1 Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark; 2 Central Institute for Animal Disease Control, Department of Bacteriology and TSEs, Houtribweg 39, PO Box 2004, 8203 AA Lelystad, The Netherlands
Received 16 February 2005; returned 15 March 2005; revised 9 May 2005; accepted 10 May 2005
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Abstract |
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Methods: Thirty-four ESBL-resistant Salmonella isolates from The Netherlands were tested towards 21 antimicrobial agents. PCR and sequencing were used to determine the underlying genetic determinants responsible for the ESBL phenotypes. The transferability of the ESBL phenotypes was tested by conjugation to a susceptible Salmonella enterica serovar Dublin and plasmid purification, restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) were employed to further characterize a subset of the isolates.
Results: A great genetic diversity was seen among the isolates. The blaTEM-52 gene was most predominant and was found among Salmonella enterica serovars Blockley, Thomson, London, Enteritidis phage type 14b, Paratyphi B, Virchow and Typhimurium phage types 11 and 507. We also found the blaTEM-20 gene in S. Paratyphi B var. Java and the blaTEM-63 gene in S. Isangi. Furthermore, we detected the blaCTX-M-28 gene in S. Isangi and the blaCTX-M-3 gene in S. Typhimurium phage type 507. The blaCTX-M-2 gene was identified in S. Virchow, which also contained a copy of the blaSHV-2 gene and a copy of the blaTEM-1 gene. The blaSHV-12 gene was found alone in S. Concord and together with the blaTEM-52 gene in S. Typhimurium. Finally, the blaACC-1 gene was cloned from a S. Bareilly isolate and was found to be present on indistinguishable plasmids in all S. Bareilly isolates examined as well as in a S. Braenderup isolate and a S. Infantis isolate.
Conclusions: Our data underscore the diversity of ESBL genes in Salmonella enterica isolated from animals, food products and human patients.
Keywords: AAC-1 , TEM-52 , plasmids , CTX-M-3 , CTX-M-28 , SHV-12
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Introduction |
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Resistance to ß-lactams in S. enterica is primarily caused by the production of acquired ß-lactamases. More than 340 ß-lactamases have been described and many of these have been identified in Salmonella.4,5 A large number of studies have investigated the occurrence of different ß-lactamases in Gram-negative bacteria isolated from clinical infections in humans. In contrast, there are only a limited number of studies that have focused on the occurrence of ß-lactamases among isolates from food animals and food products. Briñas et al. examined the occurrence of blaTEM-, blaSHV- and blaOXA-type ß-lactamases among 55 ampicillin-resistant Escherichia coli from healthy animals in Spain and found the resistance to be almost exclusively encoded by blaTEM-1b.6 Olesen et al. determined the genetic background for ß-lactamase-mediated resistance in 109 E. coli and 51 Salmonella isolates obtained from healthy and diseased food animals in Denmark.7 They also found blaTEM-1b to be the most frequently detected ß-lactamase, whereas only a few isolates expressed blaTEM-30, blaOXA or blaPSE ß-lactamases.
Salmonella isolates harbouring ESBLs have emerged worldwide during the last decade. This has caused concern since cephalosporins are drugs of choice for the treatment of salmonellosis in children. Different blaSHV, blaTEM, blaCTX and blaCMY genes have been found to encode ESBL resistance in Salmonella.810 Also, a few variants of the blaOXA genes have been identified in Salmonella. These all belong to the blaOXA-1 group (comprising blaOXA-1, blaOXA-4, blaOXA-30, blaOXA-31 and blaOXA-47 genes) and blaOXA-2 group (comprising blaOXA-2, blaOXA-3, blaOXA-15, blaOXA-32, blaOXA-34 and blaOXA-53 genes).
Identical plasmid-mediated ß-lactamase genes have been detected in Enterobacteriaceae in different countries, which could indicate a plasmid-borne spread of these genes between these countries. Nonetheless, no systematic determination of the prevalence of the different genes has been performed.
This study was conducted to investigate the genetic background responsible for ESBL resistance in Salmonella isolated from poultry, poultry products and human patients in The Netherlands.
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Materials and methods |
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A collection of 34 non-duplicate amoxicillin-resistant Salmonella isolates were examined. They were isolated in 2001 and 2002 in The Netherlands from poultry (n = 13), poultry products (n = 7) and human patients (n = 14) and showed reduced susceptibility to cefotaxime (MICs 1 mg/L).
