JAC Advance Access originally published online on June 16, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):297-304; doi:10.1093/jac/dkl242
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Characterization of antimicrobial resistance and class 1 and 2 integrons in Salmonella enterica isolates from different sources in Portugal
1 Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto Porto, Portugal 2 REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto Porto, Portugal 3 Centro Nacional de Salmonella, Instituto Nacional de Saúde Dr Ricardo Jorge Lisboa, Portugal
*Corresponding author. Tel: +351-22-2078972; Fax: +351-22-2003977; E-mail: lpeixe{at}ff.up.pt
Received 24 February 2006; returned 27 March 2006; revised 10 May 2006; accepted 17 May 2006
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Abstract |
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Objectives: The antimicrobial resistance profiles of 1183 Salmonella isolates collected during 2002–2003 from several sources (human, food products and environment) were evaluated. The occurrence, distribution and cassette content of class 1 and 2 integrons among the sulphonamide-resistant population, as well as the role of particular clones to the spread of these genetic elements, were investigated.
Methods: The isolates were examined for susceptibility to antimicrobial agents. The characterization of class 1 and 2 integrons was investigated using PCR, PCR–RFLP (restriction fragment length polymorphism) and sequencing in the sulphonamide-resistant isolates. Conjugation assays and clonality analysis by PFGE were performed.
Results: The most common resistance phenotypes were to nalidixic acid, tetracycline, streptomycin, sulfamethoxazole and ampicillin (ranging from 31% to 17%). Resistance to sulphonamides (n = 200) was associated with resistance to other antimicrobial agents, with 75% of the isolates carrying one or two class 1 integrons while only 3% simultaneously carried class 1 and 2 integrons. Integrons were observed among at least 11 serotypes (mainly Typhimurium) and in a reduced number of PFGE clones (20). Eight class 1 integron types were found, with the aadA genes (aadA1, aadA2 and aadA5) alone or downstream of a trimethoprim (dfrA1, dfrA12 and dfrA17) or a ß-lactamase resistance gene (blaOXA-30) and the blaPSE-1 gene alone. Most of the class 1 integron types were shared by several clones from the same or different serotypes obtained either from humans or food products of animal origin, especially pork products. However, some Typhimurium-specific integrons were found: aadA2 plus blaPSE-1 and blaOXA-30-aadA1.
Conclusions: Apart from the hypothetical contribution of the conjugative transfer of integrons, the incidence of Salmonella carrying these genetic units seems to rely on the ability of certain clones to spread or persist in particular animal niches. Our data suggest that food-producing animals might be simultaneously considered as a reservoir of clones and integrons carrying antibiotic resistance genes, thus making the food chain, especially pork products, a possible source of multidrug-resistant isolates in humans.
Keywords: multidrug resistance , gene cassettes , PFGE , food safety
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Introduction |
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Bacterial antimicrobial resistance has become a worldwide public health problem with direct impact on food safety, being crucial the monitoring of food-borne pathogens that possess important animal reservoirs, such as Salmonella, as proposed by the EU legislation.1
Salmonellosis is one of the most frequent food-borne diseases in almost all industrialized countries. The widespread use of antimicrobial agents in food-animal production has contributed to the occurrence of Salmonella with decreased susceptibility to antibiotics, which can be transmitted to humans through food products, particularly those of animal origin.2 The increasing number of infections with antimicrobial drug-resistant Salmonella, including the emergence of multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium phage type DT104,2 extended-spectrum ß-lactamase (ESBL)-producing Salmonella3,4 and fluoroquinolone-resistant Salmonella strains,2,5 deserves special attention. The formation of genetic elements that cointegrate antibiotic resistance and virulence determinants, which may compromise the therapeutic options in cases of invasive Salmonella infections, is also part of a worrisome trend.4
