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Detection and characterisation of integrons in Salmonella enterica serotype Enteritidis

Alistair W. Brown , Shelley C. Rankin , David J. Platt
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09331.x 145-149 First published online: 1 October 2000

Abstract

Integrons have been widely described among the Enterobacteriaceae including strains of multi-resistant Salmonella enterica serotype Typhimurium DT104; however, information with respect to the presence of integrons among S. enterica serotype Enteritidis strains is limited. Multi-resistant isolates of Enteritidis were screened for the presence of integrons using a PCR protocol. One integron was detected in all isolates that were resistant to sulfonamide and streptomycin. Characterisation of these isolates indicated an integron which ranged in size between 1000 and 2000 bp and which harboured a gene cassette encoding the ant(3″)-Ia gene specifying streptomycin and spectinomycin resistance. Further studies revealed the integrons to be located on large conjugative plasmids. This appears to be the first report of plasmid-borne integrons in Enteritidis.

Keywords
  • Salmonella enteritidis
  • Antibiotic resistance
  • Integron
  • Plasmid

1 Introduction

Bacterial resistance to an increasing number of antimicrobial agents is a well-established problem. Several mechanisms involving mobile genetic elements, such as plasmids and transposons, have been shown to contribute to the spread of this resistance [1,2]. In recent years, a novel group of DNA elements able to incorporate antibiotic resistance genes by a site-specific recombination have been identified in Gram-negative bacteria. These elements have been termed integrons [3]. Four classes of integrons have now been described among clinical bacterial isolates. Class I have been shown to be the most prevalent. Structurally, class I integrons can be divided into three discrete regions: a 5′ conserved segment that contains the integrase gene intI and an attachment site attI, a central variable region which is comprised of one or more integrated gene cassettes together with a recombination site termed the 59-base element, and a 3′ conserved segment that encodes genes for sulfonamide resistance (sulI), quaternary disinfectants and ethidium bromide (qacEΔI).

Integrons have been widely described among the Enterobacteriaceae including strains of multi-resistant Typhimurium DT104 [4]. However, information with respect to the presence of integrons among Enteritidis strains is more limited [5]. This study aimed to determine the incidence of integrons among Enteritidis isolates that showed resistance to sulfonamides and to establish the location of any integrons observed.

2 Materials and methods

2.1 Strain selection

All sulfamethoxazole-resistant Enteritidis isolates received by the Scottish Salmonella Reference Laboratory (SSRL) in 1997 were included in the study. Fifty-eight isolates from a total of 2712 Enteritidis (2%) were subcultured from Dorset's egg slopes to CLED agar, incubated at 37°C overnight and stored at 4°C until required.

2.2 Antimicrobial susceptibility

All the isolates were tested by breakpoint minimum inhibitory concentration (MIC) [6] for susceptibility to the following agents: ampicillin (50 μg ml−1), cefotaxime (1 μg ml−1), chloramphenicol (20 μg ml−1), ciprofloxacin (0.5 μg ml−1), furazolidone (20 μg ml−1), gentamicin (20 μg ml−1), kanamycin (20 μg ml−1), nalidixic acid (40 μg ml−1), netilmicin (20 μg ml−1), spectinomycin (100 μg ml−1), streptomycin (20 μg ml−1), sulfamethoxazole (100 μg ml−1), tetracycline (10 μg ml−1) and trimethoprim (10 μg ml−1).

2.3 Integron PCR

Four colonies from an overnight growth on CLED agar were suspended in 500 μl of 10 mM phosphate-buffered saline (PBS), pH 7.4 and microcentrifuged for 30 s. The pellet was then resuspended in 100 μl of TE10 buffer, pH 7.8 [7] then boiled in a water-bath for 10 min; 900 μl of sterile water was added and this template DNA was stored at −20°C until required. The primers were supplied by Oswell DNA Services (Southampton, UK) and were used at a concentration of 7.5 μg ml−1. The primers and cycles were those used in previous studies to characterise integrons in Typhimurium and to characterise TEM genes [4,8]. Annealing temperatures for primer combinations (1 and 2, 1 and 6, and 1 and 10), (3 and 4), (9 and 10) were 58°C, 63°C and 56°C, respectively.

