Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

L. P. Randall1,*, S. W. Cooles1, M. K. Osborn1, L. J. V. Piddock2 and M. J. Woodward1

1 Food and Environmental Safety Department, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB; 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Received 8 September 2003; returned 5 November 2003; revised 12 November 2003; accepted 13 November 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb2) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance.

Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb2), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow.

Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb2), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Keywords: Typhimurium, plasmids, ampicillin, streptomycin, tetracycline


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium phage type DT104 is currently the second most prevalent serotype isolated in England and Wales and is becoming increasingly common in other countries.1,2 Resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (abbreviated ACSSuT) is common, although resistance to other antibiotics and other resistance patterns occur.1,3 High level multidrug resistance is normally associated with mobile genetic elements (e.g. plasmids, transposons, integrons, phages, etc.) that encode specific resistance genes.1,4,5 Many gene cassettes within integrons such as the blaP1 cassette which encodes the Pse-1 or Carb-2 protein (different names for the same protein) have been described previously.5

The genetic make-up of many isolates of S. Typhimurium DT104 with the ACSSuT resistance phenotype is similar, comprising the floR and tetA(G) genes bracketed by two class 1 integrons carrying the aadA2, bla(Carb2) or pse1 [bla(Carb2) and pse1 are different names for the same gene] cassettes clustered on a 14 kb region of the genome.2,3,69 This region has recently been described as S. enterica genomic Island 1 (SGI1)10 and in S. Typhimurium DT104 these antibiotic resistance genes are an integral part of the chromosome.3,11 Experiments have shown that these resistance genes can be efficiently transduced by P22-like phage ES18 and by phage PDT17 that is released by all DT104 isolates so far examined.12 With this in mind, it is interesting to note that S. Typhimurium U302 and DT120 strains and S. Agona possess the same antibiotic resistance genes as MDR S. Typhimurium DT104.1,9 More recently, the DT104 MDR profile has been detected within the complete S. enterica genomic Island 1 in Salmonella serotype Paratyphi B from tropical fish in Singapore.13 These data indicate the potential for emergence of multidrug resistance in other S. enterica serotypes, possibly encoded by identical or similar gene clusters. In order to test this hypothesis, a panel of 397 strains of S. enterica that comprised 35 serotypes was examined. Specifically, each strain was tested for the presence of aadA1, aadA2, aadB, aphAI-IAB, bla(Carb2), bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, class 1 integrons, antimicrobial resistance and for the relationship between antibiotic resistance genes, antibiotic resistance and class 1 integrons.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The panel comprised 397 strains; the veterinary isolates were obtained from the Veterinary Laboratories Agency (VLA), Weybridge, UK and the human isolates from the Public Health Laboratory, Colindale, UK. Strains were selected with a bias for major serotypes and were isolated from poultry (n = 170), humans (n = 55), cattle (n = 43), the environment (n = 42, mainly farm environment such as animal housing, litter, etc.), pigs (n = 38), sheep (n = 26), domestic animals (n = 8), unknown sources (n = 10) and feed (n = 5). Sixty-five percent of the strains were isolated in 1999 with 88% of strains isolated in 1998–2000. The remaining strains were isolated between 1994 and 1997.

Strains P3749380 and P43203503 were used as positive controls for class 1 integron PCR. The following strains were used as positive controls for PCR and as DNA templates to generate antibiotic resistance gene probes: S. Enteritidis 7564/96 (aadA1), S. Typhimurium DT104 4665/99 [aadA2, bla(Carb2,), floR, sul1, tetA(G)], S. Seftenberg 7509/99 (aadB), S. Newport 6306/99 (aphAI-IAB, cat1, dhfr1), S. Liverpool 9510/97 [bla(Tem), strA, sul2], S. Typhimurium DT104 P453991 (cat2), S. Typhimurium DT193 1725/99 [tetA(A)], S. Typhimurium DT208 1859/99 [tetA(B)].

Antibiotics and chemicals

Antibiotics and chemicals used were obtained from Sigma–Aldrich (Poole, Dorset, UK) except ciprofloxacin which was kindly donated by Bayer (Newbury, Berkshire, UK).

