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Antimicrobial Agents and Chemotherapy, April 2005, p. 1319-1322, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1319-1322.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

blaCTX-M Genes in Clinical Salmonella Isolates Recovered from Humans in England and Wales from 1992 to 2003

M. Batchelor,1 K. Hopkins,2 E. J. Threlfall,2 F. A. Clifton-Hadley,1 A. D. Stallwood,1 R. H. Davies,1 and E. Liebana1*

Department of Food and Environmental Safety, Veterinary Laboratories Agency, Addlestone, Surrey,1 Laboratory for Enteric Pathogens, Health Protection Agency, London, United Kingdom2

Received 11 August 2004/ Returned for modification 4 October 2004/ Accepted 22 November 2004


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ABSTRACT
 
Cefotaximases (CTX-M) are a rapidly growing class A ß-lactamase family that has been found among a wide range of clinical bacteria. One hundred and six isolates were selected from 278,308 Salmonella isolates based on resistance to ampicillin and cephalosporins and subjected to further characterization. Fourteen isolates were blaCTX-M PCR positive, and cefotaxime MICs for these isolates were ≥16 mg/liter. Furthermore, sequence analysis revealed the presence of type CTX-M9, -15, or -17 to -18. All 14 isolates presented different PFGE restriction profiles, although six Salmonella enterica serotype Virchow isolates formed a tight cluster. The blaCTX-M genetic determinants were present in transferable plasmids of ~63, 105, and >148 kb. Plasmid restriction analysis showed that both horizontal transfer of similar plasmids among different clones and transfer of genes between different plasmids were likely mechanisms involved in the spread of blaCTX-M genes. We have found that CTX-M enzymes have emerged in community-acquired infections both linked to foreign travel and domestically acquired. This is the first report of a CTX-M enzyme in Salmonella in the United Kingdom. Also, it represents the first report of a blaCTX-M gene in Salmonella enterica serotype Stanley and a blaCTX-M-15 gene in Salmonella enterica serotypes Anatum, Enteritidis, and Typhimurium.


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INTRODUCTION
 
Antimicrobials are essential for the treatment of invasive infections caused by Salmonella spp. However, it is well known that these organisms have been successful at acquiring resistance to a broad range of commonly used antimicrobials (5, 6). Extended-spectrum cephalosporins and fluoroquinolones are often used in the treatment of invasive cases of salmonellosis. However, the utilization of these drugs in treating complicated Salmonella infections is becoming seriously compromised by the increasing development of fluoroquinolone and extended-spectrum cephalosporin resistance. A recent example of this is treatment failure due to in vivo acquisition of a plasmid containing the blaCTX-M-3 gene by Salmonella enterica serotype Anatum (12).

Cefotaximases (CTX-M) are class A ß-lactamases that in general present higher levels of hydrolytic activity against cefotaxime than against ceftazidime. Ceftazidime MICs for organisms producing these enzymes are sometimes in the susceptible range. Many laboratories use ceftazidime resistance alone as an indicator of extended-spectrum ß-lactamase production. For this reason, CTX-M-producing isolates may be missed by routine susceptibility testing performed by clinical microbiology laboratories. CTX-M enzymes comprise a rapidly growing family distributed both over wide geographic areas and among a wide range of bacteria of clinical significance. There are five major CTX-M groups (groups 1, 2, 8, 9, and 25) (3).

In order to assess the current presence of organisms and genes of concern in England and Wales, the present work aimed to screen a large collection of Salmonella isolates of human origin with for the presence of blaCTX-M genes. Subsequently, the genes and gene-bearing plasmids of the positive strains were characterized in detail.


