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Characterization of class I integrons in clinical strains of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis from Norwegian hospitals

¹Norwegian Institute of Public Health, Division of Infectious Diseases Control, N-0403 Oslo, Norway ²Department of Pharmacology, Microbiology and Food Hygiene, Norwegian School of Veterinary Sciences, N-0033 Oslo, Norway

Correspondence Bjørn-Arne Lindstedt bjorn-arne.lindstedt{at}folkehel sa.no

Received 7 May 2002 Accepted 9 October 2002

	Abstract

TOP Abstract Introduction METHODS RESULTS AND DISCUSSION REFERENCES

 
The characterization of integrons and their promoters in 156 antibiotic-resistant clinical isolates of the important zoonotic pathogens Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) from Norwegian hospitals were performed. Integrons were found in 64 of 66 S. Typhimurium isolates (97 %) and in 20 of 90 S. Enteritidis isolates (22.2 %) with the following sizes; 650, 1000, 1200, 1500, 1600, 1700, 2000 and 2100 bp. The integrons were further sequenced and the aadA1, aadA2, aadA5, aadB, pse-1, catB3, oxa1, dfrA1, dfrA12 and dfrA17 genes, as well as a fragment of the sat1 gene, were found embedded in cassettes. An internal fragment of the purG gene was additionally found as an artefact PCR amplicon.

	Introduction

TOP Abstract Introduction METHODS RESULTS AND DISCUSSION REFERENCES

 
Integrons are genetic units that include components of a site-specific recombination system enabling them to capture and mobilize genes. Integrons contain a gene of the family of integrases that carries out recombination between two distinct target sites; the attI site and the 59-base element where attI is the target site for cassette integration (Hall & Collis, 1995; Hall & Stokes, 1993). The genes captured by this system are packaged as small discrete units with essentially all flanking sequences removed, but with the 59-base element included, and are named ‘gene cassettes’ (Hall & Collis, 1995; Hall & Stokes, 1993). Integrons are believed to be ancient structures (Rowe-Magnus et al., 2001), although the mechanism that created these cassettes is at present unknown. The majority of known gene cassettes are antibiotic resistance genes, but it is likely that any gene could be included in a cassette and several uncharacterized ORFs have been found in cassettes. Integrons are classified by comparison of the amino acid sequence of their integrases, encoded by the intI genes. Four integron types have been identified, but in recent studies additional integron classes have been reported (Rowe-Magnus et al., 2001; Nield et al., 2001). The majority of integrons found in clinical isolates of enterobacteria are of class I. More than 60 different antibiotic resistance genes have been identified within cassette structures either alone or in combination (Mazel & Davies, 1999).

A promoter region is located in the 5'-conserved segment of the integron and contains two promoters named P_c (P_ant) and P2. Four different P_c and two different P2 promoters have been described in the literature (Stokes & Hall, 1989; Bunny et al., 1995) and their relative strength as compared to the Escherichia coli tac promoter has been reported (Lévesque et al., 1994; Collis & Hall, 1995). Two of the P_c promoters are designated strong (TTGACA-N₁₇-TAAACT) and weak (TGGACA-N₁₇-TAAGCT), and two are named hybrid promoters, with the strong promoter six times more effective than the E. coli tac promoter. The tac promoter is, however, more efficient than the weak cassette promoter and the two hybrid promoters, TGGACA-N₁₇-TAAACT and TTGACA-N₁₇-TAAGCT (Lévesque et al., 1994; Collis & Hall, 1995). The P2 promoter has an active version TTGTTA-N₁₇-TACAGT, but when the -35 and -10 regions are separated by 14 nt, TTGTTA-N₁₄-TACAGT, there is no P2 promoter activity.

Integrons are widespread and have been studied in great detail among the Enterobacteriaceae, with a particular focus on the multi-resistant Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) definitive type (DT) 104, while studies on integrons among Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) isolates are limited. The incidence of S. Enteritidis infections has increased in many countries, and in Europe this serotype is now predominant among human Salmonella isolates. The increase began as early as the mid-1970s and S. Enteritidis displaced S. Typhimurium as the primary cause of infection by 1990 (Kingsley & Baumler, 2000; Baumler et al., 2000; Guard-Petter, 2001). While other Salmonella serotypes have been shown to experimentally infect eggs, S. Enteritidis is the only human pathogen that causes frequent human illness associated with egg contamination (Guard-Petter, 2001). S. Enteritidis is therefore an important zoonotic pathogen in which increased resistance to several antibiotics will pose an increased threat.