MICs

MICs were determined by an agar doubling dilution method similar to that of the NCCLS,14 with the main exception that Iso-Sensitest agar (Oxoid, Hants, UK) rather than Mueller–Hinton agar (Oxoid) was used. Bacteria were grown overnight at 37°C in Luria–Bertani (LB) broth, diluted 1/10 in normal saline and inoculated using a multi-point inoculator onto the agar with suitable dilutions of the antibiotics ampicillin, chloramphenicol, gentamicin, kanamycin, spectinomycin, streptomycin, sulfadiazine, trimethoprim and tetracycline. Strains with MICs >16 mg/L of ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline and trimethoprim, >64 mg/L of spectinomycin and >1024 mg/L of sulfadiazine were taken to be resistant. These values were selected as the most suitable to separate strains with resistance genes from strains without resistance genes on the basis of their MICs.

Preparation of probe and colony dot blots

Nylon membranes (Hybond N+, Amersham Pharmacia Biotech, Bucks, UK) for colony dot blots were prepared as previously described.15 PCR amplicons (see below) comprising antibiotic resistance genes were extracted from gels using a Qiagen gel extraction kit (Qiagen, Sussex, UK). The resulting DNA was labelled using an Alkphos labelling kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions and used separately to hybridize to colony dot blots.

PCR amplification

The oligonucleotide primers for antibiotic resistance genes and for class 1 integrons are shown in Table 1 with respective annealing temperatures. Primers were synthesized by Eurogentec Laboratories, Southampton, UK.


View this table:
[in this window]
[in a new window]
 
Table 1. Primer sequences for PCR
 
All PCR amplifications contained 1x MgCl2 free buffer (Promega, Madison, USA), 100 ng of each primer, 0.5 µL of Taq DNA polymerase (Promega), 1 µL of dNTPs mix (Promega), 2 mM MgCl2 (Promega) and HPLC H2O made up to 50 µL. The DNA template (1 µL) for PCR was a washed overnight culture grown at 37°C in LB broth. The PCR conditions were similar to those previously described.9

Detection of aadA1 and aadA2 genes

The aadA1 probe was found to hybridize to aadA2 positive strains. However, by PCR aadA1 and aadA2 primers were specific for their respective genes. As such, the panel of strains was probed for the presence of aadA2 and then the streptomycin-resistant strains were checked by PCR for the presence of the aadA1 gene. Strains positive for both aadA1 and aadA2 were then tested for by PCR using aadA2 primers to confirm or otherwise the presence of the aadA2 gene.

Presence of resistance genes within integrons

The presence of resistance genes within class 1 integrons was determined as follows. Integron primer set A (Table 1) was used to amplify the variable region of InC which in S. Typhimurium DT104 contains the aadA2 gene and the variable region of InD which in S. Typhimurium DT104 contains the bla(Carb2) (also known as pse1) gene (Figure 1). To test if aadA2 and bla(Carb2) genes were contained within the amplified segment, the amplified segment was blotted onto nylon membranes (Hybond N+) as previously described.15 Membranes were then hybridized with either the aadA2 or bla(Carb2) gene using an Alkphos labelling kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Integron primer set B (Table 1) amplified the 3' conserved region of both InC and InD; the sul1 and qacE genes are contained in this region (Figure 1).2,16 Therefore a positive PCR result was taken as evidence that the sul1 gene was within the integron.



View larger version (5K):
[in this window]
[in a new window]
 
Figure 1. Schematic representation of genes within and between InC and InD integrons of Salmonella Typhimurium DT104. Adapted (not drawn to scale) from Figure 1 of Carattoli et al.16

 
Association of resistance genes with integrons

The floR and tetA(G) genes have been reported to be between the two class 1 integrons InC and InD (Figure 1).16 Having tested for the presence of the aadA2, bla(Carb2) and sul1 genes within integrons, the association of dhfr1, floR, strA and sul2 genes with the integrons was determined as follows. Chromosomal DNA was digested with XbaI (Promega), electrophoresed for 16 h in 0.7% agarose with a 1 kb plus ladder (Invitrogen) as a marker, then blotted onto membranes as described previously.15 Membranes were then hybridized with probes comprising separately dhfr1 or floR or strA or sul1 or sul2 genes and the 1 kb plus ladder. Location of genes within the same sized fragment as that obtained when blots were hybridized with the sul1 probe was taken as an indication of co-location of antibiotic resistance genes with the integrons (Figure 1).16


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance

Salmonella (n = 397) were screened for resistance to nine antibiotics and the following numbers of strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45). However, 219 strains were susceptible to all antibiotics. With the exception of the five S. Fischerkietz strains tested, which were susceptible to all antibiotics tested (Table 2), all major serotypes of Salmonella had examples of strains that showed resistance to some of the antibiotics tested.