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MATERIALS AND METHODS
 
Isolates and determination of ß-lactam resistance phenotype. Isolates from the Salmonella strain collection at the Laboratory for Enteric Pathogens, Health Protection Agency, Colindale, United Kingdom, from the period 1992 to 2003 (total = 278,308; 1992, n = 31,339; 1993, n = 29,276; 1994, n = 29,514; 1995, n = 28,588; 1996, n = 28,210; 1997, n = 31,480; 1998, n = 23,161; 1999, n = 16,957; 2000, n = 14,465; 2001, n = 16,038; 2002, n = 14,427; 2003, n = 14,853) were scrutinized for phenotypes consistent with possible extended-spectrum ß-lactamase production. These represent all human clinical and food Salmonella isolates submitted to the reference laboratory for England and Wales. Resistance to antimicrobials was determined using a breakpoint method in isosensitest agar (13). The breakpoints (in milligrams per liter) for resistance to the ß-lactams were as follows: ampicillin (Amp) (high-level resistance), >128; cephalexin (Lex), >16; cephradine (Rad), >16; cefuroxime (Cxm), >16; ceftriaxone (Cro), >1; and cefotaxime (Ctx), >1. The breakpoints for other antibiotics were as described previously (13). The full MICs of cefotaxime and ceftazidime were also determined for the blaCTX-M-positive isolates following previously described methods (7).

Genetic characterization of blaCTX-M genes. Confirmation of the presence of a blaCTX-M ß-lactamase gene was done by PCR with primers CTX-M universal F (5'-CGA TGT GCA GTA CCA GTA A-3') and R (5'-TTA GTG ACC AGA ATC AGC GG-3'), designed from the alignment of blaCTX-M sequences representative of each group. A 35-cycle program with annealing at 60°C was used, resulting in a 585-bp amplicon. Identification of the blaCTX-M gene group was carried out initially by sequencing this short fragment. Specific primers were then designed for the CTX-M-1 (F, 5'-ATG GTT AAA AAA TCA CTG CG-3', and R, 5'-TTA CAA ACC GTC GGT GAC-3') and CTX-M-9 (F, 5'-ATG GTG ACA AAG AGA GTG CAA C-3', and R, 5'-TTA CAG CCC TTC GGC GAT G-3') groups in order to amplify the complete genes. A 30-cycle program with annealing at 50 or 60°C was used, resulting in 876-bp amplicons. These were sequenced on both strands by automated PCR cycle sequencing using the Applied Biosystems Prism 3100-Avant Genetic Analyzer. PCR for detection of In60 sequences was performed with primers IATGR plus 341STOP and O3000SR plus ISTOPR specific for the novel class 1 integron In60 as described previously (10).

Molecular typing. Preparation of DNA for pulsed-field gel electrophoresis (PFGE) was as described by the Centers for Disease Control and Prevention (4).

Assessment of transferability of resistance. Conjugations were performed with a rifampin-resistant recipient, Escherichia coli K-12 20R764, using in-broth methods (7). Conjugation mixtures were plated on CHROMagar ECC (M-Tech Diagnostics) containing rifampin (100 mg/liter) and cefotaxime (1 mg/liter) and then incubated for 24 and 48 h at 37°C. When transfer was not achieved by conjugation, transformation experiments were conducted as follows. Plasmid DNA was prepared with a QIAGEN high-speed MIDI kit. ElectroMAX DH10B cells (Invitrogen) were transformed with 60 ng of DNA using a Bio-Rad GenePulser II electroporator and standard conditions (2 kV; 200 {Omega}; 25 µF). Transformants were selected on nutrient agar containing 1 mg of cefotaxime/liter after 16 h of incubation at 37°C.

Plasmid profiling and restriction fragment length polymorphism of plasmid DNA. Plasmid profiling was carried out according to methods described previously (7). Transferable plasmids were purified from recipient cells. Purified plasmid DNA (2 µg) was digested with HpaI (Promega), and the resulting products were separated by electrophoresis on 0.8% agarose gels at 45 V for 20 h at 20°C.