Several of the antibiotic resistance genes observed in Gram-negative microorganisms are part of a gene cassette inserted in an integron.6,7 The most common cassettes contain genes that confer resistance to a range of antimicrobial agents, including aminoglycosides, ß-lactams, chloramphenicol and trimethoprim, as well as genes that confer resistance to antiseptics and disinfectants.6,7 Several classes of integrons related to antibiotic resistance have been identified that can be distinguished by the nucleotide sequence of their respective integrase.7 Through incorporation into transposons and plasmids, integrons participate in the capture and dissemination of resistance genes among bacteria. The fact that genes yielding resistance to antibiotics commonly used in the treatment of human infections could be acquired by integron-harbouring strains may potentiate the possibilities of selection by a variety of different antimicrobials. Therefore, integron acquisition is considered the major cause of multiple resistance in Gram-negative microorganisms, mainly in enteric bacteria.7,8
Although several class 1 and 2 integrons have been identified in different Salmonella serotypes,9–17 the study of the dissemination of these elements among Salmonella obtained from different sources (clinical, foods and environmental samples) has not been currently explored. In addition, the contribution of clonal spread to the incidence of Salmonella carrying these genetic elements has been scarcely described. In the present study, the antimicrobial resistance profiles of Salmonella isolates collected during 2002–2003 from several sources (human clinical samples, food products and environmental samples) were evaluated. Moreover, the occurrence, distribution and cassette content of class 1 and 2 integrons among the sulphonamide-resistant population were investigated along with the role of particular clones to the spread of these genetic elements.
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Materials and methods |
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Bacterial strains
The study included 1183 Salmonella isolates collected in Portugal, during 2002–2003. The isolates were recovered from human clinical sources (867); food products (258), including poultry (98), pork (85), beef (10), other food types (27) and unknown sources (38); as well as from the environment (58). The strain collection was obtained from the National Centre of Salmonella (INSA, Lisboa, Portugal) and from geographically dispersed clinical and food microbiology laboratories. The serotypes were determined at the National Centre of Salmonella. A PCR assay for the identification of S. enterica serotype Typhimurium DT104 and U302 phage types was conducted according to Pritchett et al.18
Antimicrobial susceptibility testing
The MICs of 10 antimicrobial agents (streptomycin, kanamycin, gentamicin, ampicillin, nalidixic acid, ciprofloxacin, chloramphenicol, tetracycline, sulfamethoxazole and trimethoprim) were determined by the agar dilution method, according to the CLSI (Clinical Laboratory Standards Institute),19 using Mueller–Hinton agar 2 (bioMérieux, Marcy l'Étoile, France). The breakpoints used were those defined by the CLSI for Enterobacteriaceae,19 with the exception of streptomycin.20 Escherichia coli ATCC 25 922 was used as the reference strain.19 In all the ampicillin-resistant isolates a double-disc synergy test for the detection of ESBL production was performed by the disc diffusion method21 with an amoxicillin/clavulanic acid disc placed next to ceftazidime, cefotaxime, cefuroxime, aztreonam, ceftriaxone and cefepime discs on Mueller–Hinton agar 2 (bioMérieux).
Detection of class 1 and class 2 integrons
The presence of class 1 integrons was tested by PCR (Table 1), using the primers 5'CS–3'CS22 and the specific primer for the int1 gene23 in all the sulfamethoxazole-resistant isolates. To characterize the conserved segment 3'CS, a PCR, using specific primers,24 was performed to determine the presence of sul1 and qacE
1 genes in all those isolates. Class 2 integrons were detected by PCR (Table 1) with specific primers for the int2 gene,25 and subsequently the cassette regions were amplified using primers hep74 and hep51 for the attI2-orfX region.26
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Characterization of class 1 and class 2 integrons
To determine the content of the variable regions of the class 1 integrons, first the 5'CS primer was used in combination with reverse primers for several antibiotic resistance genes (Table 1). Second, typing of each class 1 and class 2 integron was performed by a PCR–RFLP analysis. PCR products corresponding to the amplification of the 5'CS–3'CS region of the class 1 integrons, and of the attI2-orfX region of the class 2 integrons, were purified using the GFX PCR DNA and gel band kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and further digested with 5 U of TaqI endonuclease (New England BioLabs, Ipswich, MA, USA). The restriction products were analysed by electrophoresis on 2% agarose gels. Integron types were designated by roman numerals. Third, PCR products representing the different amplicons were sequenced by the dideoxy-chain termination method with an ALF Express automatic DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden). Obtained sequences were compared with those registered in GenBank, using the Blast program.
