusions
The genetic characteristics of class 1, SAI and class 2 integrons and their association with antibiotic resistance were examined in clinical Shigella isolates collected in Hangzhou, China. It was observed that the gene cassettes of class 1, SAI and class 2 integrons are responsible for mediating resistance to commonly used antibiotics, such as trimethoprim, streptomycin and ampicillin, in these Shigella spp. isolates. Our data indicate that multiple and complex mechanisms involving class 1 and class 2 integrons and antibiotic resistance have been developed in the evolution of Shigella strains.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
We thank D. Mazel for kindly providing plasmids and E. coli strains used in the conduct assay and Kebin Liu for critical review of the manuscript. This work was supported by Grant for Youth from Health Bureau of Zhejiang Province, China.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
|---|
1 Kotloff KL, Winickoff JP, Ivanoff B, et al. (1999) Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 77:651–66.[Web of Science][Medline]
2 Anon. (2005) Shigellosis: disease burden, epidemiology and case management. Wkly Epidemiol Rec 80:94–9.[Medline]
3
Ashkenazi S, Levy I, Kazaronovski V, et al. (2003) Growing antimicrobial resistance of Shigella isolates. J Antimicrob Chemother 51:427–9.
4
Oh JY, Yu HS, Kim SK, et al. (2003) Changes in patterns of antimicrobial susceptibility and integron carriage among Shigella sonnei isolates from southwestern Korea during epidemic periods. J Clin Microbiol 41:421–3.
5 Qu F, Bao CM, He JY, et al. (2004) Distribution and antimicrobial resistance of Shigella spp isolated from diarrheal patients between 1993 and 2002 in Beijing. J Chinese Antibiotic 29:671–4.
6
Siu LK, Lo JY, Yuen KY, et al. (2000) ß-Lactamases in Shigella flexneri isolates from Hong Kong and Shanghai and a novel OXA-1-like ß-lactamase, OXA-30. Antimicrob Agents Chemother 44:2034–8.
7
Sansonetti PJ, Kopecko DJ, Formal SB. (1981) Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. Infect Immun 34:75–83.
8 Tonin PN and Grant RB. (1987) Genetic and physical characterization of trimethoprim resistance plasmids from Shigella sonnei and Shigella flexneri. Can J Microbiol 33:905–13.[Medline]
9
Heikkila E, Skurnik M, Sundstrom L, et al. (1993) A novel dihydrofolate reductase cassette inserted in an integron borne on a Tn21-like element. Antimicrob Agents Chemother 37:1297–304.
10
Turner SA, Luck SN, Sakellaris H, et al. (2003) Molecular epidemiology of the SRL pathogenicity island. Antimicrob Agents Chemother 47:727–34.
11 Rajakumar K, Bulach D, Davies J, et al. (1997) Identification of a chromosomal Shigella flexneri multi-antibiotic resistance locus which shares sequence and organizational similarity with the resistance region of the plasmid NR1. Plasmid 37:159–68.[CrossRef][Web of Science][Medline]
12 Iversen J, Sandvang D, Srijan A, et al. (2003) Characterization of antimicrobial resistance, plasmids, and gene cassettes in Shigella spp. from patients in Vietnam. Microb Drug Resist 9:Suppl 1, S17–24.[Medline]
13
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. J Clin Microbiol 41:1919–24.
14
Navia MM, Capitano L, Ruiz J, et al. (1999) Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. J Clin Microbiol 37:3113–7.
15
Peirano G, Agerso Y, Aarestrup FM, et al. (2005) Occurrence of integrons and resistance genes among sulphonamide-resistant Shigella spp. from Brazil. J Antimicrob Chemother 55:301–5.
16
Mammina C, Pontello M, Dal Vecchio A, et al. (2005) Identification of Shigella sonnei biotype g isolates carrying class 2 integrons in Italy (2001–2003). J Clin Microbiol 43:2467–70.
17
McIver CJ, White PA, Jones LA, et al. (2002) Epidemic strains of Shigella sonnei biotype g carrying integrons. J Clin Microbiol 40:1538–40.
18 National Committee for Clinical Laboratory Standards. (2000) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Fifth Edition: Approved Standard M7-A5 (NCCLS, Wayne, PA, USA).
19 National Committee for Clinical Laboratory Standards. (2000) Performance Standards for Antimicrobial Disk Susceptibility Tests—Seventh Edition: Approved Standard M2-A7 (NCCLS, Wayne, PA, USA).