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RESULTS
 
Isolate selection process. The criterion for selection of isolates was high-level resistance to ampicillin plus at least one of the cephalosporins in our panel (Lex, Rad, Cxm, Cro, or Ctx). One hundred six isolates were selected from the total of 278,308 isolates analyzed based on the criterion and were subsequently subjected to further characterization.

Resistance characterization. The 106 isolates selected were screened by the blaCTX-M universal PCR, and 14 of them were proven to be positive. Within this group, Salmonella enterica serotype Virchow isolates were the least resistant (cefotaxime MICs, 16 to 32 mg/liter); the cefotaxime MICs for the remaining isolates were ≥128 mg/liter. The patterns of resistance to the different antimicrobials are given in Table 1. Sequencing, followed by BLAST searches, confirmed the identities of the different genes, and this information is summarized in Table 1. The In60 PCRs suggested that serotype Virchow isolates C5, C7, C9, and C16 could carry a structure similar to the In60 integron described by Sabate et al. in 2002 (10), characterized by insertion of IS3000 and the presence of orf513 and orf1005 and a second copy of the 3'-end conserved region. However, full sequencing of these elements is required to fully demonstrate the identities of these integrons. Serotype Virchow isolates C4, C6, C8, and C11 were positive only by PCR with primers 341STOP and IATGR, with the expected product of 907 bp, and were negative with the primers ISTOPR and O3000SR. This suggests the presence of a transposase-like region (orf513) normally present in complex sul1-type integrons, but the structure is different from that of In60 and will be the subject of future work.


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  		TABLE 1. Characterization of blaCTX-M-containing Salmonella isolates from humans in England and Wales

All the isolates investigated presented different PFGE restriction profiles, although isolates C4 to C9 formed a cluster with 72% similarity. Figure 1 shows the XbaI PFGE fingerprints for the 14 isolates.



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  		FIG. 1. Image generated by Bionumerics software showing the XbaI PFGE restriction profiles for 14 Salmonella CTX-M-positive isolates. The sizes (in kilobases) of the fragments generated were calculated by comparison to a serovar Braenderup PulseNet universal control strain (H9812).

Transferability of resistance. Transconjugants were obtained for nine of the strains. E. coli transformants were obtained for two of the isolates for which transfer by conjugation was not successful (Table 1).

Isolates C1, C10, C12, C13, and C15 transferred a plasmid of ~105 kb. Isolates C3, C11, and C16 transferred a plasmid of ~63 kb. Isolates C4, C6, and C8 transferred a large low-copy plasmid of >148 kb. PCR analysis of the transconjugants and transformants showed that the blaCTXM genetic determinants were present in the specific plasmids acquired by the recipient strains.

Restriction analysis of the CTX-M-containing plasmids was carried out on plasmids extracted from transconjugants or transformants and showed that two strains (C1 and C13) had the same plasmid backbone (Fig. 2). These two isolates were of different serotypes (Anatum and Typhimurium) isolated in different years (2001 and 2003). The serotype Anatum isolate was associated with a visit to Pakistan, while the serotype Typhimurium isolate was acquired domestically. Also, strains C12 and C13, both of serotype Typhimurium but isolated in different years (2002 and 2003), showed very closely related restriction fragment length polymorphism (RFLP) plasmid patterns differing in only one band. Conversely, plasmids found in C3 (serotype Stanley), C10 (serotype Enteritidis), C11 (serotype Virchow), C15 (serotype Enteritidis), and C16 (serotype Virchow) presented distinct RFLP patterns (Fig. 2). Despite several attempts to extract plasmids for RFLP analysis from isolates C4, C6, and C8, we consistently failed to obtain sufficient plasmid for further experiments. The fact that these plasmids were of the same approximate size and carried blaCTX-M9 downstream of orf513 suggests that the same plasmid may be present in the three isolates.



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  		FIG. 2. Image generated by Bionumerics software showing the HpaI restriction profile for CTX-M-containing plasmids.