View this table:
[in this window]
[in a new window]
 
Table 2. Number of strains within each serotype resistant to antibioticsa
 
There was good correlation between the presence of resistance genes and corresponding resistance phenotypes suggesting resistance genes, when present, were usually expressed. MICs of sulfadiazine against strains harbouring the sul1 or sul2 genes were >2048 mg/L. MICs of all the other antibiotics tested were 32 to >128 mg/L for strains harbouring resistance genes specific for the antibiotic, with the exception of 13/59 strains with the strA gene that were streptomycin-susceptible (Table 3). All strains that were ampicillin-resistant contained either the bla(Carb2) and/or the bla(Tem) gene and the two gentamicin-resistant strains contained the aadB gene. The aadA1 and aadA2 probes cross-hybridized and using the combined probe and PCR methodology described, it was shown that 15 strains were positive for aadA1 alone, 68 strains were positive for aadA2 alone and three strains were positive for both genes (Table 4).


View this table:
[in this window]
[in a new window]
 
Table 3. MIC range for strains with specific resistance genes
 

View this table:
[in this window]
[in a new window]
 
Table 4. Number of strains within each serotype with specific resistant genes
 
There were several occasions in which a strain was resistant to an antibiotic but the identity of the gene conferring resistance was not ascribed. For example, 24 of 85 chloramphenicol-resistant strains did not contain cat1, cat2 or floR genes, one of 14 kanamycin-resistant strains did not contain the aphAI-IAB gene, five of 81 spectinomycin-resistant strains did not contain the aadA1 or aadA2 genes, 16 of 119 streptomycin-resistant strains did not contain aadA1, aadA2 or strA genes, seven of 127 sulfadiazine-resistant strains did not contain sul1 or sul2 genes, 21 of 108 tetracycline-resistant strains did not contain tetA(A), tetA(B) or tetA(G) genes and 38 out of 45 trimethoprim-resistant strains did not contain the dhfr1 gene.

Distribution of antibiotic resistance genes between different serotypes

The genes bla(Carb2), floR and tetA(G) were associated with S. Typhimurium strains (Table 4) and one strain of S. Emek that contained the floR and tetA(G) genes (Table 4, ‘Others’). The aadA2 and sul1 genes were found mainly in S. Typhimurium isolates with both genes in ~85% of DT104, in ~65% of U302 and ~20% non-DT104 and U302 S. Typhimurium, but these genes were also found in other serotypes (Table 4). The tetA(G) gene was the most prevalent tetracycline resistance determinant in S. Typhimurium DT104 and U302 strains, whereas the tetA(A) and tetA(B) genes were relatively common in other serotypes and phage types (Table 4). Ampicillin-resistant strains that were not positive for bla(Carb2) were positive for bla(Tem), although there were two instances of S. Typhimurium DT104 positive for both bla(Carb2) and bla(Tem). Of the three streptomycin resistance genes tested for (aadA1, aadA2, strA), the aadA2 gene was the prevalent resistance determinant for S. Typhimurium DT104, the strA gene was the prevalent resistance determinant for non-Typhimurium strains and the aadA1 gene was only detected in non-S. Typhimurium isolates (Table 4). There were instances of the aadA1 and aadA2, the aadA1 and strA, and the aadA2 and strA genes occurring together (data not shown).

The dhfr1 gene was present in only seven strains and was not present in any of the trimethoprim-resistant S. Typhimurium isolates. The aphaA1-IAB gene was detected in most of the kanamycin-resistant strains of different serotypes and the aadB gene was detected in the two gentamicin-resistant strains. The cat genes were only found in S. Dublin, Newport, Typhimurium DT104 and Virchow and only one strain in each of the four serotypes was positive (Table 4).

The more common resistance genes such as aadA2, bla(Carb2), bla(Tem), floR, tetA(G), strA and sul1 were present in Salmonella strains from cattle, the environment, humans, pigs, poultry and sheep with the exception that the bla(Tem) gene was not found in isolates from cattle (data not shown).

Multiple antibiotic resistance

For strains which had class 1 integrons, multiple antibiotic resistance (resistance to at least three and up to seven antibiotics) was associated with the serotypes S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Typhimurium DT104, U302 and other phage types of Typhimurium and Virchow (Table 5). For strains that did not have class 1 integrons, multi-resistance was associated with the serotypes S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow (Table 6). The most common DT104 resistance profile was AMP-CHL-SPT-STR-SDZ-TET. A strain of S. Newport that was resistant to AMP-CHL-KAN-STR-SDZ-TET-TMP and had two integrons (~0.5 and 1.5 kb) was not resistant to cefoxitin (results not shown).


