Abstract

Objectives: The purpose of this work was to study the genetic determinants responsible for extended-spectrum β-lactamase (ESBL) resistance of Salmonella isolated from Dutch poultry, poultry meat and hospitalized humans.

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.

Introduction

Salmonella enterica is one of the most common causes of human gastroenteritis worldwide and improper handling and digestion of inadequately cooked food primarily cause the infections. A large number of different food animal sources have been identified as reservoirs.1 β-Lactams are widely used to treat infections with Salmonella in both animals and humans. As a consequence, widespread resistance to different β-lactams has emerged.2,3

Resistance to β-lactams in S. enterica is primarily caused by the production of acquired β-lactamases. More than 340 β-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 β-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 β-lactamases among isolates from food animals and food products. Briñas et al. examined the occurrence of blaTEM-, blaSHV- and blaOXA-type β-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 β-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 β-lactamase, whereas only a few isolates expressed blaTEM-30, blaOXA or blaPSE β-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 β-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.

Materials and methods

Bacterial isolates

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 Mueller–Hinton 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 Mueller–Hinton 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 β-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).

Table 1.

Primers used in this study

Primer
 
Gene
 
Sequence
 
TAnneal (°C)
 
TiElongate (s)
 
Size (bp)
 
Reference
 
757 blaTEM 5′-GCGGAACCCCTATTTG-3′ 50 60 964 
821  5′-TCTAAAGTATATATGAGTAAACTTGGTCTGAC-3′     
1113 PampC 5′-GTGAATACAGAGCCAGACGC-3′ 50 60 343 this study 
796  5′-GTTGTTTCCGGGTGATGC-3′     
1436 blaSHV 5′-TTCGCCTGTGTATTATCTCCCTG-3′ 50 60 854 this study 
1437  5′-TTAGCGTTGCCAGTGYTCG-3′     
1354 blaCTX 5′-ATGTGCAGYACCAGTAARGTKATGGC-3′ 60 60 593 35 
1355  5′-TGGGTRAARTARGTSACCAGAAYCAGCGG-3′     
1004 blaCMY-1 group 5′-GTGGTGGATGCCAGCATCC-3′ 58 60 915 this study 
1005  5′-GGTCGAGCCGGTCTTGTTGAA-3′     
1006 blaCMY-2 group 5′-GCACTTAGCCACCTATACGGCAG-3′ 58 60 758 this study 
1007  5′-GCTTTTCAAGAATGCGCCAGG-3′     
1062 blaOXA-1 5′-ATGAAAAACACAATACATATCAACTTCGC-3′ 62 60 820 
1063  5′-GTGTGTTTAGAATGGTGATCGCATT-3′     
1420 blaOXA-2 5′-ACGATAGTTGTGGCAGACGAAC-3′ 62 60 602 this study 
1421  5′-ATYCTGTTTGGCGTATCRATATTC-3′     
1359 blaACC-1 5′-AGCCTCAGCAGCCGGTTAC-3′ 53 60 818 this study 
1360  5′-GAAGCCGTTAGTTGATCCGG-3′     
Primer
 
Gene
 
Sequence
 
TAnneal (°C)
 
TiElongate (s)
 
Size (bp)
 
Reference
 
757 blaTEM 5′-GCGGAACCCCTATTTG-3′ 50 60 964 
821  5′-TCTAAAGTATATATGAGTAAACTTGGTCTGAC-3′     
1113 PampC 5′-GTGAATACAGAGCCAGACGC-3′ 50 60 343 this study 
796  5′-GTTGTTTCCGGGTGATGC-3′     
1436 blaSHV 5′-TTCGCCTGTGTATTATCTCCCTG-3′ 50 60 854 this study 
1437  5′-TTAGCGTTGCCAGTGYTCG-3′     
1354 blaCTX 5′-ATGTGCAGYACCAGTAARGTKATGGC-3′ 60 60 593 35 
1355  5′-TGGGTRAARTARGTSACCAGAAYCAGCGG-3′     
1004 blaCMY-1 group 5′-GTGGTGGATGCCAGCATCC-3′ 58 60 915 this study 
1005  5′-GGTCGAGCCGGTCTTGTTGAA-3′     
1006 blaCMY-2 group 5′-GCACTTAGCCACCTATACGGCAG-3′ 58 60 758 this study 
1007  5′-GCTTTTCAAGAATGCGCCAGG-3′     
1062 blaOXA-1 5′-ATGAAAAACACAATACATATCAACTTCGC-3′ 62 60 820 
1063  5′-GTGTGTTTAGAATGGTGATCGCATT-3′     
1420 blaOXA-2 5′-ACGATAGTTGTGGCAGACGAAC-3′ 62 60 602 this study 
1421  5′-ATYCTGTTTGGCGTATCRATATTC-3′     
1359 blaACC-1 5′-AGCCTCAGCAGCCGGTTAC-3′ 53 60 818 this study 
1360  5′-GAAGCCGTTAGTTGATCCGG-3′     

