Antimicrobial susceptibility and occurrence of resistance genes among Salmonella enterica serovar Weltevreden from different countries

Frank M. Aarestrup1,*, Monton Lertworapreecha2, Mary C. Evans1, Aroon Bangtrakulnonth3, Thongchai Chalermchaikit2, Rene Sjøgren Hendriksen1 and Henrik Caspar Wegener1

1 WHO Collaborating Centre for Antimicrobial Resistance in Bacteria from Food animals and Food of Animal Origin, Danish Veterinary Institute, Bülowsvej 27, DK-1790 Copenhagen V, Denmark; 2 Center for Antimicrobial Resistance Monitoring in Food-borne Pathogens. Faculty of Veterinary Science, Chulalongkorn University, Bangkok; 3 WHO International Salmonella and Shigella Centre, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand

Received 16 June 2003; returned 27 June 2003; revised 17 July 2003; accepted 23 July 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: This study was conducted to investigate the occurrence of antimicrobial resistance among Salmonella Weltevreden isolates from different sources in South-East Asia (Indonesia, Laos, Malaysia, Taiwan, Thailand, Vietnam), Australia, Denmark, New Zealand and the USA.

Methods: A total of 503 isolates were examined for susceptibility to antimicrobial agents, and resistant isolates were examined for the presence of selected resistance genes by PCR.

Results: Only 48 (9.5%) of the isolates were resistant to one or more of the antimicrobial agents tested. A low frequency of resistance was found towards ampicillin (1.8%), chloramphenicol (1.6%), florphenicol (0.4%), nalidixic acid (1.6%), neomycin (0.6%), streptomycin (4.4%), sulfamethoxazole (4.2%), tetracycline (4.0%) and trimethoprim (1.4%), whereas all isolates were susceptible to co-amoxiclav, ceftiofur, ciprofloxacin, colistin and gentamicin. All nine ampicillin-resistant isolates contained a sequence similar to the blaTEM-1b gene, one of the eight chloramphenicol-resistant isolates a sequence similar to the catA1 gene, all three neomycin-resistant isolates a sequence similar to the aphA-2 gene, 16 (73%) of the 22 streptomycin-resistant isolates a sequence similar to the aadA gene, the remaining six (27%) a sequence similar to the strA gene, and all 21 sulfamethoxazole-resistant isolates a sequence similar to the sul2 gene. Thirteen (65%) of the 20 tetracycline-resistant isolates contained the tet(A) gene, four (20%) the tet(B) gene, and one (5%) the tet(C) gene.

Conclusions: This study showed a low frequency of resistance among Salmonella Weltevreden isolated from humans and other reservoirs in South-East Asia and elsewhere. There was no major difference in the occurrence of resistance between source or geographical origin.

Keywords: Salmonella Weltevreden, antimicrobial resistance, genes, South-East Asia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Salmonella enterica is one of the most common causes of human gastroenteritis worldwide. More than 2500 different serovars of S. enterica have been identified, and most of them have been described as the cause of human infections. A few serovars are responsible for the majority of human infections. S. enterica serovar Typhimurium and S. enterica serovar Enteritidis have been implicated, in particular, as causes of human salmonellosis. However, in South-East Asia, S. enterica serovar Weltevreden has been reported as a frequent and increasing cause of human infection, and is the predominating serovar in both Malaysia and Thailand.1, 2

In recent years, an increase in the occurrence of antimicrobial resistance among S. enterica has been observed in several countries, but there is only limited information on the occurrence of antimicrobial resistance among Salmonella Weltevreden. Boonmar et al.2 reported a very low frequency of resistance among 111 Salmonella Weltevreden from human infections in 1993 in Thailand, which was in contrast to the frequent occurrence of resistance observed among other serovars. However, in 1994, they reported a much higher occurrence of resistance among 139 isolates. Thong et al.3 examined 95 isolates from different sources in Malaysia and found a low frequency of resistance.

This study was conducted to investigate the occurrence of antimicrobial resistance, and the distribution of resistance genes, among 503 Salmonella Weltevreden isolates from different sources in 10 countries, most of them from South-East Asia.


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

A total of 503 isolates were received from laboratories in South-East Asia [Indonesia (45), Laos (6), Malaysia (105), Taiwan (7), Thailand (197), Vietnam (19)], Australia (46), Denmark (16), New Zealand (40) and the USA (22). The isolates were collected during 1995–2001. Most isolates originated from humans (282), but some were from animals (59), food products (114) and water, the environment or other sources (48).

Susceptibility testing

Susceptibility to antimicrobial agents was performed as MIC determinations. As described in NCCLS guidelines,4 a commercially prepared, dehydrated panel (Sensititre) was used for the following antimicrobial agents: ampicillin, ceftiofur, chloramphenicol, ciprofloxacin, co-amoxiclav, colistin, florphenicol, gentamicin, nalidixic acid, neomycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim.

