National Salmonella Reference Laboratory, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, 12277 Berlin, Germany
Received 22 April 2005; returned 5 June 2005; revised 15 July 2005; accepted 13 September 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: A total of 319 epidemiologically independent multidrug-resistant isolates from German foodstuffs comprising 25 different serovars were tested for their antimicrobial susceptibility by broth microdilution. The presence of antimicrobial resistance genes, integrons of classes 1 and 2 and their integrated resistance gene cassettes as well as the Salmonella genomic island 1 (SGI1) was investigated by PCR and DNA sequencing. Localization of integrons and relevant resistance genes was done by Southern hybridization. Sequence analysis revealed mutations in the quinolone resistance-determining region of the gyrA gene.
Results: The most prevalent resistances found in the multidrug-resistant serovars of Salmonella enterica from foods were to streptomycin (94%), sulfamethoxazole (92%), tetracycline (81%), ampicillin (73%), spectinomycin (72%), chloramphenicol (48%) and trimethoprim (27%). Twenty-four resistance genes covering six antimicrobial families (ß-lactams, aminoglycosides, phenicols, sulphonamides, tetracycline, and trimethoprim) were identified in the food isolates, many of them integrated as gene cassettes in class 1 and class 2 integrons. Class 1 integrons were detected in 65% of the multidrug-resistant Salmonella isolates comprising 16 different serovars, while class 2 integrons were found in 10% of the isolates belonging to two serovars only. The results demonstrate a clear predominance of both SGI1-borne resistance genes and class 1 integrons in Salmonella serovar Typhimurium DT104 and of class 2 integrons in Salmonella serovar Paratyphi B (D-tartrate positive). Nalidixic acid resistance found in 15% of the isolates was associated with single mutations in the gyrA gene.
Conclusions: This study confirms the role of foods of animal and other origin as a reservoir of multidrug-resistant Salmonella and underlines the need for continuing surveillance of food-borne zoonotic bacterial pathogens along the food chain.
Keywords: multidrug resistance , resistance genes , integrons , class 1 integrons , class 2 integrons , gene cassettes , food safety
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the last two decades, the emergence and spread of antimicrobial-resistant pathogens, among them Salmonella, has become a serious health hazard worldwide. The routine practice of giving antimicrobial agents to domestic livestock as a means of preventing and treating diseases, as well as promoting growth, is an important factor in the emergence of antibiotic-resistant bacteria that are subsequently transferred to humans by the food chain.2 Of particular concern is the increasing association of human infections with multidrug-resistant Salmonella strains. In recent years, numerous outbreaks have been caused by the multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type DT104 with resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulphonamides and tetracycline, but increasingly other multidrug-resistant Salmonella serovars have been responsible for infections in humans.35
Mobile genetic elements such as plasmids, transposons, and the more recently explored integrons, which are able to disseminate antibiotic resistance genes by horizontal or vertical transfer, have been shown to play an important role in the evolution and dissemination of multidrug resistance in Gram-negative bacteria.68 Integrons are genetic elements that are able to capture gene cassettes from the environment and incorporate them by using site-specific recombination. Class 1 integrons, mostly found as part of the Tn21 or Tn402 transposon family are the most widely disseminated ones among the members of the family Enterobacteriaceae,9 including many of the S. enterica serovars.10,11 Integrons of this class contain a 5' conserved segment (5'-CS) with the integrase gene intI1, the attI1 recombination site and the promoter, generally followed by a variable region that consists of an array of one or more resistance gene cassettes. The majority of the class 1 integrons also contain a 3' conserved segment (3'-CS) of variable length but in most cases consisting of qacE1, encoding marginal resistance to some antiseptics; the sul1 gene, encoding sulphonamide resistance; and two open reading frames, orf5 and orf6.12 Class 2 integrons are associated with the Tn7 transposon family and have been identified in Salmonella so far only in the serovars Typhimurium and Paratyphi B [D-tartrate positive (dT+)].13,14