TAnneal is the annealing temperature and TiElongate is the elongation time used in each case (see the Materials and methods section for details).

Sequencing of PCR-amplified β-lactamase genes

Before sequencing, all PCR products were purified using the ‘GFX™ 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 (λ = 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 β-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 β-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

Results

MIC determination

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).

Table 2.

Salmonella isolates used in this study

Name
 
Date
 
Serovar
 
Origin
 
AMX
 
CEF
 
CFF
 
CXM
 
CAZ
 
AMC
 
FOX
 
CTX
 
Additional resistances
 
bla gene(s)
 
Group
 
24.73 Mar-2001 S. Isangi patient >32 >64 >64 >32 16 >64 GEN, STR, SMX, TET CTX-M-28 ESBL-I 
37.49 Aug-2001 S. Isangi patient >32 >64 >64 >32 16 >64 GEN, STR, SMX, TET, TMP CTX-M-28 ESBL-I 
55.10 Apr-2002 S. Isangi patient >32 >64 >64 >32 16 >64 STR, SMX, TET, TMP CTX-M-28 ESBL-I 
59.45 Aug-2002 S. Typhimurium PT 507 patient >32 >64 >64 32 32 16 >64 GEN, NAL, SPE, STR, SMX, TET, TMP CTX-M-3 ESBL-I 
59.70 Sep-2002 S. Typhimurium PT 507 patient >32 >64 >64 32 16 >64 SPE, STR, SMX, TET, TMP CTX-M-3 ESBL-I 
24.26 Feb-2001 S. Bareilly poultry >32 >32 32 >32 >32  ACC-1 ESBL-II 
31.65 May-2001 S. Bareilly patient >32 >64 32 >32 >32 32  ACC-1 ESBL-II 
31.72 May-2001 S. Bareilly patient >32 >64 32 >32 >32  ACC-1 ESBL-II 
32.69 Jun-2001 S. Bareilly patient >32 >64 32 >32 >32  ACC-1 ESBL-II 
35.04 Jun-2001 S. Braenderup broiler 16 >64 >64 32 >32  ACC-1 ESBL-II 
48.75 Jan-2002 S. Infantis layer >32 >64 32 >32 >32  ACC-1 ESBL-II 
50.32 Feb-2002 S. Bareilly poultry >32 >64 32 >32 >32 32  ACC-1 ESBL-II 
51.43 Feb-2002 S. Bareilly poultry >32 >64 32 >32 >32 16  ACC-1 ESBL-II 
60.50 Sep-2002 S. Bareilly broiler >32 >64 32 >32 >32 16 GEN, NEO, SPE, STR, SMX ACC-1 ESBL-II 
34.60 Jun-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
36.04 Jul-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
36.52 Aug-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
37.56 Aug-2001 S. Blockley meat product >32 >64 >64 32 16  TEM-52 ESBL-III 
40.26 Sep-2001 S. Thompson patient >32 >64 >64 32 16  TEM-52 ESBL-III 
40.33 Sep-2001 S. Blockley patient >32 >64 >64 32 16  TEM-52 ESBL-III 
42.16 Aug-2001 S. London patient >32 >64 >64 32 16  TEM-52 ESBL-III 
42.32 Aug-2001 S. Enteritidis PT 14b patient >32 >64 >64 32 16 SPE, SMX, TMP TEM-52 ESBL-III 
44.02 Aug-2001 S. Paratyphi B poultry >32 >64 >64 32 16 SPE, SMX, TMP TEM-52 ESBL-III 
46.20 Nov-2001 S. Blockley poultry meat >32 >64 >64 32 32 SPE, SMX, TMP TEM-52 ESBL-III 
51.09 Feb-2002 S. Blockley poultry >32 >64 >64 >32 16  TEM-52 ESBL-III 
54.12 Apr-2002 S. Virchow poultry >32 >64 >64 32 32 SPE, STR, SMX, TMP TEM-52 ESBL-III 
44.78 Nov-2001 S. Typhimurium PT 11 poultry >32 >64 >64 32 16 NEO, SPE, SMX, TET, TMP TEM-52 ESBL-IV 
48.78 Jan-2002 S. Typhimurium PT 507 poultry >32 >64 >64 32 16 SPE, STR, SMX, TMP TEM-52 ESBL-IV 
63.48 Oct-2002 S. Paratyphi B var. Java poultry meat >32 >64 ≤16 SPE, TMP TEM-20 ESBL-V 
63.71 Oct-2002 S. Paratyphi B var. Java broiler >32 >64 ≤16 NAL, SPE, TMP TEM-20 ESBL-V 
58.67 Aug-2002 S. Virchow broiler >32 >64 >64 32 16 >64 NAL, SPE, SMX, TET, TMP CTX-M-2 + SHV-2 + TEM-1 ESBL-VI 
38.47 Aug-2001 S. Concord patient >32 >64 >64 >32 >64 SMX, TMP SHV-12 ESBL-VII 
46.72 Jan-2002 S. Typhimurium ARSa poultry meat >32 >64 >64 >32 >64  TEM-52 + SHV-12 ESBL-VIII 
61.12 Sep-2002 S. Isangi patient >32 32 >64 >32 16 FFN, GEN, SPE, STR, SMX, TET, TMP TEM-63 ESBL-IX 
Name
 