Detection of resistance genes

PCR was used to detect the presence of antimicrobial resistance genes. Ampicillin-resistant isolates were examined for blaTEM genes using primer pairs 5'-ATGAGTATTCAACATTTCCG-3' (blaTEM) and 5'-AC-CAATGCTTAATCAGTGAG-3' (blaTEM); chloramphenicol-resistant isolates were examined for catA1 using primers 5'-CGCCTGATGAATGCTCATCCG-3' (catA1) and 5'-CCTGCCACTCATCGCAG TAC-3' (catA1); neomycin-resistant isolates were examined for aphA-2 using primer pairs 5'-GCTATTCGGCTATGACTGGGC-3' (aphA-2) and 5'-CCACCATGATATTCGGCAAGC-3' (aphA-2); streptomycin-resistant isolates were examined for strA and aadA using primer pairs 5'-CCAATCGCAGATAGAAGGC-3' (strA), 5'-CTTGGTGATAACG-GCAATTC-3' (strA), 5'-ATCCTTCGGCGCGATTTTG-3' (aadA) and 5'-GCAGCGCAATGACATTCTTG-3' (aadA); sulfamethoxazole resistant isolates were examined for sul1 and sul2 using primer pairs 5'-CTTCGATGAGAGCCGGCGGC-3' (sul1), 5'-GCAAGGCGGAAACCCGC GCC-3' (sul1), 5'-GCGCTCAAGGCAGATGGCATT-3' (sul2) and 5'-GCGTTTGATACCGGCACCCGT-3' (sul2); and tetracycline-resistant isolates were examined for tet(A), tet(B), tet(C) and tet(G) using primer pairs 5'-GTAATTCTGAGCACTGTCGC-3' [tet(A)], 5'-CTGCCTGGACAACATTGCTT-3' [tet(A)], 5'-CTCAGTATTCCAAGCCTTTG-3' [tet(B)], 5'-ACTCCCCTGAGCTTGAGGGG-3' [tet(B)], 5'-GGTTGAAGGCTCTCAAGGGC-3' [tet(C)], 5'-CCTCTTGCGGGAAT- CGTCC-3' [tet(C)], 5'-GCAGCGAAAGCGTATTTGCG-3' [tet(G)] and 5'-TCCGAAAGCTGTCCAAGCAT-3' [tet(G)].

Using the same primers as in the PCR analysis, DNA sequencing verified the identity of the gene products in selected isolates. The gene product of all blaTEM-positive isolates was sequenced.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Only 48 (9.5%) of the 503 isolates were resistant to one or more of the antimicrobial agents tested. All isolates were susceptible to co-amoxiclav, ceftiofur, ciprofloxacin, colistin and gentamicin. A low frequency of resistance was found towards ampicillin (1.8%), chloramphenicol (1.6%), florphenicol (0.4%), nalidixic acid (1.6%), neomycin (0.6%), streptomycin (4.4%), sulfamethoxazole (4.2%), tetracycline (4.0%) and trimethoprim (1.4%) (Table 1). Resistance was observed most frequently among isolates from Laos and Malaysia, but otherwise the occurrence of resistance was so low that no major difference between country or isolate origin could be found.


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Table 1. Occurrence of antimicrobial resistance among Salmonella Weltevreden isolates from different countries
 
All nine ampicillin-resistant isolates gave positive amplicons for the blaTEM gene (Table 2). An 820 bp sequence was obtained from the 859 bp amplicon in all isolates. A comparison with the GenBank revealed 100% identity to the sequence of blaTEM-1b. One of the eight chloramphenicol-resistant isolates contained the catA1 gene, all three neomycin-resistant isolates the aphA-2 gene, 16 (73%) of the 22 streptomycin-resistant isolates the aadA gene and six (27%) the strA gene, and all 21 sulfamethoxazole-resistant isolates the sul2 gene. Thirteen (65%) of the 20 tetracycline-resistant isolates contained the tet(A) gene, four (20%) the tet(B) gene, and one (5%) the tet(C) gene. None of the four tetracycline-resistance genes could be detected in two tetracycline-resistant isolates.


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Table 2. Occurrence of resistance genes among antimicrobial-resistant Salmonella Weltevreden isolates from different countries
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A very low frequency of resistance was detected among the 503 Salmonella Weltevreden isolates. This could be because this serovar does not easily acquire resistance, or because the natural reservoirs were not exposed to large amounts of antimicrobial agents. It is not known which hypothesis is correct. Boonmar et al.2 reported a low occurrence of resistance among Salmonella Weltevreden isolated from humans in Thailand in 1993, and a major increase in resistance in 1994. Such an increase was not observed by us among isolates from Thailand in 1994. We found an infrequent occurrence of resistance, more in agreement with the observed frequency in Thailand in 1993, and that reported by Thong et al.3 from different sources in Malaysia.