This study presents the result of characterization of integrons and their promoters in 156 multi-resistant strains of the important zoonotic pathogens S. Typhimurium and S. Enteritidis from human clinical isolates (hospitalized patients) submitted to the Reference Laboratory for enteropathogenic bacteria at the Norwegian Institute of Public Health from hospitals throughout Norway.

	METHODS

TOP Abstract Introduction METHODS RESULTS AND DISCUSSION REFERENCES

 
Strain selection

S. Typhimurium.

In all 66 antibiotic-resistant isolates of S. Typhimurium from 22 cases of domestically acquired infections and 44 cases of infections acquired abroad, were obtained from the strain collection at the reference laboratory for enteropathogenic bacteria at the Norwegian Institute of Public Health, Oslo, Norway. The strains were isolated from 1996 to 2001. A total of 53 isolates were phage-typed as DT104 and 22 of these had the ACSSuT (see below for definition of abbreviations) resistance profile (Tables 1 and 2).

View this table: [in this window] [in a new window]   Table 3.Characteristics of the S. Enteritidis isolates containing integrons All the integron-positive isolates were positive for intI1, qacE1 and sulI. All the integrons were localized on plasmids. NDA, Not domestically acquired, but country of origin uncertain.

Antimicrobial resistance.

Antimicrobial susceptibility was tested by a tablet diffusion method according to the manufacturer's guidelines (Rosco Diagnostics). The isolates were screened for resistance to ampicillin (A), ciprofloxacin (Cx), tetracycline (T), chloramphenicol (C), nalidixic acid (Na), trimethoprim-sulfamethoxazole (Ts), sulphadiazine (Su), streptomycin (S), gentamicin (Gn) and doxycycline (Dx). Only S. Enteritidis strains that were resistant to two or more antibiotics, and S. Typhimurium strains resistant to four or more antibiotics were included in the study.

Integron PCR.

A number of PCR primers were used for amplification of gene cassettes and genes known to be associated with integrons as well as a gene associated with transposons, including Tn21, Tn501 and Tn1696. These genes include intI1, intI2, intI3, sulI1, qacE1, merA and tniA. All strains were tested with markers for the integrase gene intI1, the quaternary ammonium compound resistance gene qacE1 and the sulphonamide resistance gene sulI, as well as for the integrated gene cassettes. Integrons have been located within transposons and all isolates were additionally tested for the presence of the transposase gene tniA that can be associated with integron In2 (Partridge et al., 2001) and the mercury resistance gene merA associated with the transposon Tn21 as well as others. Primers for amplification of the antibiotic resistance gene floR were included (Table 4). To examine the cassette promoters, an upper primer situated at the 3' end of the intI1 gene coupled with lower primers situated in the aadA1, aadA2, aadB, pse-1, oxa1, dfrA1, dfrA12 and dfrA17 genes were used. See Figs 1 and 2 for the location of the most used primer sets. The primer in the intI1 gene was placed such that the P_c and the P2 promoter sequences, as well as the binding site for the integron cassette-A primer, would be included in the amplified PCR product. Furthermore, primers for fully sequencing the middle regions of the longer cassettes were designed from the end sequences generated by the vector-localized T7 and T3 universal primers.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Location of the PCR primers used to amplify the integron variable regions containing the inserted gene cassettes (cassette-A and cassette-B), the primer set to amplify the disinfectant resistance gene qacE1 and the primer set for the sulI1 gene. Black circles denote the location of the 59-base element, attI is the cassette integration site and P denotes promoters.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Location of the PCR primers used to amplify genes associated with the S. Typhimurium DT104 integron region. The genes are aadA2, floR and pse-1.

The temperature profile was: 94 °C denaturation for 5 min followed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, then a 7 min extension step at 72 °C on a Perkin-Elmer GeneAmp PCR system 9700. The primers in the floR, sulI1, merA and tniA genes had the same profile but with annealing at 60 °C.

Cloning and sequencing.