Most of the studies on the prevalence and characterization of antimicrobial resistance genes and integrons in Salmonella spp. reported during the last few years focused on isolates from clinical or animal sources.10,11,15,16 Studies on food isolates are rare and limited to selected food products,17,18 and as a result, at present there is a paucity of data regarding the antimicrobial resistance and molecular mechanisms involved in Salmonella and other pathogen strains from foodstuff. Thus, this study was initiated to: (i) determine antimicrobial susceptibility of German multiresistant food-borne Salmonella strains of different serovars; (ii) identify the antimicrobial resistance genes involved; and (iii) characterize integrons and the resistance gene cassettes therein and their relationship to the Salmonella genomic island 1 with the aim of advancing our understanding of the molecular mechanisms of antimicrobial resistance.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 810 epidemiologically unrelated Salmonella isolates obtained from a variety of German foodstuffs during the year 2001 were selected from routine submissions to the National Salmonella Reference Laboratory by previously described criteria.19 Serotyping was performed according to the Kauffmann-White scheme,20 phage typing was done as described before.21
Antimicrobial susceptibilities to ampicillin, chloramphenicol, ceftiofur, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim were assessed by determining the minimum inhibitory concentration using the NCCLS broth microdilution method.22 Breakpoints given from the NCCLS and from DANMAP were used.23 Of the 810 Salmonella food isolates, 319 (39.4%) were multidrug-resistant (defined as isolates that were resistant to two or more antimicrobial agents) and selected for further molecular investigations. The isolates originated from food of animal origin mainly: 220 isolates were recovered from foodstuffs of both cattle and swine (beef, pork, ground meat, different sausages and meat products, innards), 80 isolates from foodstuffs of poultry (chicken, turkey, goose, duck, giblets, eggs and egg products); 19 isolates originated from other food sources (sweets, cereal products, vegetables, game).
PCR analysis, DNA purification and DNA sequencing
DNA templates used for PCR were prepared by boiling bacterial cultures or by using the QIAGEN Genomic-tip System (QIAGEN GmbH, Hilden, Germany). The following genes implicated with antimicrobial resistance were detected by PCR amplification and DNA sequencing: blaPSE-1, blaOXA-1-like and blaTEM-1-like encoding ß-lactam resistance; aadA1-like, aadA2, strAstrB, aac(3)-II, aac(3)-IV, aadB, aphA-1 and kn encoding aminoglycoside resistance; catA1, cmlA1-like and floR encoding chloramphenicol resistance; sul1, sul2 and sul3 encoding sulphonamide resistance; tet(A), tet(B), tet(C), tet(D) and tet(E) encoding tetracycline resistance; dfrA1-like, dfrA7, dfrA12, dfrA14, and dfrA17 encoding trimethoprim resistance. The primer sets and the assay conditions used for amplification were described elsewhere.2433 Amplifications were performed in 25 µL reaction mixtures containing 2.5 µL of DNA, 2.5 µL 10x PCR buffer, 1.5 mM MgCl2, 200 µM each deoxynucleoside triphosphate (Invitrogen GmbH, Karlsruhe, Germany), 1 µM each primer (BioTEZ Berlin Buch GmbH, Berlin, Germany), 1 U of Platinum Taq DNA Polymerase (Invitrogen GmbH). The PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis.
The detection of mutations in the quinolone resistance-determining region (QRDR) of the subunit A of DNA gyrase of nalidixic acid-resistant isolates was performed as described previously.34
Class 1 and class 2 integrons were screened for by PCR with primers specific for the intI130 and intI235 integrase genes. Primers 5'-CS and 3'-CS described by Lévesque et al.28 targeting the inserted gene cassette regions of class 1 integrons were used to determine these regions. The PCR of White et al.36 using the primers hep74 and hep51 was performed to amplify the class 2 integron gene cassette regions. To determine the gene order in the cassette region, PCR amplification was carried out using primers for sequences located at the ends of the inserted gene cassettes in combination with those specific for other gene cassettes, as well as with those for the conserved segments.28
Amplification products were extracted from the gel, purified with the GFXTM DNA and Gel Band Purification Kit (Amersham Biosciences, Freiburg, Germany) and sequenced on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Sequences obtained were compared with those registered in the GenBank of the National Center for Biotechnology Information via the BLAST network service.
