To characterize plasmids and resistance genes of multidrug-resistant (MDR) Salmonella Senftenberg and Salmonella Concord isolated from patients in the Netherlands.
The resistance genes of four MDR Salmonella isolates (three Salmonella Concord and one Salmonella Senftenberg) were identified by miniaturized microarray, PCR and sequencing. Plasmids were characterized by S1 nuclease-PFGE and PCR-based replicon typing (PBRT). Linkage between plasmids and genes was determined by conjugation experiments and microarray analysis. The genetic relationship between the three Salmonella Concord isolates was determined by XbaI-PFGE.
A large variety of resistance genes was detected, including qnrB2 and the β-lactamase genes blaTEM-1 and blaSHV-12 in all isolates; moreover all Salmonella Concord isolates also harboured blaCTX-M‐15. Salmonella Senftenberg harboured a large IncHI2 plasmid. The three Salmonella Concord isolates harboured two large plasmids typed as IncHI2 and IncA/C.
We detected the first plasmid-mediated MDR Salmonella isolates in the Netherlands harbouring both qnr and extended-spectrum β-lactamase (ESBL) genes. In Salmonella Senftenberg one large plasmid (IncHI2) and in Salmonella Concord two large plasmids (IncHI2 and IncA/C) were responsible for the multidrug resistance.
Worldwide, Salmonella is one of the major causes of foodborne infections in humans. In the majority of the cases these infections are self-limiting. However, for patients at risk and for invasive or prolonged infections antibiotic treatment is indicated. Fluoroquinolones and third-generation cephalosporins are drugs of choice for these cases.1 Infections caused by multidrug-resistant (MDR) Salmonella will affect the available treatment options. This may result in treatment failure and an increase in complications.
Although extended-spectrum β-lactamase (ESBL)-producing Salmonella Concord isolates from adopted Ethiopian children have been reported previously from different European countries including the Netherlands,2–4 there is still scarce information about the genetic background of Salmonella Concord isolates carrying both qnr and ESBL genes. In addition, no information on the characterization of MDR Salmonella Senftenberg isolates is available to date.
The aim of the study was to characterize genes and plasmids of the first qnr-positive, ESBL-producing MDR Salmonella isolated from patients in the Netherlands.
Materials and methods
Susceptibility tests and detection of resistance genes
In 2007, four Salmonella isolates expressing a remarkable type of multidrug resistance were identified. The isolates were selected for further study, since all four strains showed resistance to third-generation cephalosporins and exhibited an unusual quinolone resistance phenotype; being low-level resistant to ciprofloxacin, but still susceptible to nalidixic acid. In addition, all isolates were resistant to most classes of antibiotics tested. Three Salmonella Concord isolates (199.69, 206.54 and 210.52) originated from adopted Ethiopian children and a Salmonella Senftenberg isolate (200.27) was obtained from a male adult patient who had recently travelled to Egypt. Susceptibility to antimicrobials was tested by broth microdilution according to ISO standards (ISO 20776-1: 2006) in microtitre trays with a custom-made dehydrated panel of antibiotics (Sensititre©, Trek Diagnostic Systems, UK). The results were interpreted using epidemiological cut-off values as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast.org). The panel included the following antibiotics: ampicillin, cefotaxime, ceftazidime, tetracycline, sulfamethoxazole, trimethoprim, ciprofloxacin, nalidixic acid, chloramphenicol, florfenicol, gentamicin, kanamycin, streptomycin and colistin.
To detect antimicrobial resistance genes a miniaturized microarray (AMR04, Identibac, Veterinary Laboratories Agency, UK)5 was used followed by PCR for confirmation of the detection of plasmid-mediated quinolone resistance genes6–10 and of the β-lactamase genes blaTEM,11blaCTX-M12 and blaSHV (www.medvetnet.org/pdf/Reports/Appendix_2_Workpackage_9.doc). PCR products were purified by the QIAquick PCR Product Purification Kit (Qiagen GmbH, Germany). Sequences were determined by using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, USA) on a 3100-Avant Genetic Analyzer (Applied Biosystems). Sequence data were analysed with the Sequencher 4.6 program. The BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search for gene sequences homologous to the nucleotide sequences found.