Phage typing
Phage typing was performed by the National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands. The Colindale phage typing system was used for S. Enteritidis and the Dutch system was used for S. Typhimurium.
Susceptibility testing
Susceptibility was tested quantitatively by broth microdilution with cation-adjusted MuellerHinton broth, according to NCCLS guidelines.11 For broth microdilution, microtitre trays were used with dehydrated dilution ranges of custom-made panels of antibiotics (Trek Diagnostic Systems, Basingstoke, UK). ATCC strain E. coli 25922 was used daily to monitor the quality of the results. The following antimicrobial agents were included in the panels: amoxicillin, amoxicillin + clavulanate, apramycin, cefalothin, cefuroxime, ceftiofur, chloramphenicol, ciprofloxacin, colistin, gentamicin, imipenem, nalidixic acid, neomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim and florfenicol. Also, MICs for all isolates were determined for cefotaxime, cefoxitin and ceftazidime by the agar dilution assays in MuellerHinton agar according to NCCLS guidelines.11
Detection of blaTEM, blaSHV, blaCTX, blaCMY-1 group, blaCMY-2 group, blaOXA-1 and blaOXA-2
Rapid degradation of the Salmonella PCR products was prevented by using the High Pure PCR Template Preparation Kit (Roche Applied Science, catalogue no. 1796828) as suggested by the manufacturer, with an additional phenolization step. Here, 200 µL of phenol/chloroform/isoamylalcohol (25:24:1, by vol.) was added to the cell lysates immediately before they were transferred to the spin columns and the tubes were mixed thoroughly. The primers used were either adapted from previously published ones (Table 1) or designed using computer analysis of all available ß-lactamase sequences (GenBank) with the Vector NTI v8.0 program (Informax, Inc.). Primers and amplification conditions for each PCR are listed in Table 1. Each PCR test used the same basic set-up: 94°C for 3 min followed by 25 cycles of 1 min at 94°C, 1 min at TAnneal°C and TiElongate min at 72°C, where TAnneal is the specific annealing temperature and TiElongate is the specific elongation time for each reaction (see Table 1 for values) and one final step with 10 min of extension at 72°C. The following strains were used as controls for PCR: E. coli K-12 XL1-blue harbouring the plasmid pBR322 (blaTEM), S. Keurmassar DAK-2 (blaSHV), S. Virchow 75-22438-1 (blaCTX-M group), Klebsiella pneumoniae MISC339 (blaMOX-1 of the blaCMY-1 group), S. Newport S05127-02 (blaCMY-2 group), E. coli Co365 (blaOXA-30 of the blaOXA-1 group) and E. coli JS3 (blaOXA-2 group).
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Before sequencing, all PCR products were purified using the GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences, catalogue no. 27-9602-01). Sequencing was performed with the ABI PRISM Bigdye® Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, catalogue no. 4337450) using the same primers as were used to generate the PCR product (Table 1). Sequence analysis was performed on an ABI 377 DNA Sequencer (Perkin-Elmer, Applied Biosystems) and analysed using the computer program Vektor NTI Suite 8 (InforMax, Inc.). The obtained nucleotide sequences and the derived amino acid sequences were compared with previously described sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/) and www.lahey.org/studies/webt.html, respectively.
Transferability of ESBL phenotype
All Salmonella isolates were grown overnight in BHI medium at 37°C without shaking. A plasmid-free amoxicillin-susceptible Salmonella Dublin isolate JEO66, which was made resistant to nalidixic acid and rifampicin (called JEO66 RN), was used as recipient for the mating experiments.12 From overnight cultures in BHI broth incubated aerobically at 37°C, 100 µL of each ESBL-positive isolate was transferred to 5 mL of fresh BHI broth and incubated at 37°C until an optical density ( = 550 nm) of
0.5 was reached. Then 500 µL of each ESBL isolate was mixed with 500 µL of the recipient and the entire volume was inoculated on a fresh 5% calf blood agar plate. The blood agar plates were incubated aerobically for 5 h at 37°C. Transconjugants were recovered by pipetting 1 mL of BHI broth on the calf blood agar plates. After gentle mixing, 10 µL was transferred to selective BHI agar plates containing cefalothin (32 mg/L), nalidixic acid (50 mg/L) and rifampicin (50 mg/L). JEO66 RN, described above, served as a negative control.