The PCR amplicons from the cassette primers were cloned directly into the vector PCR-Script Amp using the PCR-Script Amp Electroporation-competent Cell Cloning Kit (Stratagene) after purification with the QIAquick PCR purification kit (Qiagen). In some cases bands were cut out from the agarose gel and DNA was extracted with the QIAquick gel extraction kit (Qiagen) before cloning. The ligated vector + amplicon construct was transformed into Epicurian Coli XL-1 Blue MRF' electroporation competent cells (Stratagene) with a Gene Pulser II (Pulse Controller Plus; Bio-Rad) set at 400 resistance, 2.5 kV voltage and 25 µF capacitance using a 0.2 cm cuvette. The transformed cells were incubated for 1 h at 37 °C in SOC medium before plating on IPTG/X-Gal plates with 50 µg ampicillin ml^-1 for blue-white screening of positive inserts. The plates were incubated overnight at 37 °C and cooled 1 h at 4 °C to intensify the blue-coloured colonies. White colonies were picked and incubated overnight in LB medium with 50 µg ampicillin ml^-1 added. Plasmids were then isolated with the Wizard Plus SV Minipreps DNA Purification System (Promega). All the isolated plasmids were digested for 1 h at 37 °C with the restriction endonucleases PstI and NotI (New England Biolabs) and separated on a 1 % GTG agarose gel to confirm insert sizes. Plasmids containing inserts were subjected to cycle sequencing with the BigDye Terminator kit (ABI biosystems) from both ends with the T7 and T3 universal sequencing primers. The sequence reaction was separated by capillary electrophoresis on an ABI-310 Genetic Analyser (ABI biosystems) with POP-6 polymer. Some of the amplicons from the cassette primers were in addition subjected to direct sequencing after purification with the QIAquick PCR Purification kit (Qiagen) using the BigDye Terminator kit (ABI biosystems) with 3.2 pmol of either the cassette-A or cassette-B primer (Table 4). All sequencing reactions were purified with the DyeEx Spin Kit (Qiagen) to remove unincorporated dye terminators prior to electrophoresis. The promoter regions for all the found cassette amplicons were subjected to direct sequencing of both strands with 3.2 pmol of the appropriate primer sets.

Sequence analysis.

The sequence raw data were exported from the ABI-310 into the Lasergene software package (DNASTAR). All sequence comparisons and alignments were done using this software which additionally performed on-line searches for related sequences via the National Center for Biotechnology Information (NCBI) BLAST server.

Plasmid isolation and hybridization.

Plasmids were isolated from all strains as detailed by Kapperud et al. (1989) and separated by 1 % agarose gel electrophoresis. The gels were photographed under UV light after ethidium bromide staining. Plasmid DNA was transferred to Hybond-N+ membranes (Amersham-Pharmacia Biotech) by vacuum blotting and hybridized at 42 °C using synthetic oligonucleotide probes directed at the intI1, sulI and qacE1 genes with the ECL 3'-oligolabelling and detection system (Amersham-Pharmacia Biotech).

	RESULTS AND DISCUSSION

TOP Abstract Introduction METHODS RESULTS AND DISCUSSION REFERENCES

 
Integron markers

Sixty-six antibiotic-resistant S. Typhimurium and 90 S. Enteritidis isolates, all of human origin, were tested for the presence of known markers for integrons by PCR amplification. The integrase intI1 gene was detected in 91 % (60/66) of the S. Typhimurium isolates; only strains 10403, 700/98, 5031/00, 6347/00, 4805/00 and 5790/00 failed to give an amplicon after repeated attempts with the intI1 primer set. The isolates were tested with primers for two additional known integron integrases, intI2 and intI3, which both failed to amplify a fragment. In the S. Typhimurium isolates 5031/00 and 6347/00 no amplicons from the gene cassette primers were detected. In isolates 10403, 700/98, 4805/00 and 5790/00 gene cassettes were found with the cassette primers, indicating a possible deletion in the intI1 gene upstream from the 5' conserved segment cassette primer-binding site, or the presence of a different class of integrase. The most common amplicons were 1000 and 1200 bp that predominantly occurred in combination and were detected in 51 of the 66 (77.3 %) S. Typhimurium isolates. The 1000 bp amplicon occurred alone in two S. Typhimurium isolates (3 %) and the remaining S. Typhimurium amplicons had sizes of 200, 1500, 1600, 1700, 2000 or 2100 bp. A notable feature is that the vast majority of S. Typhimurium isolates carried two gene cassettes; only 10 S. Typhimurium isolates (15.2 %) displayed single bands from the cassette primers. The disinfectant resistance gene qacE1 was absent in one S. Typhimurium isolate (1.5 %) and the sulphonamide resistance gene sulI was absent in seven S. Typhimurium isolates (10.6 %). Only one S. Typhimurium (1.5 %) isolate (2755/97) was positive for the tniA gene which has been associated with the In0, In2, In5 and Tn402(In16) integrons (Radstrom et al., 1994; Brown et al., 1996; Hall et al., 1994).