A number of different resistance genes were detected among the 48 resistant isolates. All ampicillin-resistant isolates contained a sequence similar to blaTEM-1b. blaTEM genes have been found previously among Salmonella isolates.5 The catA1 gene has been detected previously in Salmonella isolates,6 but is also widespread among other Gram-negative bacteria. Tetracycline resistance was mainly mediated by tet(A), but a few isolates contained tet(B) and a single isolate contained tet(C). tet(A) is located frequently on transposons such as Tn1721, and the gene has been found widespread among Gram-negative bacteria including salmonella.7 Resistance to sulfamethoxazole was exclusively mediated by sul2. In the past, the sul1 gene has been found mainly in connection with integrons, whereas sul2 is located usually on a variety of plasmids.8 All neomycin-resistant isolates contained aphA-2, which also has been found in several Gram-negative species. Both aadA and strA have been found frequently among streptomycin-resistant isolates, as has Salmonella Typhimurium in Denmark.9 Class I integrons containing the aadA gene have been observed often among clinically important Enterobacteriaceae.10 However, the class I integrons normally contain sul1; this gene was not observed in any strains in this study.

In conclusion, this study showed a low frequency of resistance among Salmonella Weltevreden isolated from humans and other reservoirs in South-East Asia and elsewhere. Resistance was encoded by genes previously widespread in other Enterobacteiaceae. No major differences between country or source could be observed.


    Acknowledgements
 
We are grateful to the following persons and institutes for providing isolates for this study. Australia: Dianne Davos, Institute of Medical and Veterinary Science, Australian Salmonella Reference Centre, Adelaide. Denmark: Steen Ethleberg, Statens Serum Institute, Copenhagen. Indonesia: Sri Poernomo, Research Institute for Veterinary Science, Bogor. Lao People’s Democratic Republic: Keophilavane Vorakoummane and Latsamy Vongsack, Food and Drug Quality Control Center, Vietiane. Malaysia: Mohamid Zainuldin and Rohani Yasin, Institute for Medical Research, Bacteriology Unit, Kuala Lumpur. Son Radu, Noorzaleha Awang Salleh and Gulam Rusul, University of Putra Malaysia, Department of Biotechnology, Selangor. Maria Jamli and Johara Mohd Yob, Veterinary Research Institute, Bacteriology Section, Ipoh. New Zealand: Carolyn Nicol and David Duncan, ESR Enteric Reference Laboratory, Porirua. Taiwan: Tsai-Ling Lauderdale, National Health Research Institutes, Taipei, Taiwan. USA: Shannon Rossiter, Kevin Joyce and Fred Angulo, Centers for Disease Control and Prevention, Atlanta. Vietnam: Nguyen Binh Minh, National Institute of Hygiene and Epidemiology, Department of Enteric Pathogens, Hanoi.


    Footnotes
 
* Corresponding author: Tel: +45-35-30-01-00; Fax: +45-35-30-01-20; E-mail: faa{at}vetinst.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Yasin, R. M., Tiew, C. C. & Jegathesan, M. (1995). Human salmonellosis in Malaysia for the period 1989–July 1994. Southeast Asian Journal of Tropical Medicine and Public Health 26, 457–60.

2 . Boonmar, S., Bangtrakulnonth, A., Pornruangwong, S. et al. (1998). Predominant serovars of Salmonella in humans and foods from Thailand. Journal of Veterinary Medical Sciences 60, 877–80.[CrossRef]

3 . Thong, K. L., Goh, Y. L., Radu, S. et al. (2002). Genetic diversity of clinical and environmental strains of Salmonella enterica serotype Weltevreden isolated in Malaysia. Journal of Clinical Microbiology 40, 2498–2503.[Abstract/Free Full Text]

4 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Second Edition: Approved Standard M31-A2. NCCLS, Villanova, PA, USA.

5 . Gallardo, F., Ruiz, J., Marco, F. et al. (1999). Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance. Journal of Medical Microbiology 48, 367–74.[Abstract]

6 . Guerra, B., Soto, S., Helmuth, R. et al. (2002). Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrobial Agents and Chemotherapy 46, 2977–81.[Abstract/Free Full Text]

7 . Frech, G. & Schwarz, S. (2000). Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes. Journal of Applied Microbiology 89, 633–41.[CrossRef][ISI][Medline]

8 . Radstrom, P., Swedberg, G. & Skold, O. (1991). Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution. Antimicrobial Agents and Chemotherapy 35, 1840–8.[ISI][Medline]

9 . Madsen, L., Aarestrup, F. M. & Olsen, J. E. (2000). Characterisation of streptomycin resistance determinants in Danish isolates of Salmonella Typhimurium. Veterinary Microbiology 75, 73–82.[CrossRef][ISI][Medline]

10 . Sallen, B., Rajoharison, A., Desvarenne, S. et al. (1995). Molecular epidemiology of integron-associated antibiotic resistance in clinical isolates of Enterobacteriaceae. Microbial Drug Resistance 1, 195–202.[ISI][Medline]