PFGE
Clonality among isolates was assessed by PFGE following XbaI digestion of genomic DNA according to the standard 1 day protocol of the CDC.27 DNA fragments were subjected to PFGE in agarose (Seakem Gold Agarose, Cambrex BioScience Rockland, Inc., USA) (1.2%, w/v) in 0.5x TBE buffer in a CHEF-DR III system (Bio-Rad Laboratories, Richmond, CA, USA), with the following running conditions: 6 V/cm at 14°C, 120°, with pulse times from 2.2 to 63.8 s for 19 h. The addition of thiourea (10 mg/mL) to 0.5x TBE buffer was performed in PFGE of non-typeable strains.27 Genomic DNA from Salmonella serotype Braenderup H9812, obtained from the CDC, was also restricted with XbaI and used as a molecular size marker. Isolates with electrophoretic patterns that differed by three bands, at most, were assigned to the same clone, as in a previous report.28 Clones were designated by capital letters and subtypes were defined by a subindex that indicates the number of bands that differed from the strain considered to be the initial PFGE type.
Conjugation assays
Conjugative transfer of plasmids from Salmonella isolates into E. coli K802N (nalidixic acid and rifampicin resistant) was attempted using agar plates. Transconjugants were selected on Mueller–Hinton agar 2 (bioMérieux) plates containing sulfamethoxazole (256 mg/L) plus nalidixic acid (64 mg/L) (or 100 mg/L rifampicin if the donor was nalidixic acid resistant).
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Antimicrobial susceptibility
Forty-six per cent of the isolates were susceptible to all tested antimicrobial agents, 33% were resistant to a single antibiotic and 21% were multiresistant (ranging from 2–8 antibiotics). This high incidence of resistance was observed in isolates from all sources: a total of 178 out of 258 food-borne isolates (69%), 437 out of 867 human clinical isolates (50%) and 18 out of 58 environmental isolates (31%). The most common resistance phenotypes detected were to nalidixic acid (31%), tetracycline (19%), streptomycin (18%), sulfamethoxazole (17%) and ampicillin (17%). It is important to note that some of the ampicillin-resistant isolates also exhibited reduced susceptibility to amoxicillin/clavulanate, but remained susceptible to expanded-spectrum cephalosporins. Resistance to chloramphenicol (9%), trimethoprim (7%), gentamicin (1%) and kanamycin (0.2%) was also observed (Table 2). In general, the incidence of resistance to the tested antibiotics was higher among the food-borne isolates than among those of human origin, mainly to tetracycline, streptomycin and sulphonamides. It should be noted that 69 (22%) isolates from non-human origin and 176 (20%) from clinical samples with resistance to nalidixic acid also exhibited decreased susceptibility to ciprofloxacin (MIC 0.25–0.5 mg/L).
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Class 1 integrons
Resistance to sulphonamides was associated with resistance to other antimicrobial agents, a fact observed in 200 out of the 243 MDR isolates. Of the sulfamethoxazole-resistant strains 77% (n = 154) carried one or two class 1 integrons, with detection of resistance genes in variable regions of integrons in 150 (75%) of those isolates. In four isolates the array of gene cassettes was undetermined. Integrons were detected in 79% (59/75), 77% (93/121) and 50% (2/4) of food product, human and environmental isolates tested, respectively (Table 3). Sulphonamide resistance and class 1 integrons were more frequently found among isolates from pork products (49/85 and 39/85, respectively) than among isolates from poultry products (10/98 and 9/98, respectively). Apart from other less frequent serotypes, Salmonella serotype Enteritidis, the predominant serotype in human infection, was rarely found among the sulphonamide-resistant isolates (23/200) and isolates carrying class 1 integrons (19/154). In contrast, the high prevalence of Salmonella Typhimurium among the isolates with sulphonamide resistance (122/200) and those carrying class 1 integrons (88/154) is remarkable (Table 4).
