JAC Advance Access published online on July 6, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm249
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Emergence of multidrug-resistant clones of Salmonella Infantis in broiler chickens and humans in Hungary
1 Department of Phage-typing and Molecular Epidemiology, National Center for Epidemiology, Gyáli u. 2-6, H-1097 Budapest, Hungary 2 Department of Bacteriology, National Center for Epidemiology, Gyáli u. 2-6, H-1097 Budapest, Hungary 3 Microbiology Department, National Food Investigation Institute, Mester u. 81, H-1095 Budapest, Hungary 4 Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Hungária krt. 21, H-1143 Budapest, Hungary
* Corresponding author. E-mail: nogradyn{at}oek.antsz.hu
Received 9 February 2007; returned 5 April 2007; revised 8 June 2007; accepted 11 June 2007
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Abstract |
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Objectives: The characterization of a Salmonella Infantis strain collection that was set up from isolates of animal and human origin obtained in Hungary in recent years.
Methods: All isolates were phage typed. Antimicrobial resistance was tested by the disc diffusion method, while the presence of the antimicrobial resistance genes and class 1 integrons was investigated by PCR. Genetic relatedness of the isolates was tested by PFGE and plasmid profiling.
Results: The majority of the isolates representing different parts of Hungary are characterized by phage types 213 and 217 and the nalidixic acid–streptomycin–sulphonamide–tetracycline resistance type. They harbour a class 1 integron with an aadA1 gene in the 855 bp variable region, a tet(A) gene, a >168 kb plasmid and 66% of them represent one genetic clone as determined by XbaI PFGE fingerprinting.
Conclusions: It seems that broiler chickens constitute a reservoir for one large and a few smaller multidrug-resistant Salmonella Infantis clones in Hungary, which might have spread to humans through chicken meat.
Key Words: multidrug resistance , class 1 integron , plasmid
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Introduction |
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Salmonellosis is one of the most important enteric diseases affecting many people worldwide. Being a zoonotic disease, the economic loss it causes to the industry, and especially for the poultry sector, is also significant. In Hungary, the serotypes Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Typhimurium had traditionally been predominant among poultry and humans. However, during the last decade the prevalence of these serotypes decreased, possibly due to the massive use of Salmonella Enteritidis and Salmonella Typhimurium vaccines as part of the Salmonella eradication programmes. In contrast, the proportion of Salmonella Infantis has increased both in the poultry flocks and among the human cases, and in the last few years Salmonella Infantis became the most frequently isolated serotype from meat-producing (broiler) chickens and the third most frequent serotype causing human infections in Hungary in 2004.1 Similar, but less striking, developments have been reported recently from some other European countries.1
In order to characterize these emerging Salmonella strains, we have set up a Salmonella Infantis collection of isolates from recent years (2004–05, n = 132) originating from human stool (n = 56), raw and processed broiler chicken meat (n = 31), broiler chicken faeces (n = 29) and from other animal or feed sources (n = 16). Antibiotic resistance, genetic diversity and the possible epidemiological links among the isolates were studied. In order to test the genetic alteration that might have occurred as compared with the earlier years, one human isolate and five chicken isolates originating from 1994 (n = 6) were also included in the study.
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Materials and methods |
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Bacterial isolates, phage-typing and antimicrobial susceptibility testing
The human isolates were received during 2004–05 by the Phage-typing and Molecular Epidemiology Department of the National Center for Epidemiology from several public health laboratories representing the whole territory of Hungary. The animal isolates were collected by the Microbiology Department of the National Food Investigation Institute from different parts of the country during the same time period. All strains were serotyped according to the Kauffmann–White scheme,2 phage typed according to the Hungarian scheme for Salmonella Infantis as described by Laszlo et al.3 and investigated for antibiotic susceptibility by the disc diffusion method on Mueller–Hinton agar using antibiotic discs (Oxoid Ltd, Basingstoke, UK) as follows: ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulphonamide, sulfamethoxazole/trimethoprim and tetracycline. MICs of ciprofloxacin were determined by broth microdilution. The methods were carried out and the zones of growth inhibition were evaluated according to the recommendations of the CLSI (2005).4 The Escherichia coli strain ATCC 25922 was used as a reference strain.