Date
 
Serovar
 
Origin
 
AMX
 
CEF
 
CFF
 
CXM
 
CAZ
 
AMC
 
FOX
 
CTX
 
Additional resistances
 
bla gene(s)
 
Group
 
24.73 Mar-2001 S. Isangi patient >32 >64 >64 >32 16 >64 GEN, STR, SMX, TET CTX-M-28 ESBL-I 
37.49 Aug-2001 S. Isangi patient >32 >64 >64 >32 16 >64 GEN, STR, SMX, TET, TMP CTX-M-28 ESBL-I 
55.10 Apr-2002 S. Isangi patient >32 >64 >64 >32 16 >64 STR, SMX, TET, TMP CTX-M-28 ESBL-I 
59.45 Aug-2002 S. Typhimurium PT 507 patient >32 >64 >64 32 32 16 >64 GEN, NAL, SPE, STR, SMX, TET, TMP CTX-M-3 ESBL-I 
59.70 Sep-2002 S. Typhimurium PT 507 patient >32 >64 >64 32 16 >64 SPE, STR, SMX, TET, TMP CTX-M-3 ESBL-I 
24.26 Feb-2001 S. Bareilly poultry >32 >32 32 >32 >32  ACC-1 ESBL-II 
31.65 May-2001 S. Bareilly patient >32 >64 32 >32 >32 32  ACC-1 ESBL-II 
31.72 May-2001 S. Bareilly patient >32 >64 32 >32 >32  ACC-1 ESBL-II 
32.69 Jun-2001 S. Bareilly patient >32 >64 32 >32 >32  ACC-1 ESBL-II 
35.04 Jun-2001 S. Braenderup broiler 16 >64 >64 32 >32  ACC-1 ESBL-II 
48.75 Jan-2002 S. Infantis layer >32 >64 32 >32 >32  ACC-1 ESBL-II 
50.32 Feb-2002 S. Bareilly poultry >32 >64 32 >32 >32 32  ACC-1 ESBL-II 
51.43 Feb-2002 S. Bareilly poultry >32 >64 32 >32 >32 16  ACC-1 ESBL-II 
60.50 Sep-2002 S. Bareilly broiler >32 >64 32 >32 >32 16 GEN, NEO, SPE, STR, SMX ACC-1 ESBL-II 
34.60 Jun-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
36.04 Jul-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
36.52 Aug-2001 S. Blockley poultry meat >32 >64 >64 32 16  TEM-52 ESBL-III 
37.56 Aug-2001 S. Blockley meat product >32 >64 >64 32 16  TEM-52 ESBL-III 
40.26 Sep-2001 S. Thompson patient >32 >64 >64 32 16  TEM-52 ESBL-III 
40.33 Sep-2001 S. Blockley patient >32 >64 >64 32 16  TEM-52 ESBL-III 
42.16 Aug-2001 S. London patient >32 >64 >64 32 16  TEM-52 ESBL-III 
42.32 Aug-2001 S. Enteritidis PT 14b patient >32 >64 >64 32 16 SPE, SMX, TMP TEM-52 ESBL-III 
44.02 Aug-2001 S. Paratyphi B poultry >32 >64 >64 32 16 SPE, SMX, TMP TEM-52 ESBL-III 
46.20 Nov-2001 S. Blockley poultry meat >32 >64 >64 32 32 SPE, SMX, TMP TEM-52 ESBL-III 
51.09 Feb-2002 S. Blockley poultry >32 >64 >64 >32 16  TEM-52 ESBL-III 
54.12 Apr-2002 S. Virchow poultry >32 >64 >64 32 32 SPE, STR, SMX, TMP TEM-52 ESBL-III 
44.78 Nov-2001 S. Typhimurium PT 11 poultry >32 >64 >64 32 16 NEO, SPE, SMX, TET, TMP TEM-52 ESBL-IV 