Detection of Salmonella genomic island 1 (SGI1) and its location was performed using primers corresponding to left and right junctions in the chromosome as described previously.37,38
Pulsed-field gel electrophoresis (PFGE), plasmid analysis and Southern blot hybridization
Genomic DNA suitable for PFGE was prepared according to standard methods outlined by PulseNet.39 PulseNet S. enterica serovar Braenderup H9812 and both Lambda Ladder PFG and Low Range PFG markers (New England Biolabs, Schwalbach, Germany) were used as molecular reference markers. The DNA embedded in agarose was digested with 0.4 U/µL XbaI (Amersham Biosciences) and electrophoresed on 1% agarose gels in 0.5x TrisborateEDTA buffer at 14°C in a CHEF DR-III system (Bio-Rad, Munich, Germany) using the following conditions: initial switch time, 2 s; final switch time, 64 s at an angle of 120° at 6 V/cm for 19 h (for 13 x 14 cm gels) or 26 h (for 20 x 14 cm gels).
Plasmid DNA was prepared according to the alkaline denaturation procedure of Kado and Liu40 and separated on 0.7% agarose gels. High molecular weight plasmids that were not discovered by that method, were identified using nuclease S1 digestion prior to PFGE as previously described.41 Briefly, DNA embedded in agarose was digested with 0.1 U/µL nuclease S1 (Amersham Biosciences) and electrophoresed on 1% agarose gels in a CHEF DR-III system. Electrophoresis was carried out at an angle of 120° at 6 V/cm with pulses ramping from 2 to 30 s over 19 h in 0.5x TrisborateEDTA buffer at 14°C.
Southern blot hybridizations of plasmid patterns, of nuclease S1-PFGE patterns and of XbaI-PFGE patterns were performed by standard methods.42 In order to determine the location of identified class 1 and class 2 integrons on plasmids and/or the chromosome intI1- and intI2-specific probes labelled with digoxigenin using the PCR DIG Labeling Mix (Roche Diagnostics GmbH, Mannheim, Germany) were used. For determining the location of selected integron- or non-integron-borne resistance genes, gene-specific sequences were labelled in the same way and used as probes. Hybridization procedures and conditions were performed as specified with a non-radioactive labelling and detection kit (Roche Diagnostics GmbH).
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 319 multidrug-resistant Salmonella food isolates could be assigned to 25 different S. enterica serovars (Table 1). The remarkably high prevalence of Salmonella serovar Typhimurium is in concordance with the predominance of this serovar in German livestock, particularly in cattle and swine.23 In contrast, Salmonella serovar Enteritidis, the predominant serovar in human infections, was rarely found among the multiresistant isolates. Because of its epidemiological importance, Salmonella serovar Typhimurium phage type DT104 (hereafter referred to as DT104) was separated from the other Salmonella serovar Typhimurium phage types (hereafter referred to as non-DT104) and each group was evaluated independently. Representing nearly 45% of all food isolates investigated, DT104 turned out to be the most common serovar. However, its prevalence in food originating from cattle and swine was substantially higher (51.4%) than in food originating from poultry (15.0%). As a result, DT104 ranks highest in food originating from cattle and swine, but only comes second in food from poultry, where the most common serovar has been Paratyphi B dT+ (41.2%).
|
Using the NCCLS broth microdilution method, 93.7% of the 319 isolates were found to be resistant to streptomycin, 92.5% to sulfamethoxazole, and 80.9% to tetracycline. The rates of resistance to ampicillin, spectinomycin, chloramphenicol, trimethoprim and nalidixic acid were 73.0%, 72.4%, 48.3%, 27.3% and 15.0%, whereas only 4.4% and 3.1% of the isolates were resistant to kanamycin and gentamicin, respectively (Table 1). None of the isolates was resistant to ceftiofur. Isolates were resistant to two and up to eight antimicrobial agents. Reflecting the high prevalence of multidrug-resistant (MDR) DT104, the most common antimicrobial resistance pattern observed included ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole and tetracycline (resistance type ACS/SpSuT). In addition, a wide variety of resistance phenotypes was found, and the most important of them are shown in Table 2.
|
Twenty-eight genes implicated with antimicrobial resistance and conferring resistance to six families of antimicrobials, including ß-lactams, aminoglycosides, phenicols, sulphonamides, tetracycline, and trimethoprim, had been chosen to determine their distribution in different food-associated Salmonella serovars. Twenty-four resistance genes were identified among the isolates investigated. Table 1 shows a good correlation between PCR detection of genes and corresponding individual resistance phenotypes. Results indicate that Salmonella isolates from food may contain multiple genes specifying identical resistance phenotypes. In 49 cases, such genes were present even in one and the same isolate: blaPSE-1 and blaTEM-1 in one, aadA1 and aadA2 in three, aadA1 and strA-strB in 15, aadA2 and strA-strB in five, sul1 and sul2 in 22, sul1, sul2 and sul3 in one, tet(A) and tet(B) in two case(s). In 16 cases, the genes responsible for resistance could not be identified, indicating other resistance mechanisms.