View this table:
[in this window]
[in a new window]
 
Table 5. Strains positive for class 1 integrons, approximate size of integron PCR productsa and resistance genes within or associated with integrons
 

View this table:
[in this window]
[in a new window]
 
Table 6. Multi-resistancea in strains without class 1 integrons
 
Integrons and resistance genes

Of the 397 strains tested, 81 were positive for class 1 integrons primarily associated with S. Typhimurium DT104, U302 and other S. Typhimurium but also with several other serotypes (Table 5). Where they occurred, the aadA2, bla(Carb2) and sul1 genes were found to be within integrons using the methods described (Table 5 and Figure 1). Results showed that the floR, dhfr1 and tetA(G) genes were co-located with the sul1 gene on XbaI fragments, suggesting that these genes were co-located with the integrons (Table 5 and Figures 1 and 2). The strA and sul2 genes did not appear to be co-located with the integrons except possibly in one of the S. Newport strains (Table 5).



View larger version (46K):
[in this window]
[in a new window]
 
Figure 2. Southern hybridization of genomic DNA from 10 strains of S. Typhimurium (DT104, U302 and other phage types) digested to completion with XbaI and probed with sul1 (a) and tetA(G) (b). (a) With sul1, two hybridizing fragments were seen. The one of 12 kb is associated with sul1 contained in the InC integron7 and the other fragment is associated with sul1 on the InD integron.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The emergence of MDR S. Typhimurium, Paratyphi and Agona suggests that this multi-antibiotic-resistant phenotype may emerge in other S. enterica serotypes.1,13 It may also be that some of the resistance genes in the ACSSuT-resistant S. Typhimurium DT104 strains can transfer to other S. enterica serotypes. Although there was evidence for all or some of the ACSSuT resistance profile being in a range of serotypes other than S. Typhimurium DT104 and U302, the bla(Carb2), floR and tetA(G) genes were exclusively associated with S. Typhimurium with the exception of one strain of S. Emek which harboured the florR and tet(G) resistance genes. The aadA2 gene was found mainly in S. Typhimurium strains as was the sul1 gene, but the sul1 gene was also found in several other serotypes apart from S. Typhimurium. Other resistance genes such as aadA1, aphA-IAB, cat1, cat2, dhfr1, strA, sul2, tetA(A) and tetA(B) were found in a wide range of serotypes, although the aadA1 and dhfr1 genes were not found in any S. Typhimurium strains tested. Multi-resistance was shown to be associated with strains positive and negative for class 1 integrons. The involvement of integrons in multi-resistance in S. Typhimurium DT104 isolates has been studied extensively and such integrons are reported to be 1 and 1.2 kb in size.1,2,4,6,7,10,16 In S. Typhimurium DT104, the aadA2 and sul1 genes are located on the InC integron whereas the bla(Carb2) or pse1 gene and sul1 gene are located on the InD integron and the region between these two integrons contains the floR and tetA(G) genes.2 Our studies confirmed the presence of aadA2 and or bla(Carb2) and or sul1 within the class 1 integrons of all strains tested including those integron-positive non-Typhimurium strains, with the exception of two S. Newport strains and these warrant further investigation. Additionally, the dhfr1, floR and tetA(G) genes were found to be associated with an XbaI fragment containing sul1 for all strains tested, suggesting that these genes were also associated with class 1 integrons. With the exception of the S. Newport strain, these data indicated that the strA and sul2 genes were associated with plasmids. The S. Newport strain that was resistant to seven antibiotics and which contained eight resistance genes and two integrons (approximate sizes of 0.5 and 1.5 kb) warrants further investigation. However, this strain, which was of porcine origin, was not resistant to cefoxitin and as such does not warrant the concern currently shown for cefoxitin-resistant strains of S. Newport.17 In the USA, multi-resistant strains of S. Newport with the ACSSuT resistance profile have increasingly been isolated from clinical disease in humans and animals.17 Some strains also show resistance to third generation cephalosporins such as ceftriaxone and this is of particular concern as it is one of the antibiotics for treating Salmonella in children.17

It was interesting to note that some strains harboured two different resistance genes for the same antibiotic, such as aadA2 and strA. It is possible that for class 1 integron-positive strains, one gene was associated with the integron, but that the strain also harboured a plasmid containing the other resistance gene. However, in some instances, strains negative for class 1 integrons also contained two resistance genes for the same antibiotic. These alternative possibilities are worthy of further investigation.