One microlitre of each primer, 22 μl of sterile water and 1 μl of template DNA were added to a Ready-To-Go™ (Pharmacia Biotech) PCR tube on ice. The tubes were immediately placed into a Biometra® Personal Cycler and run on various programs that were appropriate for the primers used. PCR products (25 μl) were electrophoresed on a 1.5% agarose minigel in TBE buffer [7] for 40 min at 80 mA, stained in ethidium bromide solution (1.5 μg ml−1) and photographed.

2.4 Conjugation and restriction enzyme fragmentation pattern (REFP) analysis of plasmid DNA

Plasmid REFP with the restriction enzyme SmaI was performed on all isolates that were shown to be positive for integron DNA [9]. To determine whether the integrons were plasmid- or chromosomally located, conjugation to a plasmid-free strain of Escherichia coli K12, J53-2, was performed as previously described [10]. Potential transconjugants were lactose-fermenting colonies on CLED agar and resistant to rifampicin and ampicillin or streptomycin.

3 Results

3.1 Integron detection

The relationship between the integron and PCR products is shown in Fig. 1. A positive PCR result with primers sulI B and qacEΔI F (primers 3 and 4, Table 1) was indicated by a product of approximately 800 bp. Of the 58 isolates screened with these primers, 11 (19%) gave an 800-bp product (Fig. 2), indicating that these strains harboured integron DNA. Characteristics of these integron-harbouring isolates are shown in Table 2.

1

Diagrammatic integron to show the approximate position of primer (Table 1) sites and regions of amplified DNA.

View this table:
1

PCR primers used for identification and characterisation of integrons [4]

Primer numberAssociated genePrimer sequence
1intI F (5′-CS)GGC ATC CAA GCA GCA AGC
2intI B (3′-CS)AAG CAG ACT TGA CCT GAT
3sulI BGCA AGG CGG AAA CCC GCG CC
4qacEΔI FATC GCA ATA GTT GGC GAA GT
5ant(3″)-Ia FGTG GAT GGC GGC CTG AAG CC
6ant(3″)-Ia BATT GCC CAG TCG GCA GCG
7pseI FCGC TTC CCG TTA ACA AGT AC
8pseI BCTG GTT CAT TTC AGA TAG CG
9TEM-FATA AAA TTC TTG AAG ACG AAA
10TEM-BGAC AGT TAC CAA TGC TTA ATC A
F=forward primer; B=backward primer; CS=conserved segment.
2

Integron detection by PCR of Enteritidis DNA using primers for sulI B and qacEΔI F. Lane 1 contains a 100-bp DNA ladder (Life Technologies™), lanes 2, 4 and 5 illustrate positive results for the 797-bp product.