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Taking into account the number and size of the amplicons associated with an isolate, the RFLP patterns and sequencing data, eight class 1 integron profiles (IPs) were defined: IP I included two integrons (1000 and 1200 bp) and IP II to IP VIII included one integron each of different size (1000–2000 bp), whose variable region carried one or two resistance genes (Table 5). Eight different gene cassettes were detected, encoding resistance to aminoglycosides, trimethoprim and ß-lactams. A single gene cassette was present in integrons from 65 isolates and two gene cassettes were identified in integrons from 85 isolates (Table 5). The aminoglycoside adenyltransferase genes (aadA), which confer resistance to streptomycin and/or spectinomycin, were the most prevalent among gene cassettes. The aadA genes (aadA1, aadA2 and aadA5), alone or in combination with other resistance genes, were observed in variable regions of all but one integron-containing isolate and were always located close to the 3'CS. As a common feature, 65 of those integrons included an aadA gene downstream of a trimethoprim resistance gene (dfrA1, dfrA12 or dfrA17). An integron-associated ß-lactamase gene was identified in 67 isolates, conferring resistance to ampicillin: blaPSE-1 in 47 integrons of 1200 bp and blaOXA-30 in 20 integrons of 2000 bp, upstream of the aadA1 gene. Several integrons shared identical gene cassettes: IP I, IP II and IP VII, aadA2; IP III, IP V and IP VIII, aadA1; and IP I and IP IV, blaPSE-1. As described previously,28 the sul1 gene was located in 149 of the class 1 integrons and the other sul genes (sul2 or sul3) were found in five strains whose integrons lacked the qacE
1 and sul1 genes. In four isolates whose integrons lacked the qacE1 and sul1 genes, the sul3 gene conferred the sulphonamide resistance and the two gene cassettes typical of the IP VII (dfrA12 and aadA2) were detected (Table 5).
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Class 2 integrons
Resistance to trimethoprim was observed in 84 isolates, being associated with resistance to sulphonamides in 83 of those isolates. Five out of 200 (3%) sulfamethoxazole-resistant strains simultaneously presented class 1 and class 2 integrons. The RFLP profiles of variable regions (PCR product of 
2200 bp) of the five class 2 integrons were similar and sequencing of one of the PCR products revealed the presence of three resistance genes: dfrA1, sat1 and aadA1. One of those isolates was from the Typhimurium serotype and four isolates presented a clonal relationship and were classified as serotype Muenchen, all isolated from humans.
Relationships of integrons, serotypes, origins and PFGE clones
Although the spread of the class 1 integrons occurred among several Salmonella serotypes (Typhimurium, Enteritidis, Rissen, Derby, Saintpaul, Brandenburg, Muenchen, Bredeney, Brikama, IIIb:65:Iv:enxz15 and Heidelberg) a reduced number of PFGE clone types carrying class 1 integrons (20 clones/150 isolates) was observed (Table 5 and Figure 1). It is important to note that the integron-carrying Salmonella isolates were from diverse sources and regions. The presence of class 1 integrons was mainly associated with serotype Typhimurium (88/154) (Tables 4 and 5). Two 1000 bp (aadA2) and 1200 bp (blaPSE-1) integrons were specific for the MDR DT104 serotype Typhimurium clone, and one of 2000 bp (blaOXA-30-aadA1) was carried only by another serotype Typhimurium clone, previously published,29 both obtained from geographically distant locations and sources, including animal food products and humans. In contrast, the other profiles appeared dispersed through several serotypes, with the integrons carrying aadA (aadA1 or aadA2) downstream of the dfrA (dfrA1 or dfrA12) as the most disseminated among different serotypes and sources, including humans, environment, pork and poultry products (Table 5). It is important to stress that all integron types were shared by isolates obtained either from humans or food products, geographically dispersed. The presence of class 1 integrons was more frequent among isolates from pork products (39/154) than from poultry (9/154) (Tables 3 and 5). In fact, all integron types, with the exception of IP IV, were observed in human and pork isolates, in contrast with a lower diversity in poultry (IP V, IP VII and IP VIII).