Table 1 also summarizes the results regarding the prevalence and the serovar distribution of the genes. The high prevalence of the genes blaPSE-1 (61.8%), aadA2 (53.5%), floR (90.9%), tet(G) (54.3) and sul1 (69.8%) was expected and is, above all, attributed to the high prevalence of MDR DT104 among the foodstuffs investigated. Of interest, during this study we found 10 additional isolates, of them four serovar Typhimurium phage type U302, two serovar Typhimurium phage type DT120 and four serovar Subspecies I-Rough, which revealed MDR DT104-like resistance genes and traits. In fact, recently, MDR DT104-like traits have been reported in serovar Typhimurium phage types other than DT104 and in other S. enterica serovars.37,38 Genes blaPSE-1, floR and tet(G) were found in MDR DT104-like isolates exclusively, while aadA2 was found, in addition, in serovars Agona, Derby, Heidelberg, Schwarzengrund and non-DT104. Sul1 was detected both in MDR DT104-like isolates and in 15 other S. enterica serovars (Table 1). A wide distribution among 11 to 19 S. enterica serovars has been shown for genes dfrA1-like, blaTEM-1-like, sul2, strA-strB, tet(A), and aadA1 with prevalences of 75.9%, 38.6%, 36.9%, 30.4%, 28.7%, and 26.1%, however, in MDR DT104-like isolates, these genes were detected in a few cases only (Table 1). The dihydrofolate reductase gene dfrA1 was found to be the most common trimethoprim resistance mechanism in the food isolates, followed by dfrA14 (17.2%) and dfrA12 (5.7%). Kanamycin resistance was conferred by aphA-1 in 12, and by kn in one of the 14 resistant isolates. Mechanisms of gentamicin resistance deserve further study, because genes aac(3)-II, aac(3)-IV or aadB were detected in only four of the 10 resistant isolates.
The major mechanism of resistance to the quinolone nalidixic acid is known amino acid substitutions in the gyrA QRDR region at positions 83 and 87. The nucleotide sequence analysis of this region of the 48 nalidixic acid-resistant food isolates (32 from poultry) revealed two different single point mutations at positions 83 and three at position 87, described in detail previously.34 Serovar Paratyphi B dT+ isolated from poultry meat was the main contributor (45.8%) to nalidixic acid resistance.
Class 1 and class 2 integrons and their distribution among different serovars
Two hundred and eight (65.2%) of the multidrug-resistant Salmonella food isolates were found to carry class 1 integrons, and 33 (10.3%) to carry class 2 integrons. Among these isolates, four (1.2%) carried a class 1 and a class 2 integron simultaneously. Sixteen types of class 1 integrons and two types of class 2 integrons were detected, each type characterized by the size of its amplicon and the type and number of antibiotic resistance gene cassette(s) integrated. These features are summarized in Table 2. Class 1 integrons were detectable in 16 different Salmonella serovars (Agona, Derby, Enteritidis, Goldcoast, Haifa, Heidelberg, Infantis, Livingstone, London, Mbandaka, Paratyphi B dT+, Saintpaul, Schwarzengrund, Subspecies I-Rough and both MDR DT104 and non-DT104), while class 2 integrons were found in Salmonella serovars Paratyphi B dT+ and non-DT104 only. Results indicate that integron-borne multidrug resistance was not solely associated with a few particular serovars.