Detection of antimicrobial resistance genes
For the PCR detection of class 1 integrons and sul1, tet(A) and tet(B) genes, bacterial DNA was prepared by boiling a bacterial culture in 200 µL of F1 buffer (20 mM Tris, 2 mM EDTA, pH = 8.0, 0.5% Triton X-100) for 10 min. Class 1 integron carriage was determined as described by Levesque et al.5 For sul1 the PCR method described by Sandvang et al.6 was used, whereas for tet(A) and tet(B) the PCR methods described by Guerra et al.7 and Guillaume et al.,8 respectively, were used. The gene encoded in the variable region of the detected class 1 integron was identified by sequencing in the case of three representative isolates obtained from human stool, chicken faeces and chicken meat, using the 5'CS and 3'CS PCR primers and an ABI Prism 377 DNA sequencer. DNA sequences were compared with those in the GenBank database (NCBI) using the BLAST program (http://ncbi.nih.gov).
Pulsed-field gel electrophoresis
PFGE was carried out according to the standardized Salmonella protocol of the CDC PulseNet. The Salmonella Braenderup H9812 strain was used as a molecular standard. PFGE-generated DNA profiles were entered into the Fingerprinting II Software (Bio-Rad Laboratories, Ventura, CA, USA) for analysis. Cluster analysis was performed by the unweighted pair-group method with arithmetic averages; DNA relatedness was calculated on the basis of the Dice coefficient. A 1.0% position tolerance and 1.5% optimization setting were applied.
Plasmid preparation was conducted by the alkaline lysis method of Kado and Liu.9 Agarose gel electrophoresis was performed using 0.75% agarose in a vertical system. The approximate sizes of plasmids were estimated by comparing them with the reference plasmids of E. coli V517 (2.0–53.7 kb) and E. coli R27 (168 kb) using the Quantity One software (Bio-Rad Laboratories).
Conjugation and restriction enzyme analysis of the large plasmid
To test the transferability of the large plasmid, three representative strains (human stool, 2/04; chicken meat, 15/04; and chicken faeces, 54/04), all belonging to pulsotype B2, having nalidixic acid–streptomycin–sulphonamide–tetracycline resistances and only harbouring the >168 kb plasmid were selected as donors. The plasmid-free E. coli J 5-3 RifR strain was used as recipient. Transconjugants (TCs) were selected on Mueller–Hinton agar plates supplemented with 10 mg/L tetracycline and 300 mg/L rifampicin and tested by API 20E (bioMérieux, Marcy l'Étoile, France). The transfer of the plasmid and the presence of the transmitted antimicrobial resistance genes were tested by the methods described above. The plasmids from the TCs were purified and restricted according to standard methods.