48.78 Jan-2002 S. Typhimurium PT 507 poultry >32 >64 >64 32 16 SPE, STR, SMX, TMP TEM-52 ESBL-IV 
63.48 Oct-2002 S. Paratyphi B var. Java poultry meat >32 >64 ≤16 SPE, TMP TEM-20 ESBL-V 
63.71 Oct-2002 S. Paratyphi B var. Java broiler >32 >64 ≤16 NAL, SPE, TMP TEM-20 ESBL-V 
58.67 Aug-2002 S. Virchow broiler >32 >64 >64 32 16 >64 NAL, SPE, SMX, TET, TMP CTX-M-2 + SHV-2 + TEM-1 ESBL-VI 
38.47 Aug-2001 S. Concord patient >32 >64 >64 >32 >64 SMX, TMP SHV-12 ESBL-VII 
46.72 Jan-2002 S. Typhimurium ARSa poultry meat >32 >64 >64 >32 >64  TEM-52 + SHV-12 ESBL-VIII 
61.12 Sep-2002 S. Isangi patient >32 32 >64 >32 16 FFN, GEN, SPE, STR, SMX, TET, TMP TEM-63 ESBL-IX 

For β-lactamase resistance determination, MICs are given in mg/L. Additional resistance phenotypes are given according to the NCCLS standards. AMX, amoxicillin; CEF, cefalothin; CFF, ceftiofur; CXM, cefuroxime; CAZ, ceftazidime; AMC, amoxicillin + clavulanate; FOX, cefoxitin; CTX, cefotaxime; GEN, gentamicin; STR, streptomycin; SMX, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim; NAL, nalidixic acid; SPE, spectinomycin; NEO, neomycin; FFN, florfenicol.

a

Atypical reaction strain.

Detection of genes responsible for ESBL resistance

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).

Figure 1.

Organization of the genetic environment surrounding the blaACC-1 gene of S. Bareilly 60.50. Regions identical with previously published sequences are indicated including the corresponding GenBank accession number.

Figure 1.

Organization of the genetic environment surrounding the blaACC-1 gene of S. Bareilly 60.50. Regions identical with previously published sequences are indicated including the corresponding GenBank accession number.

Detection of the blaACC-1 in inhibitor-resistant isolates

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.

Discussion

The isolates in this study showed a high degree of genetic diversity between the bla genes. This may not be surprising, as the isolates originate from healthy animals, meat products as well as hospital patients. In general, the isolates can be divided into three large sets (ESBL-I, ESBL-II and ESBL-III) and six minor subsets (ESBL-IV, ESBL-V, ESBL-VI, ESBL-VII, ESBL-VIII and ESBL-IX) based on the bla gene found in them (Table 2).

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 β-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.