View this table:
2

Properties of integron-harbouring Enteritidis isolates

SSRL reference numberPhage typeBreakpoint resistance patternaPlasmid profile (kb)Amplicon size (bp)
Primers 1 and 2Primers 1 and 6
9710394Gen, Spc, Stm, Smx, Tet100; 571000750
9725004Amp, Spc, Stm, Smx, Tri100; 57; 3.217001350
9720224Amp, Stm, Smx, Tet, Tri100; 5717001350
9760884Amp, Gen, Spc, Stm, Smx, Tet100; 57; 3.21000750
9712624Stm, Smx, Tet, Tri100; 57; 5017001350
9742504Spc, Stm, Smx100; 57; 4520001700
9717205aAmp, Spc, Smx, Tet, Tri100; 5717001350
9721396aAmp, Spc, Stm, Smx, Tet100; 5717001350
974565UntypeableAmp, Cm, Spc, Stm, Smx, Tet, Tri100; 4.88001700
97381821Amp, Stm, Smx, Tri100; 57; 3.317001350
9738304Spc, Stm, Smx, Tri100; 5717001350
  • aAntibiotic abbreviations and breakpoint MICs: Amp, ampicillin (>50 μg ml−1); Cm, chloramphenicol (>20 μg ml−1); Gen, gentamicin (>20 μg ml−1); Spc, spectinomycin (>100 μg ml−1); Stm, streptomycin (>20 μg ml−1); Smx, sulfamethoxazole (>100 μg ml−1); Tet, tetracycline (>10 μg ml−1); Tri, Trimethoprim (>10 μg ml−1). The 57-kb plasmid present in 10 of the 11 isolates is the Enteritidis serotype-associated plasmid.

3.2 Determination of integron frequency and size of amplicon

The 11 isolates that contained integron DNA were further investigated by PCR using the primers intI F (5′ conserved sequence) and intI B (3′ conserved sequence) to determine the number and size of integron(s) present (Fig. 3). All 11 isolates produced a single amplicon, which confirmed the presence of a single integron in each.

3

PCR of Enteritidis DNA to determine the number of integrons present per isolate and size of amplicon product. Seven isolates yielded a 1700-bp product (lanes 4, 6 and 7), two yielded a 1000-bp product (lanes 5 and 8), one yielded a 2000-bp product (lane 3) and one yielded an 800-bp product (not shown). A laboratory strain of Typhimurium DT104 known to contain two integrons was used as a positive control (lane 2).

3.3 Aminoglycoside resistance

The 11 positive isolates were tested by PCR with the primers intI F and ant(3″)-Ia B (primers 1 and 6), to detect the presence of the streptomycin/spectinomycin resistance gene ant(3″)-Ia within an integron. All 11 isolates were found to be resistant to these agents in susceptibility tests and PCR products confirmed the presence of the ant(3″)-Ia gene on an integron.

3.4 Ampicillin resistance

Seven of the 11 isolates were shown to be resistant to ampicillin and therefore the presence of pseI and TEM β-lactamase genes within and external to integrons was investigated. No isolate yielded an amplicon product with the pseI primers.

All seven isolates yielded a PCR amplicon of approximately 1100 bp with TEM primers (primers 9 and 10), indicating the presence of a TEM β-lactamase gene. When the ampicillin-resistant isolates were tested for presence of the TEM gene within an integron using primers 1 and 10 (Table 1), no PCR product was detected which suggested that this gene was not located within a class I integron.

3.5 Plasmid analysis

All 11 isolates harboured one or more plasmids in addition to the serotype-associated plasmid (SAP) [11]. Table 2 indicates the number and sizes of plasmids found. No isolates had identical plasmid REFPs although some were very similar (Fig. 4).

4

SmaI plasmid REFPs of integron-harbouring Enteritidis isolates. Plasmid transfers were successful with eight of the 11 isolates. This figure illustrates four of these in conjunction with an Enteritidis control strain in lane 2 (SR 16485), which harbours only the serotype-associated plasmid.

Plasmids were successfully transferred to E. coli by conjugation from eight of the 11 isolates. Fig. 5 illustrates several of these and also shows that the SAP (lane 2) was not mobilised by the conjugative R-plasmids.

5

SmaI plasmid REFPs of Enteritidis isolates paired with their plasmid transconjugants. Lane 1, PstI digest of lambda phage DNA; lane 2, Enteritidis SAP control (SR16485). Tx=SmaI digests of transconjugant plasmids.

3.6 Analysis of E. coli transconjugants for presence of integrons

PCR of the E. coli transconjugants was performed to determine whether the integrons were plasmid-mediated. All transconjugants produced an amplicon of approximately 800 bp following PCR with primers 3 and 4. This confirmed that these E. coli transconjugant isolates had acquired plasmids that carried integrons.