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Conjugation assays
Out of 108 Salmonella isolates tested 50 transferred their plasmids containing integrons to E. coli; the resistance profiles acquired by the transconjugants are shown in Table 5. The conjugation results obtained with four isolates supported the chromosomal location of the class 1 integrons in the prevalent Typhimurium DT104 clone, as no transconjugants were obtained on the selection plates containing sulfamethoxazole. In contrast with the antimicrobial resistance determinants in Salmonella Typhimurium DT104, most of the integrons in other Salmonella isolates were encoded in a transferable plasmid and could be transferred to E. coli by conjugation.
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Discussion |
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In the present study, the widespread occurrence of resistance to several groups of antibiotics in Portuguese Salmonella isolates was demonstrated, especially among those of food-borne origin. The most frequent resistance phenotypes observed were to tetracycline, streptomycin, ampicillin and sulphonamide. These findings may not be surprising, as these antimicrobials have been widely used in modern animal production systems, both in Portugal and other countries.11,25,30 As an interesting example, it seems important to consider the high prevalence of resistance to sulfamethoxazole, mainly associated with the serotype Typhimurium, which accounts for 29% of resistance when only food products are considered, as a possible reflection of high usage of sulphonamides in food-producing animals, as verified in our country by Pena et al.31 In opposition with current reports on non-typhoid Salmonella strains isolated in various countries worldwide,3,4,17 the dissemination of ESBL-producing isolates has not yet been documented in Portugal, in spite of the high percentage of isolates exhibiting ampicillin resistance. Additional information also worthy of attention seems to be the high incidence of nalidixic acid-resistant isolates with decreased susceptibility to fluoroquinolones, as observed in this study, due to the possibility of treatment failures in invasive gastrointestinal infections when these agents are used.5 This worrisome feature, observed in human and food-borne isolates, especially in poultry isolates, seems to be mainly associated with serotype Enteritidis (data not shown), an observation that is in close agreement with other European studies.32
It has been argued that a substantial proportion of antimicrobial resistance to several antibiotics is the responsibility of integron structures.7,8 In the present study, the presence of gene cassettes coding for a particular antibiotic resistance was only demonstrated for streptomycin, trimethoprim, ampicillin and sulphonamides. In addition, the results of conjugation experiments also suggested the structural association of resistance genes in mobile elements, including those encoded by integrons, which might account for the increase in MDR Salmonella. Therefore, there seems to exist a strong association between multiresistance and the presence of integrons,8,26 a fact that can be easily confirmed when analysing the present results where a high (77%) proportion of sulphonamide-resistant (which are also MDR isolates) Salmonella isolates presented integrons.
The analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to aminoglycosides (aadA genes) and trimethoprim (dfrA genes), with aadA genes carried by all the integron-containing Salmonella serotypes. The persistence of these genes, which have been reported worldwide in isolates of different origins,9–13,17 might be associated with the extensive use of streptomycin in food-producing animals, or is most likely the result of their structural association with other resistance genes (co-selection), such as the sul1 gene, for which selective pressure is continuously observed or with genetic elements whose extreme versatility promotes their conservation.33 However, not all the Enteritidis serotype strains with the dfrA1-aadA1 integron were resistant to streptomycin even though they harboured the aadA1 gene, as also described by White et al.17 It is also interesting to note that five strains harboured two different integrons (class 1 and class 2) containing the aadA1 gene cassette, as observed by other authors,26 although the benefit of having these two identical genes has not yet been fully understood.