Class 1 integron-associated gene cassettes within Salmonella genomic island 1 (SGI1)
It is obvious that the majority of class 1 integron-positive isolates (74.0%) in this study revealed a clear specificity to the epidemic MDR DT104 and its relatives, such as MDR U302, MDR DT120 and MDR Subspecies I-Rough (Table 2). In all these isolates, the presence of the Salmonella genomic island 1 (SGI1) and its location in the chromosome could be confirmed by PCR analysis using primers corresponding to the left and right junctions of SGI1 in the chromosome.37 The complete SGI1 with its complex class 1 integron structure that belongs to the In4 group,37 was detected in 137 food isolates of resistance type ACS/SpSuT and in three isolates of resistance type ACS/SpSuTTp. In SGI1, there is a duplication of the 5'-CS, each one followed by a gene cassette. The first one is the aadA2 cassette, which confers streptomycin/spectinomycin resistance, the second is the pse-1 cassette conferring ampicillin resistance. This arrangement produced the characteristic pair of amplicons of 1000 and 1200 bp using primers 5'-CS and 3'-CS. The cassettes bracket the floR and tet(G) genes which confer resistance to chloramphenicol and tetracycline, respectively.
Ten isolates of S/SpSu resistance type generating a 1000 bp amplicon and three isolates of ASu resistance type generating a 1200 bp amplicon were found to contain SGI1 variants with only one 5'-CS. Hence, they contain a single gene cassette, either aadA2 (SGI1-C) or pse-1 (SGI1-B).37 Genes floR and tet(G) were not present. A DT104B low isolate of AS/SpSuTTp resistance type, which produced two amplicons of 1200 bp and 1600 bp, contained the pse-1 cassette (SGI1-B), too. The second amplicon corresponded to the dfrA1aadA1 gene cassette array (conferring trimethoprim and streptomycin/spectinomycin resistance) and had no relation to SGI1.
Class 1 integron-associated gene cassettes not related to SGI1
The remaining class 1 integron-positive isolates (26.0%) harboured no SGI1. They comprised 13 non-DT104 isolates and 41 isolates representing 14 other S. enterica serovars (Table 2). The isolates showed a variety of resistance phenotypes, but all of them except two had the core S/SpSu resistance type in common (Table 2). Integrons of these isolates contained an aadA1 (84.6%), aadA2 (19.2%) or aadA5 (1.9%) gene cassette encoding resistance to streptomycin/spectinomycinalone (in 23.1% of the cases) or in combination with other gene cassettes. Probably, the high prevalence of the aadA1 cassette among these isolates is related to its location on Tn21-transposable elements.6,8 Thirty-four of the isolates (65.4%) carried upstream of the aadA cassette a dfrA1, dfrA12 or dfrA14 cassette encoding trimethoprim resistance, three (5.8%) a sat cassette encoding streptothricin resistance, one (1.9%) an aadB cassette encoding gentamicin resistance, and one (1.9%) a catB3 cassette encoding chloramphenicol resistance. Amplicons generated were 150, 1000, 1600, 1700, 1900, 1950, 2200, 2300 and 2400 bp in size. Both 150 bp amplicons were found to be a class 1 integron element without any cassette in it. The subsequent amplicons were shown to correspond to aadA1 and/or aadA2, dfrA1aadA1, aadBaadA2, dfrA12orfaadA2 and sataadA1 cassette regions, respectively (Table 2). Such integrons have been already described in studies on animal and clinical isolates.8,10,11,15,16,36 In contrast, the last specified amplicons revealed the presence of novel variants of gene cassette arrays, so far not detected in Salmonella: dfrA1orfaadA1 in Salmonella serovar London, dfrA14aadA1catB2 in Salmonella serovar Derby and dfrA1catB3aadA5 in Salmonella serovar Livingstone. Cassettes catB2 and catB3 confer chloramphenicol resistance through chloramphenicol acetyltransferases.7
While all class 1 integrons mentioned above possess the sul1 gene as part of the 3'-CS, two Salmonella serovar Heidelberg isolates of CS/SpSu resistance type lacked it, and no amplicon was generated. Such integrons, which are missing parts of the 3'-CS, have been described before.12 By PCR analysis, these isolates revealed a linkage of 5'-CS to a domain consisting of a cmlA1 cassette flanked by the aadA1 and aadA2 cassettes (Table 2). The cmlA1 cassette is a rare cassette, which mediates a non-enzymic chloramphenicol resistance mechanism through a drug efflux pump.43 Of interest, sulphonamide resistance in the isolates is conferred by the recently described dihydropteroate synthase gene sul3.32 Additional investigations are under way in order to complete the organization of this class 1 integron and to compare it with the unusual class 1 integron structure recently detected in swine Escherichia coli.43
Class 2 integron-associated gene cassettes
All class 2 integron-positive isolates showed a core S/SpTp resistance type combined in many cases with additional resistance determinants (Table 2). The major class 2 integron type comprised all but one isolate and revealed a definite specificity to Salmonella serovar Paratyphi B dT+. The isolates were from food of poultry origin and amplicons of 2200 bp size corresponded to the known Tn7-like array of gene cassettes, dfrA1sat1aadA1orfX, recently described as a characteristic feature of the predominant Salmonella serovar Paratyphi B dT+ clone in poultry in Germany.14 The presence of this array accounts for the resistance to trimethoprim, streptothricin and streptomycin/spectinomycin. The second integron type encompassed one isolate only, a non-DT104 isolate from pork generating an amplicon of about 2400 bp, consistent with the presence of the cassette array dfrA1orfaadA1. No sat gene was found suggesting that possibly this integron type is not part of Tn7 or related genetic elements.