Previous workers have shown that non-S. Typhimurium isolates (such as S. Agona, Enteritidis, Chomedey, Djugu, Infantis and Oranienburg) can have integrons ranging in size from 0.65 to 2.7 kb and these integrons were associated with the presence of various resistance genes including aadA1, aadA2, aadA5, aadB, bla(Carb2) (also known as pse1), catB3, oxa1, dhfrA1, dhfrA12, dhfrA17 and dfrXIII.1820 This study confirms the presence of resistance genes associated with integrons in additional non-Typhimurium serotypes to those reported above including S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley and Virchow.

Multi-resistance in strains that did not have class 1 integrons was associated with the serotypes S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport and Typhimurium DT193. These strains sometimes contained the aadA2 and sul1 genes common to S. Typhimurium DT104 and U302 strains. As the sul1 gene is the backbone of class 1 integrons,2,3 it was of some concern that six strains negative for class 1 integrons were positive for the sul1 gene and these strains warrant further investigation. However, resistance determinants for ampicillin, chloramphenicol and tetracycline in class 1 integron-negative strains were bla(Tem), cat1, tetA(A) and tetA(B) instead of the bla(Carb2), floR and tetA(G) genes. Additionally, the aadA1, sul2 and strA genes were often found in these strains rather than the aadA2 and sul1 genes. Analysis of XbaI fragments suggested that the sul2 and strA genes were plasmid located, possibly.

The trimethoprim-resistant strains that lacked the dhfr1 gene usually contained the sul2 gene, and this may suggest that a trimethoprim resistance gene other than dhfr1 was co-located on a plasmid with the sul2 and strA genes. However, it is possible that integrons other than class 1 integrons were also involved in multi-resistance and a number of different class A dihydrofolate reductase genes within gene cassettes have been described by others.5

Overall the data did not suggest any difference in the distribution of resistance genes between human and animal isolates of Salmonella. As Salmonella is a zoonotic organism, it is to be expected that many of the strains isolated from humans originated from animals. As such, it would be expected that in general the same resistance genes would be found in strains from both animals and humans.

It has been shown that antibiotic resistance genes can be silent in Salmonella. For example, three Salmonella enterica isolated from retail ground meat samples were susceptible to streptomycin even though they harboured the aadA2 gene.20 In this study, 13/59 strains that contained the strA gene were susceptible to streptomycin. It would be of interest to determine the genetic basis for susceptibility and to evaluate the risk that might be associated with reversion to resistance. It is possible that these strains did not contain the strB gene which is also required for resistance, but this was not established.

Overall, the data indicate that transfer of the resistance genes bla(Carb2), floR and tetA(G) from strains such as S. Typhimurium DT104 or U302 to other serotypes is rare on the basis of the strains studied in this panel. However, the data illustrate the involvement of a range of different resistance genes in both class 1 and non-class 1 integron associated resistance. Whilst resistance genes occurred in a wide range of Salmonella serotypes, results illustrated the specificity of some genes to either S. Typhimurium or non-S. Typhimurium serotypes. Finally, the presence of class 1 integrons is described in serotypes for which it is believed class 1 integrons have not previously been reported and the location of some resistance genes within or associated with the integrons is verified.


    Acknowledgements
 
We are grateful to James Cariss, University of Birmingham, UK, for designing the primers to detect cat1, cat2, tetA(A) and tetA(B) genes. This work was supported by DEFRA UK, grant reference number OD2004.


    Footnotes
 
* Corresponding author. Tel: +44-1932-357582; Fax: +44-1932-347046; E-mail: l.randall{at}vla.defra.gsi.gov.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Boyd, D. A., Peters, G. A., Cloeckaert, A. et al. (2001). Complete nucleotide sequence of a 43-kilobase genomic island associated with the multi-drug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. Journal of Bacteriology 183, 5725–32.[Abstract/Free Full Text]

2 . Sandvang, D., Aarestrup, F. M. & Jensen, L. B. (1997). Characterisation of integrons and antibiotic resistance genes in Danish multi-resistant Salmonella enterica Typhimurium DT104. FEMS Microbiology Letters 157, 177–81.[CrossRef][ISI][Medline]