All the integron-positive S. Enteritidis isolates (Table 3) contained the intI1, qacE1 and sulI genes. The merA marker was found in all the S. Enteritidis isolates with the dfrA1-aadA1 integron, but none of the S. Enteritidis isolates were positive for the tniA marker.

S. Typhimurium gene cassettes

All the 1000 bp amplicons were shown to contain the aadA2 gene that confers resistance to streptomycin and spectinomycin with a perfect match (100 % similarity) to the aadA2 gene from S. Typhimurium DT104 (accession no. AF071555; Briggs & Fratamico, 1999). The 1200 bp amplicons contained the pse-1 ß-lactamase gene with a perfect match to the pse-1 gene from S. Typhimurium DT104 (accession no. AF071555; Briggs & Fratamico, 1999). The aadA2 gene cassette was seen alone in two strains (4380/00 and 7192/00) which also failed to amplify products with pse-1-specific primers. The pse-1 cassette thus appears to be lost in these strains. No isolates carried the pse-1 gene cassette alone without an accompanying aadA2 cassette. The 1500 bp amplicon was found to carry two gene cassettes in tandem, consisting of the aadB and the catB3 genes with a perfect match to the integron In-t1 (accession no. AJ009818; Tosini et al., 1998). The 2000 bp amplicon carried two known genes, dfrA12 and aadA2, as well as an ORF and was present in a DT104a isolate. This integron is discussed below in the S. Enteritidis section. The 2100 bp amplicon carried two tandemly inserted gene cassettes consisting of the oxa1 and the aadA1 genes with a perfect match to the In-t2 integron (accession no. AJ009819; Tosini et al., 1998). Tosini et al. (1998) found a third integron (In-t3) of 3200 bp that was localized to an IncL/M plasmid. This 3200 bp integron was absent in our isolates, which appear to contain only the IncFI plasmid. The 1700 bp amplicon revealed two gene cassettes consisting of the dfrA17 and the aadA5 genes. The 1600 bp amplicon contained gene cassettes with the dfrA1 and aadA1 genes and was found in one isolate (7542/00) from Belgium. This dfrA1-aadA1-containing integron was confirmed to be plasmid-located.