Plasmid characterization and location of resistance genes
Transfer of resistance was tested by standard broth mating experiments using a rifampicin-resistant, indole-negative Escherichia coli K12 as recipient. Transconjugants were selected on MacConkey agar with 75 mg/L rifampicin and 1 mg/L cefotaxime. The plasmids in the donor strains and the transconjugants were analysed by PCR-based replicon typing (PBRT).13 IncHI2-positive plasmids were further typed using 10 different PCRs, based on the sequences of the IncHI2 plasmids R478 and pAPEC-01-R.14 The sizes of the plasmids were determined using PFGE of S1 nuclease digests of total DNA.15 Linkage between plasmids and resistance genes was determined by miniaturized microarray analysis of the transconjugants. The location of blaCTX-M-15 genes in the Salmonella Concord isolates was determined by PFGE of I-CeuI and XbaI digests followed by Southern blot hybridization using a PCR-generated digoxigenin-labelled CTX-M-15 probe. To determine the genetic relationship between the three Salmonella Concord isolates, XbaI-PFGE was performed according to the PulseNet protocol (www.pulsenet.com).
Susceptibility tests and detection of resistance genes
All Salmonella isolates were resistant to ampicillin, ceftazidime, cefotaxime, tetracycline, sulfamethoxazole, trimethoprim, chloramphenicol, gentamicin and streptomycin; the Salmonella Concord isolates were also resistant to florfenicol and the Salmonella Senftenberg isolate was resistant to kanamycin. Furthermore, all isolates were low-level resistant to ciprofloxacin (MIC: 0.12–0.5 mg/L), but still susceptible to nalidixic acid (MIC: 8–16 mg/L). The resistance genes qnrB2, blaTEM-1, blaSHV-12, sul1, dfrA19, tet(D), strA and strB were detected in all isolates. Some resistance genes were only detected in Salmonella Concord, including blaCTX-M-15, floR, sul2 and tet(A). The aminoglycoside resistance gene aac(6′)-1b was only detected in Salmonella Senftenberg. This classical variant of the gene was confirmed by sequencing the amplicon. In addition, the resistance genes qnrC, qnrD and qepA (not included in the microarray) were not detected by PCR in any of the four isolates.
Plasmid characterization and location of resistance genes
Salmonella Senftenberg 200.27 harboured one 310 kb IncHI2 plasmid, which was transferred to the E. coli K12 strain by conjugation. All nine resistance genes identified in the donor strain were detected in transconjugant 200.27-T1 (Table 1). The IncHI2 plasmid of the Salmonella Senftenberg isolate was characterized as an R478-like plasmid.
Replicon types of plasmids and resistance genes detected in donor strains (D) and transconjugants (-T) of the Salmonella Senftenberg isolate (200.27) and Salmonella Concord isolates (199.69, 206.54 and 210.52).
Both Salmonella Concord 199.69 and Salmonella Concord 206.54 harboured two plasmids identified by PBRT as IncHI2 (200 kb) and IncA/C (230 kb). Transconjugant 199.69-T1 harboured both plasmids including all 12 resistance genes identified in the donor strain. Transconjugants harbouring IncA/C lacked two resistance genes identified in the donor strain [blaSHV and tet(D)]. Transconjugants with only IncHI2 were not obtained. Salmonella Concord 210.52 also harboured IncHI2 and IncA/C plasmids, but of different sizes: 170 kb (IncA/C); and 290 kb (IncHI2). Conjugation experiments with Salmonella Concord 210.52 resulted in transconjugants with either IncHI2 or IncA/C plasmids. Transconjugant 210.52-T1 harbouring an IncHI2 plasmid lacked blaCTX-M-15, tet(A) and floR, whereas transconjugant 210.52-T7 harbouring an IncA/C plasmid lacked blaSHV-12, qnrB2, tet(D) and dfrA19 (Table 1). All IncHI2 plasmids in the three Salmonella Concord isolates were characterized as R478-like plasmids. However, all plasmids lacked three genes present in R478 (arsB, smr136 and tnsD) and harboured the 01R_160 locus as in pAPEC-O1-R.