Genetic localization of class 1 and class 2 integrons
Class 1 integrons that are part of SGI1 or SGI1 variants were shown to be located in the chromosome.37 As a confirmation, Southern blot hybridization using an intI1 probe was performed and revealed the expected 12 kb XbaI fragment in isolates containing the complete SGI1, the expected 7 kb XbaI fragment in those containing SGI1-C and the expected 2.5 kb XbaI fragment in those containing SGI1-B (Table 2).
In contrast, the vast majority of class 1 integrons in non-SGI1 isolates mapped to high molecular weight plasmids of sizes ranging between 50 and 350 kb (Table 2). However, the aadA2-carrying integron in isolates of S/SpSuT resistance type was localized to a chromosomal XbaI fragment of 60 kb, and the 3'-CS-defective integron in isolates of CS/SpSu resistance type mapped to a plasmid of about 110 kb and to the chromosome as well. Moreover, unlike other integrons of this type, the integron carrying dfrA1aadA1 in the MDR DT104B low isolate of AS/SpSuTTp resistance type was located on the chromosome. Another chromosomally located dfrA1aadA1-carrying integron has been described previously.44
Class 2 integrons were localized to the bacterial chromosome, exclusively. In all isolates of Salmonella serovar Paratyphi B dT+, the intI2 probe hybridized to the same two chromosomal XbaI fragments of about 78 and 410 kb, indicating that two copies were present and confirming previously described data.14 The distinct class 2 integron of non-DT104 is located on a single chromosomal XbaI fragment of 30 kb.
Non-integron-borne resistance genes
In isolates of resistance types ACS/SpSuT, SSpSu and ASu, the entire multiresistance phenotype was encoded by resistance genes within the class 1 integron, while in isolates of resistance type ACS/SpSuTTp, additional non-integron-borne genes dfrA14, strAstrB and sul2 were detected, all co-located on a 7 kb plasmid. In non-SGI1 isolates, the following resistance determinants in the different multiresistance phenotypes were found to be independent of a class 1 or class 2 integron: the ampicillin resistance was encoded by blaTEM-1, exclusively, the kanamycin resistance by the aphA1 or kn genes, the tetracycline resistance by tet(A), tet(B), tet(C) or tet(D) genes. Resistance to chloramphenicol, gentamicin, streptomycin, and sulphonamide, respectively, was encoded by catA1, aac(3)-IV, strAstrB, sul2 or sul3, alone or together with the relevant genes within integrons.