Cloning of unknown ESBL resistance gene of S. Bareilly isolates
A transconjugant from the mating experiment described above between JEO66 RN and the ESBL-positive S. Bareilly strain 60.50 as a donor was selected for further investigation. Plasmid DNA was partially digested with HindIII and PstI and ligated into a HindIII- and PstI-digested cloning vector pLOW1. The ligation product was electroporated into electrocompetent E. coli XL1-blue (Stratagene, catalogue no. 200228) cells.13 Cloning of the S. Bareilly ESBL determinant was achieved by selecting transconjugants on agar plates containing ceftiofur (8 mg/L). An insert with a size of 7 kb was cloned and sequenced. The result of the sequencing has been submitted to GenBank (accession no. AY856832). The 7 kb fragment was analysed for possible open reading frames (ORFs) using the program Vector NTI version 8.0 (Informax, Inc.).
Detection of blaACC-1 based on the cloned sequence
Based on the sequence of the cloned fragment, primers (Table 1) were designed to amplify the ß-lactamase ACC-1, which was identified on the fragment. These primers were used to test for the presence of blaACC-1 in the remaining eight strains with unknown ß-lactamase resistance. Isolate 60.50 served as a positive control.
Restriction fragment length polymorphism (RFLP) analysis of ACC-1-positive strains
Plasmids were purified with the QIAGEN plasmid midi kit (Qiagen, catalogue no. 12145) as suggested by the manufacturer. RFLP analysis of plasmid DNA was then conducted with the restriction nuclease EcoRI and run on a 0.8% agarose gel at 40 V for 16 h.
Pulsed-field gel electrophoresis (PFGE) analysis of S. Bareilly and S. Blockley isolates
PFGE was carried out on selected Salmonella isolates using XbaI as previously described.14
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Results |
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All 34 strains examined (Table 2) were resistant to amoxicillin and cefalothin and all but two isolates were resistant to ceftiofur (MIC 8 mg/L), cefuroxime (MIC
32 mg/L) and ceftazidime (MIC
32 mg/L). Nine isolates were fully resistant to amoxicillin + clavulanate, while seven showed intermediate resistance. Eight isolates were resistant to cefotaxime (MIC
64 mg/L). Finally, 33 isolates were susceptible (MIC
8 mg/L) and one showed intermediate resistance (MIC = 16 mg/L) to cefoxitin (isolate 59.45 in Table 2) and all isolates were fully susceptible to imipenem (MIC
0.5 mg/L).
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All isolates were tested by PCR for the presence of blaTEM, blaSHV, blaCTX, the blaCMY-1 group, the blaCMY-2 group, the blaOXA-1 group and the blaOXA-2 group. This approach made it possible to detect the genetic background for many of the isolates (Table 2). However, negative results were obtained for nine isolates (all seven S. Bareilly isolates as well as the S. Braenderup and the S. Infantis isolates). All of these were inhibitor-resistant (amoxicillin + clavulanate > 32 mg/L). From one of these isolates (S. Bareilly 60.50), a 7176 bp fragment containing the genetic determinant responsible for the ESBL phenotype was cloned. Sequencing of the cloned fragment revealed the presence of five ORFs as well as two partial ORFs (Figure 1). One of these ORFs showed 100% identity to the blaACC-1 gene of Klebsiella pneumoniae (AJ270942).
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The remaining eight inhibitor-resistant isolates were tested for the presence of the blaACC-1 gene and all contained this gene (Table 2). To further establish the clonal relation between these isolates, plasmid purification was performed on the transconjugants from all nine blaACC-1-containing isolates, which were then subjected to RFLP analysis using EcoRI. All plasmid profiles were indistinguishable.
Transferability of ESBL resistance to a S. Dublin recipient
Mating experiments of all Salmonella isolates to the plasmid-free and amoxicillin-susceptible Salmonella Dublin recipient JEO66 RN were conducted. All isolates were able to transfer the ESBL phenotype to the recipient. However, even though the four strains 38.47, 59.45, 59.70 and 61.12 did lead to transconjugants on the selective plates when conjugated to JEO66 RN, it was not possible to maintain the ESBL resistance phenotype on fresh plates.