S. Typhimurium integron-combinations and structure

The most common feature in S. Typhimurium was the presence of two integrons, one containing aadA2 and the other the pse-1 gene. This combination has been observed several times and is characteristic for the genetically homogeneous S. Typhimurium DT104 strain which has emerged to become the most common type of S. Typhimurium isolate in several countries and is predominantly associated with resistance to multiple antibiotics. We failed to get any integron variable region amplicons for only two isolates (5031/00 and 6347/00) using the cassette primers, but isolate 6347/00 was positive for both aadA2 and pse-1 when primers directed at these genes were used, and isolate 5031/00 was positive for aadA2. It is likely that these genes are located as cassettes, but deletions in the 5' integron structures that affect the upper cassette primer possibly prevent PCR amplification. These two strains also carried the integron-associated qacE1 gene and one strain (6347/00) also carried the sulI gene, which may further indicate the presence of a 5' deleted integron structure. Another possible, but less likely explanation would be that the aadA2 and pse-1 genes in these isolates are not localized within integrons. Isolate 5031/00 (DT104) is additionally negative for the sulI and the floR genes, which again suggest extensive deletions in the integron region of this isolate. Both domestically acquired isolates and isolates from patients that have been abroad show the same dominance of the DT104 integron combination as would be expected from the known frequency of this phage type worldwide. The 1600 bp amplicon from isolate 7542/00 with the dfrA1-aadA1 gene combination, conferring resistance to trimethoprim and streptomycin, has recently been seen in Irish S. Typhimurium animal and food isolates (Daly & Fanning, 2000) as well as in Norwegian animal isolates (Sunde & Sørum, 1999). Two isolates (3428/98 and 900/98, Table 2) from infants that had returned from travelling in Africa showed two recently published integron structures, In-t1 and In-t2 (Tosini et al., 1998) with sizes of 1500 and 2100 bp, respectively. These two integrons have previously been identified in S. Typhimurium isolates from Albanian infants with acute gastroenteritis (Tosini et al., 1998). The 1500 bp In-t1 amplicon contained the aadB gene that has been shown to confer resistance to kanamycin and low levels of gentamicin (Shaw et al., 1993), and the catB3 gene that encodes the enzyme chloramphenicol acetyltransferase (CatB3) (Bunny et al., 1995). The 2100 bp In-t2 amplicon contained the oxa1 gene that encodes a ß-lactamase and the aadA1 gene that encodes the aminoglycoside adenyltransferase AAD(3'')-Ia enzyme which confers streptomycin resistance. These two integrons were both located on an IncFI plasmid in the Albanian isolates (Tosini et al., 1998; Carattoli et al., 2001), and also in our two isolates the integrons were confirmed by plasmid isolation and hybridization to be located on plasmids. This is the second report confirmed by sequencing of S. Typhimurium isolates that carry a plasmid with both In-t1 and In-t2 where all isolates in both studies originated from infants with gastroenteritis. One isolate (1864/96) carried only a plasmid-borne In-t1 integron. This was a 28-year-old patient infected in India. The second IncL/M plasmid was absent in our isolates with the In-t1 and In-t2 integrons. This can possibly be ascribed to the higher conjugation frequency of the IncFI plasmid as compared to the IncL/M plasmid, giving it a higher potential to transfer between isolates. Isolate 3428/98 is additionally positive for the mercury resistance gene merA that is associated with transposon Tn21 and some other known transposons, while isolate 900/98 is not. The In-t2 integron of 3428/98 is thus likely to be part of a larger transposon structure. The 200 bp amplicon was not consistently detected and was both present and absent in parallels of the same isolate, and after gel electrophoresis this 200 bp amplicon was always a fainter band than the amplicons of 1000 bp or longer. The 200 bp amplicon contained an ORF that was an internal segment of the purG gene encoding phosphoribosylformylglycinamide synthetase and has been described as a small integron in S. Typhimurium isolates from the Republic of Ireland amplified with the cassette primers (Daly et al., 2000). We found that this 200 bp amplicon was a PCR artefact on closer examination of the sequence and after negative test PCRs with a primer set with one primer in the intI1 gene and one in the purG fragment (IPUR-U/IPUR-L) (Table 4). The absence of the disinfectant gene qacE1 in one strain and the sulphonamide resistance gene in seven, indicates a possible increase in gene loss at the integron 3'-conserved segment region as noted by Hall et al. (1994). Isolate 8709/00 is a DT104 strain with an ACSSuT resistance profile, but the Pse-1 protein in this isolate is truncated as a consequence of a frameshift caused by a T insertion in the coding sequence just downstream for the ATG initiation codon. This truncated protein is believed to be non-functional. The 8709/00 isolate is still resistant to ampicillin which can only be explained by the presence of another functional ß-lactamase, either chromosomal or plasmid-borne. The integron in strain 5180/00 was shown to contain the dfrA17 and aadA5 genes. The dfrA17 gene confers resistance to trimethoprim and the recently described aadA5 gene (Sandvang, 1999) confers resistance to spectinomycin, but not to streptomycin (White et al., 2000). A sequence homology search in GenBank revealed three homologous integron sequences where two were from E. coli isolates and one was an unpublished sequence from a Klebsiella pneumoniae isolate. The first E. coli dfrA17-aadA5 integron was from Australia and most likely had a chromosomal location (accession no. AF169041), the strain having been isolated from the urinary tract of a hospitalized patient (White et al., 2000). The other E. coli dfrA17-aadA5 integron was from a clinical urine specimen isolated in Taiwan; this integron (accession no. AF170088) was located on a 119 kb transferable plasmid (Chang et al., 2000a). The third homologue to the 5180/00 dfrA17-aadA5 integron was an unpublished sequence from a K. pneumoniae isolate (accession no. AF220757). The 5180/00 S. Typhimurium strain was isolated from a patient infected in the Mediterranean region and carries the merA gene, which suggests that this integron could be transposon-associated. The plasmid preparation from strain 5180/00 in our study was negative for the integron-associated intI1, qacE1 and sulI genes, making the integron most likely chromosomal in our isolate. This appears to be the first finding of this integron structure in S. Typhimurium, and the possible association with a merA-containing transposon will facilitate its further movement between strains.