Most of the class 1 integrons, especially IP V (dfrA1, aadA1) and IP VII (dfrA12, orfF aadA2), were shared by several clones from the same or different serotype, which is suggestive of horizontal transfer, as confirmed by our conjugation results, and might indicate a wide dissemination of the specific structures in which integrons are located. All five class 2 integrons carried the same three cassettes as those found in Tn7, namely dfrA1, sat1 and aadA1, which could be explained by the fact that class 2 integrons are unable to excise existing cassettes or insert new ones.26 In contrast with class 1 integrons, this class of integrons was only detected in human isolates, one serotype Typhimurium isolate and in a clone of serotype Muenchen (this is the first report of a class 2 integron in the serotype Salmonella Muenchen), suggesting that class 2 integrons are less commonly found among Salmonella isolates from different origins. In addition, the low diversity among the integron gene cassettes in our sample suggests a great stability of several types of integron profiles, in agreement with other studies in different countries, indicating a wide distribution of some integrons in Salmonella.9–13,15–17
The presence of class 1 integrons was more frequent in serotype Typhimurium, including the pentaresistant DT104 and other worrisome MDR clones carrying different integron structures, whose dissemination seems to be associated with the food chain. Also, our study highlights the presence of resistance genes associated with integrons in additional non-Typhimurium serotypes, such as Enteritidis, a predominant serotype generally associated with low rates of resistance,32 and in less-prevalent serotypes with possible extensive reservoirs.
Serotype-specific class 1 integrons were found, such as the 1000–1200 bp, known to be part of the chromosomal located Salmonella genomic island 1 (SGI1),34 in the stable and widely disseminated clone of MDR serotype Typhimurium DT104,10,12,15,16,24 the predominant integron-carrying clone recovered in our study. In this study, apart from the presence of isolates of the widespread clone of serotype Typhimurium DT104 carrying only one of those gene cassettes (aadA2 or pse-1), it is important to note that the aadA2 gene cassette was also dispersed in another clone of a different serotype (Derby), suggesting the distribution of the SGI1 in S. enterica serotypes other than serotype Typhimurium DT104, as observed by Boyd et al.34 The presence of the 2000 bp integron carrying blaOXA-30 and aadA1 gene cassettes was specific for another MDR Typhimurium clone, disseminated in our country by food products of animal origin (pork products) and obtained from geographically dispersed sources and locations, as previously described.29 In Portugal, the ongoing spread of this MDR clone and the possible transfer of the plasmid encoding this integron to other strains is a worrisome issue, as virulence and multidrug resistance could be genetically linked, as observed recently in plasmids carrying related integrons.35,36
Although a large collection of MDR Salmonella isolates obtained from geographically dispersed regions in Portugal and from human and non-human samples was studied, only 20 PFGE types of at least 11 serotypes revealed class 1 integrons. The observation of those particular PFGE types might reflect an improved fitness of specific clones able to survive under harmful conditions, as antibiotic selective pressure or other compounds used in the veterinary field. On the other hand, the finding that no difference was observed in the distribution of integron types between human and food isolates suggests human acquisition through the food chain from several reservoirs of animal origin. However, differences in prevalence and gene cassette arrays of class 1 integrons were found in Salmonella isolates according to the food-animal sources, as also observed by other authors in E. coli isolates.37 In contrast with the higher prevalence and diversity of integron types in pork products, the lower prevalence and diversity of integron types in poultry products and the reduced number of clones within this niche may be related to the characteristics of this current animal production, where only a few different genetic lines of primary breeding flocks are used, limiting host diversity. The differences in the antibiotic selective pressure imposed through several years of intensive use might have enhanced the spread of specific clones and integrons perfectly adapted to a certain host type, within different sources.