Southern blot hybridization experiments demonstrated that the blaCTX-M-15 gene was only located on an IncA/C plasmid in all three Salmonella Concord isolates (results not shown). Finally, XbaI-PFGE revealed a unique digestion pattern for all three Salmonella Concord isolates indicative of the genetic variation of MDR Salmonella Concord strains originating from Ethiopia (results not shown).
Salmonella Senftenberg is a common serotype in the Netherlands; in the last decade, a total of 581 isolates (3%), originating from different sources, were tested for antibiotic susceptibility. Until 2007, all Salmonella Senftenberg isolates were susceptible to third-generation cephalosporins. On the contrary, Salmonella Concord is a very rare serotype; in the last decade, only nine isolates of human origin (<0.01%) were tested. Moreover, eight of these nine isolates were resistant to third-generation cephalosporins. However, until 2007, all Salmonella Concord isolates were susceptible to ciprofloxacin. These figures show the rarity of Salmonella isolates that are resistant to both third-generation cephalosporins and fluoroquinolones in the Netherlands. Although a simultaneous increase in qnr-positive Salmonella16 and ESBL-producing Salmonella has been reported in our national surveillance programme since 2003 (www.cvi.wur.nl/NL/publicaties/rapporten/maranrapportage/), this is the first report of both qnr- and ESBL-positive Salmonella isolated from human patients in the Netherlands.
The dissemination of qnr genes in Enterobacteriaceae including Salmonella of human origin is reported with increasing frequency. Recently, an IncHI2 plasmid associated with qnrB2 and blaSHV-12 was identified in a human Salmonella Bredeney isolate.17 In our study qnrB2 was detected on two different types of conjugative plasmids: IncHI2; and IncA/C. To our knowledge, this is the first description of a qnrB2 gene on an IncA/C plasmid in Salmonella enterica.
Fabre et al.2 detected blaCTX-M-15 genes in Salmonella Concord isolates on chromosomal DNA, but also on an InHI2 plasmid and on a fusion plasmid of IncY and IncA/C2. However, in our study we detected blaCTX-M-15 genes only on InA/C plasmids (negative for IncY) and not on chromosomal DNA. In addition, we identified blaSHV-12 genes only on IncHI2 plasmids. A study by Hendriksen et al.4 included four Dutch Salmonella Concord isolated from 2001 to 2006, which showed resistance to third-generation cephalosporins, but were completely susceptible to ciprofloxacin. In this Danish study the co-existence of blaCTX-M-15 and blaSHV-12 genes was reported on a single plasmid. Nonetheless, our study revealed the co-existence of these resistance genes on two different plasmids in all Salmonella Concord isolates; blaCTX-M-15 on IncA/C plasmids and blaSHV-12 on IncHI2 plasmids. The microarray revealed that the smaller IncA/C plasmid of transconjugant 210.52-T7 lacked a class 1 integron (intI1) and two resistance genes (dfrA19 and qnrB2) compared with the plasmids of transconjugants 199.69-T2 and 206.54-T1. This indicates that a fragment harbouring a complex integron is lacking on the IncA/C plasmid of Salmonella Concord 210.52. Finally the IncHI2 plasmids of all Salmonella Concord isolates were identically characterized as R478-like plasmids, all lacking the arsB gene. To our knowledge, this is the first description of such an R478-like plasmid.
The findings of this study provide additional information on the genetic background of ESBL-producing, qnr-positive Salmonella Concord and Salmonella Senftenberg isolates. The potential human health impact of infections with such MDR Salmonella emphasizes the need to monitor these resistance patterns in Salmonella carefully.
This work was supported by the Ministry of Agriculture, Nature and Food Quality (WOT-01-002-03.02).
None to declare.
Part of this work was presented at the ASM conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, Copenhagen, Denmark, 2008 (abstract number B102).
We would like to thank Dr Hilde Smith for carefully reading our manuscript and Dr Alessandra Carattoli for providing us with the R478-positive control strain.