When we only considered the non-DT104 isolates we detected the aadB-catB3 integron in three isolates, the oxa1-aadA1 integron in five isolates, the aadA2 integron in two isolates together with the pse-1 integron, the dfrA17-aadA5 integron in one isolate and the dfrA12-orf-aadA2 and dfrA1-aadA1 integrons also in one isolate each. The pse-1 sequence in the 1200 bp cassette of strain 8709/00 displayed an insertion of an extra T nucleotide at nt 466 that would cause a frameshift and thus early termination at nt 510 in the 8709/00 pse-1 gene as compared to the pse-1 sequence from GenBank (accession no. AF313472).

S. Enteritidis integrons

In this report we additionally describe the presence of class I integrons among isolates of the important zoonotic pathogen S. Enteritidis from hospitals throughout Norway, isolated from 2000 to 2001, which makes them suited for describing the present situation concerning the frequency of integron-containing isolates of S. Enteritidis in Norway. The level of S. Enteritidis infections acquired domestically is very low in Norway. The majority of patients with illness caused by S. Enteritidis are infected abroad. Twenty of the investigated resistant isolates of S. Enteritidis contained integrons (22.2 %); in 2000 16.7 % (6/36) of the isolates contained integrons while in 2001 this number had risen to 25.9 % (14/54).

Gene cassette amplicons of 650, 1600 and 2000 bp were found. The 650 bp amplicon was shown to contain a fragment of the sat1 gene, which in its full version gives resistance to streptothricin (Tschape et al., 1984; Heim et al., 1989). The 1600 bp amplicons were found to contain two integron gene cassettes, dfrA1 and aadA1. The 2000 bp amplicons were found to carry an array of three cassettes, dfrA12-orf-aadA2. The dfrA12 gene encodes a variant of the dihydrofolate reductase gene and the aadA2 gene confers resistance to streptomycin. The dfrA12 gene product confers a 10-times higher level of resistance to trimethoprim than the most common trimethoprim resistance gene dfraA1 (Heikkila et al., 1993). The dfrA12-orf-aadA2 gene cluster has previously been seen in several strains, including an E. coli O157 isolate (GenBank accession no. AF335108, unpublished), a Serratia marcescens isolate (GenBank accession no. AF284063, unpublished), a Spanish S. Typhimurium phage type U302 isolate (Guerra et al., 2001; Echeita et al., 2001), an E. coli Shiga-toxin producing O111 : H8 isolate from the USA (Zhao et al., 2001), E. coli isolates from Taiwan (Chang et al., 2000b), E. coli and Shigella isolates from Finland (Heikkila et al., 1993), an epidemic Vibrio cholerae O1 strain from Guinea-Bissau (Dalsgaard et al., 2000) and in an S. Typhimurium DT104a isolate originating from Australia (this report).

The results showed that integrons containing resistance to trimethoprim and streptomycin were frequently associated with multi-resistant S. Enteritidis isolates. Since the level of S. Enteritidis isolates containing resistance to two or more antibiotics is only about 4 % of the total isolates, the number of these mobile genetic elements brought into Norwegian hospitals by S. Enteritidis is still modest. Our results, however, indicate a possible increase in the integron-containing isolates in Norwegian hospitals from 2000 to 2001. The frequency of integron carriers among the studied isolates rose from 17 % in 2000 to 25.9 % in 2001. In a study of antimicrobial resistance in S. Enteritidis isolated between 1990 and 1998 in Southern Italy it was reported that 44 of 1889 strains (2.3 %) were resistant to at least one antibiotic (Nastasi et al., 2000). Of these 44 isolates, six were shown to carry integrons. This gives a frequency of integron carriers among resistant isolates of 13.6 % (6/44) in the strains isolated between 1990 and 1998 (Nastasi et al., 2000). Our data indicate that this number might be increasing, and our frequency of about 22 % of resistant isolates carrying integrons is most likely a slight underestimate of the total frequency, since we have only looked at isolates containing resistance to two or more antibiotics. In a screening of all sulfamethoxazole-resistant S. Enteritidis isolates received by the Scottish Salmonella Reference Laboratory in 1997, integrons were detected in 19 % of the isolates (11/58) (Brown et al., 2000), which is a frequency comparable to our data for 2000 of about 17 %. It will be interesting to see whether our increase in integron-carrying S. Enteritidis isolates from 2000 to 2001 will continue or if this was a fluctuation. Isolates from 2002 are being screened to address this in our laboratory. The only integron found in the 2000 isolates was the dfrA1-aadA1 integron, while in 2001 we also detected the aadA2 and dfrA12 genes in addition to a fragment of the sat1 gene in S. Enteritidis.