Our data suggest that food-producing animals might be simultaneously considered as a reservoir of clones and integrons carrying antibiotic resistance genes, thus making the food chain, especially pork products, a possible source of MDR isolates in humans. Intensive antibiotic use over several years may have contributed to the selection of particular clones, integrons and integron-carrying genetic elements, although other factors, not directly related to resistance, may contribute to their spread and niche specificity. Surveillance of the integron content in Salmonella populations of different sources (clinical, food and environmental samples) can provide powerful information concerning the evolutionary changes of gene cassettes, which may be fundamental to estimate health risk and to prevent the spread of particular antibiotic resistance determinants via the food chain from animals to humans.
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None to declare.
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Acknowledgements |
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We are very grateful to Centro Nacional de Salmonella (Lisboa, Portugal) for the serotyping of the strains and to CDC for the PFGE protocols and the control strain Salmonella braenderup H9812. This study was partially supported by Fundação Calouste Gulbenkian, Portugal (project no. 49975).
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1 Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the Monitoring of Zoonoses and Zoonotic Agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC. Official Journal L 325, 2003, pp. 0031–0040.
2 Threlfall EJ. (2002) Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections. FEMS Microbiol Rev 26:141–8.[CrossRef][ISI][Medline]
3 Miriagou V, Tassios PT, Legakis NJ, et al. (2004) Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int J Antimicrob Agents 23:547–55.[CrossRef][ISI][Medline]
4 Fluit AC. (2005) Towards more virulent and antibiotic-resistant Salmonella? FEMS Immunol Med Microbiol 43:1–11.[CrossRef][ISI][Medline]
5
Aarestrup FM, Molbak K, Threlfall EJ. (2003) Is it time to change fluoroquinolone breakpoints for Salmonella spp.? Antimicrob Agents Chemother 47:827–9.
6 Hall RM and Collis CM. (1998) Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updat 1:109–19.
7 Rowe-Magnus DA and Mazel D. (2002) The role of integrons in antibiotic resistance gene capture. Int J Med Microbiol 292:115–25.[CrossRef][ISI][Medline]
8 Leverstein-van Hall MA, Blok HEM, Donders ART, et al. (2003) Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis 187:251–9.[CrossRef][ISI][Medline]
9
Ahmed AM, Nakano H, Shimamoto T. (2005) Molecular characterization of integrons in non-typhoid Salmonella serovars isolated in Japan: description of an unusual class 2 integron. J Antimicrob Chemother 55:371–4.
10
Ebner P, Garner K, Mathew A. (2004) Class 1 integrons in various Salmonella enterica serovars isolated from animals and identification of genomic island SGI1 in Salmonella enterica var. Meleagridis. J Antimicrob Chemother 53:1004–9.
11
Gebreyes WA, Thakur S, Davies PR, et al. (2004) Trends in antimicrobial resistance, phage types and integrons among Salmonella serotypes from pigs, 1997–2000. J Antimicrob Chemother 53:997–1003.
12
Guerra B, Soto S, Cal S, et al. (2000) Antimicrobial resistance and spread of class I integrons among Salmonella serotypes. Antimicrob Agents Chemother 44:2166–9.
13
Lindstedt B-A, Heir E, Nygard I, et al. (2003) Characterization of class 1 integrons in clinical strains of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis from Norwegian hospitals. J Med Microbiol 52:141–9.
14 Nastasi A and Mammina C. (2001) Presence of class 1 integrons in multidrug-resistant, low-prevalence Salmonella serotypes, Italy. Emerg Infect Dis 7:455–8.[ISI][Medline]
15 Nógrády N, Gadó I, Tóth A, et al. (2005) Antibiotic resistance and class 1 integron patterns of non-typhoidal human Salmonella serotypes isolated in Hungary in 2002 and 2003. Int J Antimicrob Agents 26:126–32.[CrossRef][ISI][Medline]
16
Randall LP, Cooles SW, Osborn MK, et al. (2004) 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 53:208–16.
17
White DG, Zhao S, Sudler R, et al. (2001) The isolation of antibiotic-resistant Salmonella from retail ground meats. N Engl J Med 345:1147–54.
18
Pritchett LC, Konkel ME, Gay JM, et al. (2000) Identification of DT104 and U302 phage types among Salmonella enterica serotype Typhimurium isolates by PCR. J Clin Microbiol 38:3484–88.