We wish to thank our technician Pia Thurø Hansen for her excellent help. The work presented in this study was supported in part by a grant from the European Commission, EU 6th Framework programme, networks of Excellence, Med-Vet-Net.

References

1.
Humphrey TJ. Public-health aspects of Salmonella infections. In: Wray C, Wray A, eds. Salmonella in Domestic Animals. Wallingford, UK: CABI Publishing, CAB International,
2000
.
2.
Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat.
Clin Microbiol Rev
 
2001
;
14
:
933
–51.
3.
Gniadkowski M. Evolution and epidemiology of extended-spectrum β-lactamases (ESBLs) and ESBL-producing microorganisms.
Clin Microbiol Infect
 
2001
;
7
:
597
–608.
4.
Bush K. New β-lactamases in gram-negative bacteria: diversity and impact on the selection of antimicrobial therapy.
Clin Infect Dis
 
2001
;
32
:
1085
–9.
5.
Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for β-lactamases and its correlation with molecular structure.
Antimicrob Agents Chemother
 
1995
;
39
:
1211
–33.
6.
Brinas L, Zarazaga M, Saenz Y et al. β-Lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals.
Antimicrob Agents Chemother
 
2002
;
46
:
3156
–63.
7.
Olesen I, Hasman H, Aarestrup FM. Prevalence of β-lactamases among ampicillin resistant Escherichia coli and Salmonella isolated from food animals in Denmark.
Microb Drug Resist
 
2004
;
10
:
334
–40.
8.
Mulvey MR, Soule G, Boyd D et al. Characterization of the first extended-spectrum β-lactamase-producing Salmonella isolate identified in Canada.
J Clin Microbiol
 
2003
;
41
:
460
–2.
9.
Tzouvelekis LS, Tzelepi E, Tassios PT et al. CTX-M-type β-lactamases: an emerging group of extended-spectrum enzymes.
Int J Antimicrob Agents
 
2000
;
14
:
137
–42.
10.
Winokur PL, Brueggemann A, DeSalvo DL et al. Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-mediated CMY-2 AmpC β-lactamase.
Antimicrob Agents Chemother
 
2000
;
44
:
2777
–83.
11.
National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Second Edition: Approved Standard M7-A2. NCCLS, Villanova, PA, USA,
2003
.
12.
Olsen JE, Brown DJ, Thomsen LE et al. Differences in the carriage and the ability to utilize the serotype associated virulence plasmid in strains of Salmonella enterica serotype Typhimurium investigated by use of a self-transferable virulence plasmid, pOG669.
Microb Pathog
 
2004
;
36
:
337
–47.
13.
Hansen LH, Sørensen SJ, Jensen LB. Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system.
Gene
 
1997
;
186
:
167
–73.
14.
Ribot EM, Wierzba RK, Angulo FJ et al. Salmonella enterica serotype Typhimurium DT104 isolated from humans, United States, 1985, 1990, and 1995.
Emerg Infect Dis
 
2002
;
8
:
387
–91.
15.
Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes.
Antimicrob Agents Chemother
 
2004
;
48
:
1
–14.
16.
Acikgoz ZC, Gulay Z, Bicmen M et al. CTX-M-3 extended-spectrum β-lactamase in a Shigella sonnei clinical isolate: first report from Turkey.
Scand J Infect Dis
 
2003
;
35
:
503
–5.
17.
de Champs C, Sirot D, Chanal C et al. A 1998 survey of extended-spectrum β-lactamases in Enterobacteriaceae in France. The French Study Group.
Antimicrob Agents Chemother
 
2000
;
44
:
3177
–9.
18.
Gniadkowski M, Schneider I, Palucha A et al. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing β-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme.
Antimicrob Agents Chemother
 
1998
;
42
:
827
–32.
19.
Li CR, Li Y, Zhang PA. Dissemination and spread of CTX-M extended-spectrum β-lactamases among clinical isolates of Klebsiella pneumoniae in central China.
Int J Antimicrob Agents
 
2003
;
22
:
521
–5.
20.
Yu WL, Wu LT, Pfaller MA et al. Confirmation of extended-spectrum β-lactamase-producing Serratia marcescens: preliminary report from Taiwan.
Diagn Microbiol Infect Dis
 