All S. Enteritidis integrons in this study were localized on plasmids, indicating that they contain the potential to spread further after introduction to a hospital environment.

Integron cassette promoters

The promoter regions in front of all the gene cassettes were PCR-amplified and sequenced. The promoters were homogeneous with all isolates showing both P_c- (also called P_ant) and P2-related sequences. Only one variation of the P_c promoter was found among all the integrons sequenced. The majority of samples had the P_c promoter sequence TGGACA-N₁₇-TAAGCT, irrespective of the cassette following this promoter (Table 5). Only the 5180/00 S. Typhimurium isolate had a variant P_c promoter with the sequence TGGACA-N₁₇-TAAACT in front of the dfrA17-aadA5 gene cassettes (Table 5). In the P2-related sequences we found that all but seven isolates had the sequence TTGTTA-N₁₄-TACAGT, which is not a functional P2 promoter. The S. Typhimurium isolates 1875/96, 1550/96, 1203/97, 2339/99, 1176/96 and 900/98 had all active P2 promoters where the distances between the -35 and the -10 regions of the P2 promoters were 17 nt (Table 5). All these isolates displayed active P2 promoters in front of an oxa1-aadA1 gene cassette (Table 5). We thus found that the majority of P2 promoter signals in the In-t2 integrons (oxa1-aadA1) have a spacing between the -10 and -35 regions that is optimal for promoter function. These In-t2 isolates are thus believed to carry two active promoters (P_c and P2). This is in accordance with the study by Tosini et al. (1998) (accession no. AJ009819) that also shows a spacing of 17 nt between the -10 and -35 regions in the In-t2 P2 promoter from plasmid IncF/97, and a spacing of 14 nt between the P2 like -10 and -35 sequences in In-t1 (aadB-catB3) (accession no. AJ009818), similar to our results. Variations were noted, however: for example, the In-t2 integrons of isolate 3428/98 which had a spacing of 14 nt between the P2-like -10 and -35 signals, and isolate 900/98 which had a mutation in the P2 promoter sequence, TTGTTA-N₁₇-TACACA (the last 2 nt changed from GT) (Table 5). According to Carattoli et al. (2001) the In-t2 integron is likely to have been maintained unaltered in IncFI plasmids for nearly 30 years, while the plasmids with both In-t1 and In-t2 (IncFI/97) are considered to be new variants (Carattoli et al., 2001). It thus appears as though the IncFI/97-associated In-t2 integron exists in two variant forms, one where both promoters are active and one where only P_c is active. The In-t2 integron with only a functional P_c promoter (strain 3428/98) is believed to exhibit weaker expression of the gene cassettes. The strength of the mutated P2 promoter in 900/98 is unknown and will be investigated further.

All of the S. Enteritidis promoters consisted of only the TGGACA-N₁₇-TAAGCT P_c promoter, which is identical to the most common S. Typhimurium P_c promoters, and did not have a functional P2 promoter.

	Acknowledgments

 
Dr Truls Leergaard is gratefully acknowledged for providing data regarding antibiotic resistance of the S. Typhimurium isolates, and the staff of the National Salmonella Reference Laboratory is gratefully acknowledged for technical assistance and for providing data regarding antibiotic resistance of the S. Enteritidis isolates.

	REFERENCES

TOP Abstract Introduction METHODS RESULTS AND DISCUSSION REFERENCES