PFGE of S. Bareilly and S. Blockley strains
To further examine the clonal relationship of the S. Bareilly and S. Blockley isolates, PFGE using the XbaI restriction enzyme was performed on these strains and compared with eight amoxicillin-susceptible S. Bareilly and nine amoxicillin-susceptible S. Blockley isolated in Denmark and Thailand (data not shown). This showed the seven S. Bareilly to be closely related and to have a significantly different PFGE pattern from the Danish and Thai isolates. One of the Dutch isolates (60.50) had two additional bands compared with the six other Dutch isolates, but had otherwise the same PFGE profile as these. The seven S. Blockley isolates could neither be distinguished from the amoxicillin-susceptible isolates nor from each other, as an identical profile was seen for all isolates.
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Discussion |
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The first large ESBL set (ESBL-I in Table 2) all contained a version of the blaCTX-M gene and were all multiresistant (Table 2). All five isolates originated from human patients and belonged to two different serovars (S. Isangi and S. Typhimurium phage type 507). The S. Isangi isolates carried the blaCTX-M-28 gene and the S. Typhimurium isolates the blaCTX-M-3 gene. These variants of the blaCTX-M genes were not found in the animal or food reservoirs examined in this study, indicating that the sources of these infections in humans originate from other reservoirs or could be mainly associated with hospital infections. In fact, the blaCTX-M-3 gene is rarely, if ever, found in animal reservoirs, further supporting such a hypothesis.15 We do not have any data regarding any relationship between the five patients and cannot therefore say if the patients could have been in contact with each other. However, at least the three S. Isangi isolates were isolated over a time period of more than a year, contradicting such an event. The blaCTX-M-3 gene has previously been encountered in the most common Enterobacteriaceae.1620 This includes the Salmonella serovars Typhimurium, Enteritidis, Oranienburg, Anatum and Mbandaka.2124 In our study, we found the blaCTX-M-3 gene in S. Typhimurium as well. The blaCTX-M-28 gene is only available in the GenBank database as a direct submission from a clinical isolate of E. coli from France and has not been identified in Salmonella before. As the resistance determinants in the three S. Isangi isolates could easily be conjugated into and stably maintained in a S. Dublin recipient strain, this suggests that this gene has been acquired and that it has the potential to spread to other bacterial reservoirs in the future.
The only other blaCTX gene found in this study was from a S. Virchow isolate (the 58.67 strain), which contained the blaCTX-M-2 gene (ESBL-VI). This isolate also contained the blaSHV-2 and the blaTEM-1 genes.
Apart from the S. Virchow 58.67 isolate mentioned above, we only identified variants of the blaSHV gene in two other isolates (S. Concord 38.47 and S. Typhimurium 46.72). Both of these contained the blaSHV-12 gene and one (46.72) carried in addition the blaTEM-52 gene (ESBL-VIII). The blaSHV-12 variant seems to be relatively rare in Salmonella isolates in general, as only a few serovars have previously been associated with the blaSHV-12 gene.25,26 Again, our study is the first to describe this gene in S. Concord and S. Typhimurium.
The second main ESBL set (ESBL-II) all contained the blaACC-1 gene. This set consisted of all seven S. Bareilly isolates as well as the S. Infantis 48.75 and the S. Braenderup 35.04 isolate. In this set, the isolates were originally isolated from both poultry and human patients. It was first identified in a S. Bareilly isolated from poultry in February 2001 and 3 months later in patients. It cannot be excluded that the isolates are geographically linked, as we do not have any data regarding where they were isolated. However, our data could indicate an initial introduction of the gene with this serovar among chickens and that it subsequently clonally spread to humans. Plasmid purification revealed identical plasmid profiles for all nine isolates proving that the same plasmid is present in all three serovars. A French study from 2002 identified the blaACC-1 gene in three isolates of S. Livingstone from a Tunisian hospital.27 It would be interesting to test plasmids from these three isolates by RFLP to determine any connection between these Tunisian isolates and our isolates from The Netherlands.