Breakpoint sensitivities on the E. coli transconjugants confirmed that all resistance markers were transferred from the Enteritidis strains. This in turn indicated that all of the resistance markers were plasmid-mediated, although it does not exclude the presence of an additional chromosomal copy.

4 Discussion

In this report we describe the presence of individual class I integrons among isolates of Salmonella enterica serotype Enteritidis that specified resistance to sulfonamide and streptomycin or spectinomycin. This resistance was transferable and shown to be located on conjugative plasmids. Within the genus Salmonella, the mechanism of resistance to ampicillin is most commonly due to the TEM β-lactamase [12], however among some strains of Typhimurium, the pseI gene from Pseudomonas spp. has been found on integrons [4]. Ampicillin resistance in all the isolates investigated in this study was conferred by a TEM β-lactamase that was plasmid-mediated and not found as a cassette within an integron. To the best of our knowledge TEM genes have not been associated with integrons. The TEM PCR amplicon size of approximately 1100 bp was in agreement with results of other workers, who reported the sequenced product to be 1079 bp [8].

Given that the average size of a gene is generally estimated at around 800 bp, comparison of the ant(3″)-Ia amplicon to that of the overall integron size suggests that in most cases single gene cassettes were located within the integrons. However, one isolate (974250, Table 2) yielded an amplicon of approximately 2000 bp in size. This suggests the presence of an additional gene cassette between the integrase gene (primer 1) and the streptomycin resistance gene (primer 6).

We have demonstrated the presence of four differently sized integrons among this small group of multi-resistant Enteritidis, which suggests a degree of diversity among these recently recognised genetic elements. These strains also specified resistance to additional markers (e.g. trimethoprim) which may be present as additional cassettes. Further studies are necessary to confirm this. In addition, further work is also necessary to investigate the possibly anomalous result obtained with isolate 974565. This isolate yielded an integron size of 800 bp but an amplicon product of 1700 bp with primer 1 and primer 6.

We have also shown that among Enteritidis isolates, integrons are not limited to phage type 4, the most common phage type, but rather are found within a range of phage types. We considered that the antibiogram may be a useful indicator of integron presence since, although only 19% of all sulfonamide-resistant isolates harboured an integron, all sulfonamide- and streptomycin/spectinomycin-resistant isolates in this study harboured integrons. However, the lack of sulI in plasmid R751 and the recent findings of Rosser and Young [13] suggest that empty integrons and integrons lacking sulI may be more common than was first thought, at least among environmental isolates, and may require a different strategy for their recognition among clinical isolates. In addition to sulfonamide and streptomycin, the plasmids conferred resistance to a combination of ampicillin, chloramphenicol, gentamicin, tetracycline and trimethoprim. Whereas the genes for these determinants were not investigated in this study, other workers have previously described many of these markers as resistance cassettes [3]. Integrons have been found in Enteritidis PT4 previously [5], although these had characteristics similar to integrons found among isolates of Typhimurium DT104, and as with DT104, these integrons were chromosomally located. The integrons found in this study are distinct from those found previously and we believe this to be the first report of plasmid-borne integrons in S. enterica serotype Enteritidis.

Although most class I integrons encode qacEΔI and sulI genes and are phenotypically sulfonamide-resistant there are exceptions such as that of the plasmid R751 [3]. Thus the initial selection of isolates on the basis of sulfonamide resistance and the use of primers 3 and 4 for initial screening may have underestimated the incidence of integrons.

The detection of integrons in Enteritidis is not unexpected. Plasmids isolated during the 1970s have been shown to encode integrons, including R388, R751 and R46 [3]. Where selection pressure favours the acquisition of plasmids by Enteritidis, integrons may be expected. Such situations also provide potential for the chromosomal integration of clusters of resistance genes analogous to their occurrence in Typhimurium DT104 and to contribute to the further dissemination of the resistance genes.

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