19 National Committee for Clinical Laboratory Standards. (2003) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6 (NCCLS, Wayne, PA, USA).
20 DANMAP 2004. Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria from Food Animals, Foods and Humans in Denmark, July 2005. ISSN 1600–2032. Statens Serum Institut, Danish Veterinary and Food Administration, Danish Medicines Agency, and Danish Institute for Food and Veterinary Research.
21 National Committee for Clinical Laboratory Standards. (2002) Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement M100-S12 (NCCLS, Wayne, PA, USA).
22 Lévesque C, Piché L, Larose C, et al. (1995) PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 39:185–91.[Abstract]
23
Kerrn MB, Klemmensen T, Frimodt-Moller N, et al. (2002) Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulfonamide resistance. J Antimicrob Chemother 50:513–6.
24 Sandvang D, Aarestrup FM, Jensen LB. (1998) Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104. FEMS Microbiol Lett 160:37–41.[CrossRef][ISI][Medline]
25
Mazel D, Dychinco B, Webb VA, et al. (2000) Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene. Antimicrob Agents Chemother 44:1568–74.
26
White PA, McIver CJ, Rawlinson WD. (2001) Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 45:2658–61.
27 Centers for Disease Control and Prevention. (2002) One-Day (24–48 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli O157:H7, Non-typhoidal Salmonella Serotypes, and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE) (PulseNet PFGE Manual, CDC, Atlanta, Georgia, USA).
28
Antunes P, Machado J, Sousa JC, et al. (2005) Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica strains and relation with integrons. Antimicrob Agents Chemother 49:836–9.
29
Antunes P, Machado J, Sousa JC, et al. (2004) Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 ß-lactamase. J Antimicrob Chemother 54:429–34.
30 European Agency for the Evaluation of Medical Products (EMEA). Antibiotic Resistance in the European Union Associated with Therapeutic Use of Veterinary Medicines. Report and Qualitative Risk Assessment by the Committee for Veterinary Medicinal Products, 1999. EMEA/CVMP/342/99-corr-Final. http://www.emea.eu.int/pdfs/vet/regaffair/034299en.pdf (August 2005, date last accessed).
31 Pena A, Serrano C, Réu C, et al. (2004) Antibiotic residues in edible tissues and antibiotic resistance of faecal Escherichia coli in pigs from Portugal. Food Addit Contam 21:749–55.[CrossRef][ISI][Medline]
32 Threlfall EJ, Fisher IST, Berghold C, et al. (2003) Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance. Euro Surveill 8:41–5.[Medline]
33
Chiew Y-F, Yeo S-F, Hall LMC, et al. (1998) Can susceptibility to an antimicrobial be restored by halting its use? The case of streptomycin versus Enterobacteriaceae. J Antimicrob Chemother 41:247–51.
34
Boyd D, Cloeckaert A, Chaslus-Dancla E, et al. (2002) Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46:1714–22.
35
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. Antimicrob Agents Chemother 46:2977–81.
36
Villa L and Carattoli A. (2005) Integrons and transposons on the Salmonella enterica serovar Typhimurium virulence plasmid. Antimicrob Agents Chemother 49:1194–7.
37 Box ATA, Mevius DJ, Schellen P, et al. (2005) Integrons in Escherichia coli from food-producing animals in The Netherlands. Microb Drug Resist 11:53–7.[CrossRef][ISI][Medline]
38
Lee JC, Oh JY, Cho JW, et al. (2001) The prevalence of trimethoprim-resistance-conferring dihydrofolate reductase genes in urinary isolates of Escherichia coli in Korea. J Antimicrob Chemother 47:599–604.
39
Bert F, Branger C, Lambert-Zechovsky N. (2002) Identification of PSE and OXA ß-lactamase genes in Pseudomonas aeruginosa using PCR–restriction fragment length polymorphism. J Antimicrob Chemother 50:11–8.
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