PFGE analysis revealed identical profiles of the seven S. Bareilly isolates, which in addition differed significantly from the PFGE profiles of amoxicillin-susceptible S. Bareilly isolates from Denmark and Thailand. It is therefore most likely that the presence of the blaACC-1 gene in these three serovars is caused by a combination of both clonal and horizontal spread. This is only the second report of the blaACC-1 gene in Salmonella as K. pneumoniae and Proteus mirabilis are the common reservoirs for this gene and it is the first report of this gene in these three serovars.27,28 This could indicate that the blaACC-1 gene has now been established within the Salmonella population and has started to spread both clonally and to new serotypes, which will have to be followed closely in the future.
The remaining isolates all contained some variant of the blaTEM gene. The largest group of these all contained the blaTEM-52 gene (ESBL-III). The serotypes of this set of isolates were the most heterogenic of the different sets studied here. It contained seven different serovars isolated from poultry, poultry meat as well as human patients. The predominant serovar in ESBL-III was S. Blockley. The same serovar containing the blaTEM-52 gene has recently (July 2003) been isolated from patients with gastroenteritis in a study from France.29 The animal isolates in our study all originate from The Netherlands, and the animals could have been used for export. Also, the food products in our study could easily have been imported from neighbouring countries. Therefore, it is possible that the French isolate from a patient and the Dutch isolates from food animals and food products are related. This coincides well with the fact that we isolated the same serovar in animals and food products before it was found in patients in both The Netherlands and France. Again, our study identifies the blaTEM-52 variant in the serovars S. Thompson, S. London S. Paratyphi and S. Virchow for the first time.
Finally, we have identified other blaTEM variants in a few isolates. Two S. Paratyphi B var. Java (ESBL-V) isolated from a broiler and broiler meat, respectively, both carried the blaTEM-20 gene. These two isolates were resistant according to the NCCLS recommended breakpoint for screening of ESBL resistance (MIC 1) but only resistant to amoxicillin and cefalothin, when using the NCCLS guidelines, which is in good agreement with the detected gene. However, only one point mutation separates the blaTEM-20 gene and the blaTEM-52 gene, so this gene has a high potential to evolve into a genuine ESBL producer. The blaTEM-20 gene has previously mainly been found in E. coli, P. mirabilis and K. pneumoniae.3032 It has also been associated with Salmonella of unknown serotypes.33 Therefore, this is probably the first report of blaTEM-20 in S. Paratyphi B var. Java.
Also, one S. Isangi isolate from a patient carried the blaTEM-63 gene, which has previously been seen in S. Isangi and S. Muenchen from South Africa.34 Unfortunately, we have no knowledge about the travel history of the patient carrying the S. Isangi isolate in our study. Therefore, it cannot be excluded that the infection could have been acquired in South Africa in this incidence.
We did not find any versions of the blaOXA genes, or the genes belonging to the blaCMY-1 group or the blaCMY-2 group among our isolates. As the members of the blaOXA group are extremely diverse, we have only included primers able to detect blaOXA genes that have been found previously in Salmonella. It cannot therefore be excluded that some of our strains contain blaOXA genes not previously found in this species. Our blaCMY-1 group primers were designed to recognize blaCMY-1 as well as blaCMY-8, blaCMY-9, blaCMY-10 and blaCMY-11. They also recognize blaMOX-1, which served as a positive control in the PCR, even though the blaCMY-1 gene has one nucleotide change compared with one of the primers. However, the primers were not designed to recognize the blaMOX-2 gene, so this could in theory be present among the isolates. However, as we did not find any cefoxitin-resistant isolates, which is normally associated with the blaCMY-1 group, and as the genes we have identified in each case can explain the ESBL phenotype of the isolates examined in this study, it seems unlikely that genes belonging to the blaOXA group or the blaMOX-2 gene are present. Isoelectric focusing could be used to examine whether the Salmonella isolates investigated in this study might produce additional ß-lactamases whose genes were not detected by the PCR assay applied.
In conclusion, our data seem to confirm that many of the ESBL associated genes seem to spread rapidly into new Salmonella serovars. This makes detection of genes for epidemiological studies challenging, as prior knowledge of serovars does not always give clues to the gene responsible for the ESBL phenotype. Based on our own data in concert with other published data, some genes, like CTX-M-3, seem to be solely related to the human reservoir, while other genes are more versatile and can be found in many different reservoirs.
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Acknowledgements |
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