Taken together, the data presented in this study confirm the wide diversity of resistance mechanisms mediating antimicrobial resistance in different serovars of S. enterica isolated from German foodstuffs. Of the multidrug-resistant Salmonella isolates investigated, 75.5% were shown to carry class 1 or class 2 integrons suggesting the important role of these genetic elements in the complexity of antimicrobial resistance dynamics. Among the S. enterica serovars found in the food isolates, Salmonella serovar Typhimurium and, particularly, MDR DT104, can be considered the most important carrier of class 1 integrons and Salmonella serovar Paratyphi B dT+ the most important carrier of class 2 integrons. Because class 1 integrons have become integrated into the chromosome in MDR DT104 and its relatives, they are able to persist even in the absence of antimicrobial selection with no apparent fitness cost to the cell. The same goes for the class 2 integrons of Salmonella serovar Paratyphi B dT+. For both examples, this has led to stable and widely disseminated multidrug-resistant clones in livestock and in foods in Germany.14,23 On the other hand, the location of the non-SGI1 class 1 integrons on large transferable plasmids makes their intra- and interspecies horizontal transfer very efficient. Even if so far all of the gene cassettes found in these integrons confer resistance to the older aminoglycosides, such as streptomycin/spectinomycin and gentamicin, as well as to ß-lactams, trimethoprim and chloramphenicol, we should be aware that integration of genes conferring resistance to antimicrobials of higher generations seems a matter of time and/or selection pressure in the livestock. As expected for the zoonotic organism Salmonella, there is good agreement in the occurrence and distribution of antibiotic resistance genes and integron types in the food-borne isolates of this study compared with isolates of food-producing animal and human origin (own unpublished results).10,11,15,16,25,45 As a result, the data presented provide more evidence that foodstuffs of animal and other origin can be considered as vehicles of transmission of antimicrobial resistance.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Angulo F, Nargund V, Chiller T. Evidence of an association between use of anti-microbial agents in food animals and anti-microbial resistance among bacteria isolated from humans and the human health consequences of such resistance. J Vet Med B Infect Dis Vet Public Health 2004; 51: 3749.[ISI][Medline]
3. Threlfall EJ. Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections. FEMS Microbiol Rev 2002; 26: 1418.[CrossRef][ISI][Medline]
4.
Zansky S, Wallace B, Schoonmaker-Bopp D et al. From the Centers for Disease Control and Prevention. Outbreak of multi-drug resistant Salmonella NewportUnited States, JanuaryApril 2002. JAMA 2002; 288: 9513.
5. Brown DJ, Mather H, Browning L et al. Investigation of human infections with Salmonella enterica serovar Java in Scotland and possible association with imported poultry. Euro Surveill 2003; 8: 3540.[Medline]
6.
Liebert CA, Hall RM, Summers AO. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev 1999; 63: 50722.
7. Hall RM. Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. Ciba Found Symp 1997; 207: 192202.[ISI][Medline]
8. Carattoli A. Importance of integrons in the diffusion of resistance. Vet Res 2001; 32: 24359.[CrossRef][ISI][Medline]
9. Leverstein-van Hall MA, Blok HEM, Donders ART et al. Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis 2003; 187: 2519.[CrossRef][ISI][Medline]
10.
Guerra B, Soto S, Cal S et al. Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes. Antimicrob Agents Chemother 2000; 44: 21669.
11.
Randall LP, Cooles SW, Osborn MK et al. Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J Antimicrob Chemother 2004; 53: 20816.
12.
Partridge SR, Recchia GD, Stokes HW et al. Family of class 1 integrons related to In4 from Tn1696. Antimicrob Agents Chemother 2001; 45: 301420.
13. Sundstrom L, Roy PH, Skold O. Site-specific insertion of three structural gene cassettes in transposon Tn7. J Bacteriol 1991; 173: 30258.[ISI][Medline]
14.
Miko A, Pries K, Schroeter A et al. Multiple-drug resistance in D-tartrate-positive Salmonella enterica serovar Paratyphi B isolates from poultry is mediated by class 2 integrons inserted into the bacterial chromosome. Antimicrob Agents Chemother 2003; 47: 36403.
15.
Lindstedt BA, Heir E, Nygard I et al. Characterization of class I integrons in clinical strains of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis from Norwegian hospitals. J Med Microbiol 2003; 52: 1419.
16.
Gebreyes WA, Thakur S, Davies PR et al. Trends in antimicrobial resistance, phage types and integrons among Salmonella serotypes from pigs, 19972000. J Antimicrob Chemother 2004; 53: 9971003.
17. Zhao S, Datta AR, Ayers S et al. Antimicrobial-resistant Salmonella serovars isolated from imported foods. Int J Food Microbiol 2003; 84: 8792.[ISI][Medline]
18.