20
Ploy MC, Denis F, Courvalin P, et al. (2000) Molecular characterization of integrons in Acinetobacter baumannii: description of a hybrid class 2 integron. Antimicrob Agents Chemother 44:2684–8.
21
Dalsgaard A, Forslund A, Serichantalergs O, et al. (2000) Distribution and content of class 1 integrons in different Vibrio cholerae O-serotype strains isolated in Thailand. Antimicrob Agents Chemother 44:1315–21.
22 Martinez E and de la Cruz F. (1990) Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J 9:1275–81.[Web of Science][Medline]
23
White PA, McIver CJ, Rawlinson WD. (2001) Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 45:2658–61.
24
Guerra B, Junker E, Schroeter A, et al. (2003) Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J Antimicrob Chemother 52:489–92.
25
Chang CY, Chang LL, Chang YH, et al. (2000) Characterisation of drug resistance gene cassettes associated with class 1 integrons in clinical isolates of Escherichia coli from Taiwan, ROC. J Med Microbiol 49:1097–102.
26 Rosser SJ and Young HK. (1999) Identification and characterization of class 1 integrons in bacteria from an aquatic environment. J Antimicrob Chemother 44:11–8.[Abstract]
27
Yu HS, Lee JC, Kang HY, et al. (2003) Changes in gene cassettes of class 1 integrons among Escherichia coli isolates from urine specimens collected in Korea during the last two decades. J Clin Microbiol 41:5429–33.
28
Luck SN, Turner SA, Rajakumar K, et al. (2001) Ferric dicitrate transport system (Fec) of Shigella flexneri 2a YSH6000 is encoded on a novel pathogenicity island carrying multiple antibiotic resistance genes. Infect Immun 69:6012–21.
29
Villa L and Carattoli A. (2005) Integrons and transposons on the Salmonella enterica serovar Typhimurium virulence plasmid. Antimicrob Agents Chemother 49:1194–97.
30
Dubois V, Arpin C, Quentin C, et al. (2003) Decreased susceptibility to cefepime in a clinical strain of Escherichia coli related to plasmid- and integron-encoded OXA-30 ß-lactamase. Antimicrob Agents Chemother 47:2380–1.
31
Antunes P, Machado J, Sousa JC, et al. (2004) Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 ß-lactamase. J Antimicrob Chemother 54:429–34.
32
Hansson K, Sundstrom L, Pelletier A, et al. (2002) IntI2 integron integrase in Tn7. J Bacteriol 184:1712–21.
33 Lichtenstein C and Brenner S. (1982) Unique insertion site of Tn7 in the E. coli chromosome. Nature 297:601–3.[CrossRef][Medline]
34 Roe MT, Vega E, Pillai SD. (2003) Antimicrobial resistance markers of class 1 and class 2 integron-bearing Escherichia coli from irrigation water and sediments. Emerg Infect Dis 9:822–6.[Web of Science][Medline]
35 White PA, McIver CJ, Deng Y, et al. (2000) Characterisation of two new gene cassettes, aadA5 and dfrA17. FEMS Microbiol Lett 182:265–9.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.-C. Pan, R. Ye, H.-Q. Wang, H.-Q. Xiang, W. Zhang, X.-F. Yu, D.-M. Meng, and Z.-S. He Vibrio cholerae O139 Multiple-Drug Resistance Mediated by Yersinia pestis pIP1202-Like Conjugative Plasmids Antimicrob. Agents Chemother., November 1, 2008; 52(11): 3829 - 3836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gassama Sow, M. H. Diallo, M. Gatet, F. Denis, A. Aidara-Kane, and M.-C. Ploy Description of an unusual class 2 integron in Shigella sonnei isolates in Senegal (sub-Saharan Africa) J. Antimicrob. Chemother., October 1, 2008; 62(4): 843 - 844. [Full Text] [PDF]
|
|
|
|
|
|
A. Tsakris, A. Ikonomidis, N. Spanakis, A. Poulou, and S. Pournaras Characterization of In3Mor, a new integron carrying VIM-1 metallo-{beta}-lactamase and sat1 gene, from Morganella morganii J. Antimicrob. Chemother., April 1, 2007; 59(4): 739 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Dubois, M.-P. Parizano, C. Arpin, L. Coulange, M.-C. Bezian, and C. Quentin High Genetic Stability of Integrons in Clinical Isolates of Shigella spp. of Worldwide Origin Antimicrob. Agents Chemother., April 1, 2007; 51(4): 1333 - 1340. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||