3 . Ridley, A. & Threlfall, J. E. (1998). Molecular epidemiology of antibiotic resistance genes in multi-resistant epidemic Salmonella Typhimurium DT 104. Microbial Drug Resistance 4, 113–8.[ISI][Medline]

4 . Hall, R. M. (1997). Mobile gene cassettes and integrons: moving antibiotic resistance genes in Gram-negative bacteria. Ciba Foundation Symposium 207, 192–205.[ISI][Medline]

5 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 3015–27.[ISI][Medline]

6 . Arcangioli, M. A., Leroy-Setrin, S., Martel, J.-L. et al. (1999). A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella Typhimurium DT104. FEMS Microbiology Letters 174, 327–32.[CrossRef][ISI][Medline]

7 . Briggs, C. E. & Fratamico, P. M. (1999). Molecular characterization of an antibiotic resistance gene cluster of Salmonella Typhimurium DT104. Antimicrobial Agents and Chemotherapy 43, 846–9.[Abstract/Free Full Text]

8 . Lai-King, N. G., Mulvey, M. R., Martin, I. et al. (1999). Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrobial Agents and Chemotherapy 43, 3018–21.[Abstract/Free Full Text]

9 . Walker, R. A., Lindsay, E., Woodward, M. J. et al. (2001). Variation in clonality and antibiotic-resistance genes among multi-resistant Salmonella enterica serotype Typhimurium phage-type U302 (MR U302) from humans, animals, and foods. Microbiological Research 7, 13–21.[CrossRef]

10 . Boyd, D. A., Peters, G. A., Lai-King, N. et al. (2000). Partial characterisation of a genomic island associated with the multi-drug resistance region of Salmonella enterica Typhimurium DT104. FEMS Microbiology Letters 189, 285–91.[CrossRef][ISI][Medline]

11 . Threlfall, E. J., Frost, J. A., Ward, L. R. et al. (1994). Epidemic in cattle and humans of Salmonella Typhimurium DT104 with chromosomally integrated multiple drug resistance. Veterinary Record 134, 577.

12 . Schmieger, H. & Schicklmaier, P. (1999). Transduction of multiple drug resistance of Salmonella enterica serovar Typhimurium DT104. FEMS Microbiology Letters 170, 251–6.[CrossRef][ISI][Medline]

13 . Meunier, D., Boyd, D., Mulvey, M. R. et al. (2002). Salmonella enterica serotype Typhimurium DT 104 antibiotic resistance genomic island I in serotype Paratyphi B. Emerging Infectious Diseases 8, 430–3.[ISI][Medline]

14 . National Committee for Clinical Laboratory Standards. (2001). Methods for Dilution Antimicrobial Sensitivity Testing for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

15 . Maniatis, T., Fritsch, C. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY, USA.

16 . Carattoli, A., Filetici, E., Villa, L. et al. (2002). Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrobial Agents and Chemotherapy. 46, 2821–8.[Abstract/Free Full Text]

17 . Rankin, S. C., Aceto, H., Cassidy, J. et al. (2002). Molecular characterization of cephalosporin-resistant Salmonella enterica serotype Newport isolates from animals in Pennsylvania. Journal of Clinical Microbiology 40, 4679–84.[Abstract/Free Full Text]

18 . Lindstedt, B. A., Heir, E., Nygard, I. et al. (2003). Characterisation of class I integrons in clinical strains of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis from Norwegian hospitals. Journal of Medical Microbiology 52, 141–9.[Abstract/Free Full Text]

19 . Orman, B. E., Pineiro, S. A., Arduino, S. et al. (2002). Evolution of multi-resistance in non-typhoid Salmonella serovars from 1984 to 1998 in Argentina. Antimicrobial Agents and Chemotherapy 46, 3963–70.[Abstract/Free Full Text]

20 . White, D. G., Zhao, S., Sudler, R. et al. (2001). The isolation of antibiotic-resistant Salmonella from retail ground meats. New England Journal of Medicine 345, 1147–54.[Abstract/Free Full Text]

21 . Frana, T. S., Carlson, S. A. & Griffith, R. W. (2001). Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype Typhimurium phage type DT104. Applied and Environmental Microbiology 67, 445–8.[Abstract/Free Full Text]

22 . Daly, M. & Fanning, S. (2000). Characterisation and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype Typhimurium. Applied and Environmental Microbiology 66, 4842–8.[Abstract/Free Full Text]