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
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Abstract |
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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
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Introduction |
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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.
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Materials and methods |
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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 19982000. 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 SigmaAldrich (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 MuellerHinton agar (Oxoid) was used. Bacteria were grown overnight at 37°C in LuriaBertani (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 manufacturers 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.
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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 manufacturers 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.
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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
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Results |
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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.
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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).
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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).
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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References |
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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, 17781.[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, 1138.[ISI][Medline]
4 . Hall, R. M. (1997). Mobile gene cassettes and integrons: moving antibiotic resistance genes in Gram-negative bacteria. Ciba Foundation Symposium 207, 192205.[ISI][Medline]
5 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 301527.[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, 32732.[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, 8469.
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, 301821.
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, 1321.[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, 28591.[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, 2516.[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, 4303.[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, 28218.
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, 467984.
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, 1419.
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, 396370.
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, 114754.
21
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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, 4458.
22
.
Daly, M. & Fanning, S. (2000). Characterisation and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype Typhimurium. Applied and Environmental Microbiology 66, 48428.