2003
;
45
:
221
–4.
21.
Baraniak A, Sadowy E, Hryniewicz W et al. Two different extended-spectrum β-lactamases (ESBLs) in one of the first ESBL-producing Salmonella isolates in Poland.
J Clin Microbiol
 
2002
;
40
:
1095
–7.
22.
Gierczynski R, Szych J, Cieslik A et al. The occurrence of the first two CTX-M-3 and TEM-1 producing isolates of Salmonella enterica serovar Oranienburg in Poland.
Int J Antimicrob Agents
 
2003
;
21
:
497
–9.
23.
Gierczynski R, Szych J, Rastawicki W et al. The molecular characterisation of the extended spectrum β-lactamase (ESBL) producing strain of Salmonella enterica serovar Mbandaka isolated in Poland.
Acta Microbiol Pol
 
2003
;
52
:
183
–90.
24.
Su LH, Chiu CH, Chu C et al. In vivo acquisition of ceftriaxone resistance in Salmonella enterica serotype Anatum.
Antimicrob Agents Chemother
 
2003
;
47
:
563
–7.
25.
Villa L, Mammina C, Miriagou V et al. Multidrug and broad-spectrum cephalosporin resistance among Salmonella enterica serotype Enteritidis clinical isolates in southern Italy.
J Clin Microbiol
 
2002
;
40
:
2662
–5.
26.
Weill FX, Demartin M, Tande D et al. SHV-12-like extended-spectrum-β-lactamase-producing strains of Salmonella enterica serotypes Babelsberg and Enteritidis isolated in France among infants adopted from Mali.
J Clin Microbiol
 
2004
;
42
:
2432
–7.
27.
Rhimi-Mahjoubi F, Bernier M, Arlet G et al. Identification of plasmid-encoded cephalosporinase ACC-1 among various enterobacteria (Klebsiella pneumoniae, Proteus mirabilis, Salmonella) isolated from a Tunisian hospital.
Pathol Biol (Paris)
 
2002
;
50
:
7
–11.
28.
Bauernfeind A, Schneider I, Jungwirth R et al. A novel type of AmpC β-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia.
Antimicrob Agents Chemother
 
1999
;
43
:
1924
–31.
29.
Weill FX, Demartin M, Fabre L et al. Extended-spectrum-β-lactamase (TEM-52)-producing strains of Salmonella enterica of various serotypes isolated in France.
J Clin Microbiol
 
2004
;
42
:
3359
–62.
30.
Ben Hassen A, Fournier G, Kechrid A et al. Enzymatic resistance to cefotaxime in 56 strains of Klebsiella spp., Escherichia coli and Salmonella spp. at a Tunisian hospital (1984–1988).
Pathol Biol (Paris)
 
1990
;
38
:
464
–9.
31.
Jeong SH, Bae IK, Lee JH et al. Molecular characterization of extended-spectrum β-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey.
J Clin Microbiol
 
2004
;
42
:
2902
–6.
32.
Luzzaro F, Perilli M, Amicosante G et al. Properties of multidrug-resistant, ESBL-producing Proteus mirabilis isolates and possible role of β-lactam/β-lactamase inhibitor combinations.
Int J Antimicrob Agents
 
2001
;
17
:
131
–5.
33.
de Champs C, Chanal C, Sirot D et al. Frequency and diversity of Class A extended-spectrum β-lactamases in hospitals of the Auvergne, France: a 2 year prospective study.
J Antimicrob Chemother
 
2004
;
54
:
634
–9.
34.
Kruger T, Szabo D, Keddy KH et al. Infections with nontyphoidal Salmonella species producing TEM-63 or a novel TEM enzyme, TEM-131, in South Africa.
Antimicrob Agents Chemother
 
2004
;
48
:
4263
–70.
35.
Miro E, Navarro F, Mirelis B et al. Prevalence of clinical isolates of Escherichia coli producing inhibitor-resistant β-lactamases at a University Hospital in Barcelona, Spain, over a 3-year period.
Antimicrob Agents Chemother
 
2002
;
46
:
3991
–4.

Author notes

1Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark; 2Central Institute for Animal Disease Control, Department of Bacteriology and TSEs, Houtribweg 39, PO Box 2004, 8203 AA Lelystad, The Netherlands