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Results and discussion |
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Origin, resistance type, phage type, class 1 integron and plasmid content as well as pulsotype of the isolates are summarized in the Table 1. Two closely related phage types, PT213 and PT217 (which differ from each other in one phage sensitivity only), predominated and formed 68% (94/138) of the isolates. Out of the 138 isolates tested, 72% (n = 100) were characterized by the nalidixic acid–sulphonamide–streptomycin–tetracycline antibiotic resistance-type, and these resistances were occasionally complemented with resistance to ampicillin (n = 8), chloramphenicol (n = 1), ciprofloxacin (n = 3, MICs were 8, 16 and 64 mg/L), kanamycin (n = 2) and/or trimethoprim-sulfamethoxazole (n = 3). Interestingly, none of the isolates from 1994 showed resistance to any antibiotics tested. In contrast, there were only a few susceptible human isolates from the recent years, two from 2004 (1.45%) and five from 2005 (3.6%), and all isolates of broiler chicken faeces and chicken meat origin were resistant to nalidixic acid at least. These results are concordant with the observation of Malorny et al.10 who have also detected high percentages (35% to 74%) of quinolone resistance among recent poultry-associated Salmonella serotypes of German origin. Out of the resistant isolates, 92% (110/119) had a single class 1 integron. Sequencing of this integron purified from representative isolates showed that it has an aadA1 gene in its 855 bp variable region that codes for streptomycin–spectinomycin resistance. In the background of the sulphonamide resistance, the sul1 gene was identified in all sulphonamide-resistant strains (110/110), while tetracycline resistance was mostly encoded by the tet(A) gene (103/110) and tet(B) was not detected. Interestingly, similarly high incidence of streptomycin and tetracycline resistances and the same class 1 integron have been described recently by Japanese authors in Salmonella Infantis isolates of poultry origin.11,12 Besides the antimicrobial resistance testing, in their study Kudaka et al.11 compared the PFGE fingerprints of the broiler cloacal swab and chicken-meat isolates with those of human isolates and concluded that there was no coincidence among them. In our study, PFGE by XbaI enzyme digestion enabled discrimination of the 138 isolates into 16 distinct PFGE profiles (pulsotypes) and 5 genetic clusters (A–E). Strains with PFGE patterns of >90% similarity were considered to belong to the same genetic cluster, and within a cluster, patterns that differed from each other in at least one band were considered as subtypes (marked by digits) (Figure 1). Although all strains showed >82% similarity to each other and therefore can be regarded as closely related, a 100% identity was detected among the XbaI PFGE fingerprints of 91 of the recent isolates (pulsotype B2): 41 human, 19 chicken faeces, 25 chicken meat and 6 other isolates, clearly indicating that 66% of the Hungarian isolates tested belong to the same genetic clone. It is worth noting that the isolates from 1994 were not only susceptible to the investigated antimicrobials but were also integron- and plasmid-free, and represented clearly distinct pulsotypes (A1 or A3) compared with the recent resistant isolates (Table 1 and Figure 1). This is suggestive of the acquisition of the >168 kb plasmid that was present in all but one of the resistant isolates during the last few years, which might have resulted in the acquisition of some additional resistances. To support or reject this assumption, conjugation experiments were done using three representative donor strains that showed the nalidixic acid–streptomycin–sulphonamide–tetracycline resistance type and had this large plasmid only. The obtained TCs were tetracycline resistant and harboured the tet(A) gene but remained sulphonamide and streptomycin susceptible despite the fact that the class 1 integron was also transferred. EcoRI plasmid restriction analysis of the >168 kb plasmid purified from the TCs showed identical restriction profiles, suggesting that not only the size but also the structure of this plasmid may be identical in the different strains (data not shown). However, more detailed investigation of the structure as well as the possible contribution of this plasmid to the virulence and spread of the described isolates requires further studies.
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In summary, we report the emergence of a multidrug-resistant Salmonella Infantis clone (pulsotype B2) and of several closely related pulsotypes in recent years in Hungary, characterized by a class 1 integron and a large conjugative plasmid encoding tetracycline resistance. It seems that broiler chickens constitute a reservoir for these strains, which might have spread to humans through chicken meat. The unusually high rate of nalidixic acid resistance among these strains is a matter of concern, as they may have decreased susceptibility to ciprofloxacin, a fluoroquinolone often used in the treatment of human Salmonella infections. However, this aspect would need a more targeted study, which was beyond the scope of this short report.
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None to declare.
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Acknowledgements |
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The excellent technical assistance of Mrs Margit Király, Mrs Vera Koppány and Mrs Judit Orbán is acknowledged. Dr István Tóth provided helpful suggestions and assisted by critically reading the manuscript. These studies were partly supported by the NKFP 4/040/2001 consortium. N. N is the holder of a Bolyai János stipend from the Hungarian Academy of Sciences.
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References |
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