Chen S, Zhao S, White DG et al. Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats. Appl Environ Microbiol 2004; 70: 17.
19.
Miko A, Guerra B, Schroeter A et al. Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar Paratyphi B isolates. J Clin Microbiol 2002; 40: 318491.
20. Popoff MY, Minor LL. Antigenic Formulas of the Salmonella Serovars. Paris, France: WHO Collaborating Centre for Reference and Research on Salmonella, Institute Pasteur, 2001.
21. Anderson ES, Ward LR, Saxe MJ et al. Bacteriophage-typing designations of Salmonella typhimurium. J Hyg (Lond) 1977; 78: 297300.[Medline]
22. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA, 2000.
23. Schroeter A, Hoog B, Helmuth R. Resistance of Salmonella isolates in Germany. J Vet Med B Infect Dis Vet Public Health 2004; 51: 38992.[ISI][Medline]
24. Guerra B, Junker E, Miko A et al. Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains. Microb Drug Resist 2004; 10: 8391.[CrossRef][ISI][Medline]
25. Sandvang D, Aarestrup FM, Jensen LB. Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104. FEMS Microbiol Lett 1998; 160: 3741.[CrossRef][ISI][Medline]
26. Walker RA, Lindsay E, Woodward MJ et al. Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods. Microb Drug Resist 2001; 7: 1321.[CrossRef][ISI][Medline]
27. Madsen L, Aarestrup FM, Olsen JE. Characterisation of streptomycin resistance determinants in Danish isolates of Salmonella Typhimurium. Vet Microbiol 2000; 75: 7382.[CrossRef][ISI][Medline]
28. Levesque C, Piche L, Larose C et al. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995; 39: 18591.[Abstract]
29.
Frana TS, Carlson SA, Griffith RW. Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype typhimurium phage type DT104. Appl Environ Microbiol 2001; 67: 4458.
30.
Ng LK, Mulvey MR, Martin I et al. Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrob Agents Chemother 1999; 43: 301821.
31.
Chu C, Chiu CH, Wu WY et al. Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob Agents Chemother 2001; 45: 2299303.
32.
Perreten V, Boerlin P. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother 2003; 47: 116972.
33.
Frech G, Kehrenberg C, Schwarz S. Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources. J Antimicrob Chemother 2003; 51: 1802.
34.
Malorny B, Schroeter A, Guerra B et al. Incidence of quinolone resistance in strains of Salmonella isolated from poultry, cattle and pigs in Germany between 1998 and 2001. Vet Rec 2003; 153: 6438.
35.
Ploy MC, Denis F, Courvalin P et al. Molecular characterization of integrons in Acinetobacter baumannii: description of a hybrid class 2 integron. Antimicrob Agents Chemother 2000; 44: 26848.
36.
White PA, McIver CJ, Rawlinson WD. Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 2001; 45: 265861.
37.
Boyd D, Cloeckaert A, Chaslus-Dancla E et al. Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 2002; 46: 171422.
38.
Doublet B, Weill FX, Fabre L et al. Variant Salmonella genomic island 1 antibiotic resistance gene cluster containing a novel 3'-N-aminoglycoside acetyltransferase gene cassette, aac(3)-Id, in Salmonella enterica serovar Newport. Antimicrob Agents Chemother 2004; 48: 380612.
39. Peters TM, Maguire C, Threlfall EJ et al. The Salm-gene projecta European collaboration for DNA fingerprinting for food-related salmonellosis. Euro Surveill 2003; 8: 4650.[Medline]
40. Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 1981; 145: 136573.[ISI][Medline]
41. Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting and sizing large plasmids. Anal Biochem 1995; 226: 23540.[CrossRef][ISI][Medline]
42. Sambrook JE, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Edition. New York: Cold Spring Harbor Laboratory Press, 1989.
43. Bischoff KM, White DG, Hume ME et al. The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli. FEMS Microbiol Lett 2005; 243: 28591.[CrossRef][ISI][Medline]
44.
Daly M, Buckley J, Power E et al. Evidence for a chromosomally located third integron in Salmonella enterica serovar Typhimurium DT104b. Antimicrob Agents Chemother 2004; 48: 13502.
45.
Carattoli A, Filetici E, Villa L et al. Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrob Agents Chemother 2002; 46: 28218.
|