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DISCUSSION
 
The main enzymes found in our clinical Salmonella isolates belonged to the groups M-1 and M-9. This is in agreement with enzymes found in other countries in Europe (14). The first report of CTX-M enzyme in the United Kingdom was in a clinical isolate of Klebsiella oxytoca with blaCTX-M-9 (1). blaCTX-M-9 was the most prevalent gene, found in 8 of the 14 isolates investigated in our study, and it was found exclusively in serotype Virchow. Also belonging to CTX-M group 9, we found blaCTX-M17-18 in serotype Enteritidis and serotype Stanley. The second group of genes in our study was blaCTX-M-15, found in three serotypes: serotype Anatum, serotype Typhimurium, and serotype Enteritidis. A report from the United Kingdom of a CTX-M-15 enzyme in E. coli was published in 2001 (8), although it has never been found in Salmonella.

We have noticed that blaCTX-M determinants are often linked to infections in travelers returning to the United Kingdom. The serotype Stanley-infected individual reported traveling to Thailand, and the serotype Anatum-infected individual reported traveling to Pakistan immediately before contracting the infection. In addition, there seems to be an association between traveling to Spain and infection with serotype Virchow harboring blaCTX-M-9. Four of the eight serotype Virchow clinical cases included in our study involved individuals who reported traveling to Spain. The spread of a specific serotype Virchow clone with blaCTX-M-9 genes in different locations in Spain during 1997 and 1998 has been reported recently (11). All of the serotype Virchow isolates characterized in our study presented different PFGE profiles, suggesting that the spread of CTX-M-mediated resistance may be due to horizontal transfer rather than to the spread of a single clone. Although different, some of the serotype Virchow PFGE fingerprints (C4, C6, and C8) were highly related (similarity, 90%), which may also indicate clonal spread of this resistance. Our data show that the United Kingdom serotype Virchow strains were isolated from 1997 to 1999 and in 2001 and 2003. This demonstrates that development of CTX-M-mediated resistance within this serotype in England and Wales is not a phenomenon isolated in time.

The plasmid analysis showed that horizontal transfer of plasmids is a likely mechanism involved in the epidemiology of CTX-M-mediated resistance. The same backbone plasmids seem to be circulating among different clones and serotypes of Salmonella, and in these cases, we have shown that the plasmids are transferable by conjugation. A study from Poland has also shown that the same plasmids can be found in different species of Enterobacteriaceae (2). However, it has also been demonstrated that genes that are geographically and temporally clustered can exist on different plasmids (9, 15). Our plasmid RFLP data also suggest that transfer of the genes between different plasmids is also a mechanism that contributes to dissemination of CTX-M resistance. In our study, we found serotype Virchow clones with blaCTX-M-9 associated with a possible In60 class 1 integron in two plasmids of different sizes and RFLP patterns and blaCTXM-17-18 in two plasmids of different sizes and RFLP patterns.

In summary, we have found that CTX-M enzymes have emerged in community-acquired infections both linked to foreign travel and domestically acquired (in different regions: Oxfordshire, London, Swansea, Northamptonshire, Lancashire, Leicestershire, and Lincolnshire). This represents the first report of a CTX-M enzyme in Salmonella in the United Kingdom. Also, it represents the first published report of a blaCTX-M gene in serotype Stanley and of a blaCTX-M-15 gene in serotypes Anatum, Enteritidis, and Typhimurium.


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ACKNOWLEDGMENTS
 
This work was funded by the Department for Environment, Food and Rural Affairs, United Kingdom (project VM02136).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food and Environmental Safety, Veterinary Laboratories Agency, Addlestone, Surrey KT153NB, United Kingdom. Phone: 44-1932359582. Fax: 44-1932357595. E-mail: E.Liebana{at}VLA.DEFRA.gsi.gov.uk. Back


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Antimicrobial Agents and Chemotherapy, April 2005, p. 1319-1322, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1319-1322.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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