SHV-34: an extended-spectrum ß-lactamase encoded by an epidemic plasmid

John Heritage1,*, Philip A. Chambers1, Caroline Tyndall2 and E. Stephen Buescher2

1 Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; 2 Center for Pediatric Research, Department of Pediatrics, Eastern Virginia Medical School, Children’s Hospital of The King’s Daughters, 855 West Brambleton Avenue, Norfolk, Virginia 23510–1001, USA

Received 21 May 2003; returned 3 July 2003, revised 6 October 2003; accepted 9 October 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To elucidate the causes for treatment failure in children given extended-spectrum cephalosporins.

Methods: During April 1998–March 2000, 18 isolates of members of the family Enterobacteriaceae, fulfilling microbiological criteria for carriage of extended-spectrum ß-lactamases (ESBLs) and carrying blaSHV, were isolated from paediatric inpatients. The collection was subjected to a retrospective molecular analysis.

Results: Three species were represented in the collection: Citrobacter koseri (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (16 isolates). A common plasmid was found in these bacteria, as judged by restriction endonuclease digestion. This was able to transfer an ESBL phenotype from donors to a laboratory strain of E. coli. Nucleotide sequence analysis revealed that this phenotype was associated with a new variant in blaSHV encoding SHV-34.

Conclusions: Analysis reveals the presence of an epidemic plasmid in this collection of bacteria. This carries a gene encoding the SHV-34 ESBL, described for the first time in this report. Nucleotide sequence analysis shows that there is a mutation from A->G affecting the codon at amino acid position 64 (GAA->GGA), changing the glutamic acid typically seen in this position to glycine.

Keywords: ESBLs, Enterobacteriaceae, SHV ß-lactamase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The extended-spectrum ß-lactamases (ESBLs) are a threat to the continued use of third-generation cephalosporins such as ceftazidime and cefotaxime. The SHV family of ß-lactamases is commonly encountered in the Enterobacteriaceae and these enzymes have a global distribution. There are over 50 members in this enzyme family, related to each other by the accumulation of one or more point mutations. The consequence of mutation in blaSHV is frequently to enable the enzyme that is encoded by the mutant to display a resistance phenotype that includes the extended-spectrum cephalosporins. The genes encoding SHV ß-lactamases have been reported not just in epidemic strains, but also located on epidemic plasmids.1 This paper reports the appearance of a variant belonging to the SHV family of ß-lactamases.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Clinical isolates from an American Children’s Hospital were collected during April 1998–March 2000, when Enterobacteriaceae producing ESBLs were recognized as causing infections.

Following their isolation by standard methods, bacteria were identified by either automated (MicroScan, Dade, Inc, West Sacramento, CA, USA) or manual analysis (API 20E, BioMerieux Vitek, Hazelwood, MO, USA). Their antibiotic susceptibilities were determined by an automated microdilution method (MicroScan). For storage, organisms were grown overnight on trypticase soy agar containing 5% sheep blood. The colonies were removed by scraping, and a heavy suspension was made in sterile trypticase soy broth that included 10% glycerol. Sterile filter papers were dipped into the suspension, placed in individual sterile microfuge tubes and transferred to –70°C, where they were kept until shipped. For shipping, tubes were allowed to come to room temperature. They were wrapped and packaged, and sent without refrigeration to the UK. Upon receipt, strains were plated onto heated blood agar (Oxoid, Basingstoke, Hampshire, UK) and incubated aerobically at 37°C. A sweep was then taken for preservation in glycerol at –70°C.

Plasmid analysis

Plasmid DNA was extracted from all the selected isolates by the rapid method described by Bennett et al.,2 except that isolates of Klebsiella pneumoniae were incubated in 10 mg/L snail acetone powder (Sigma-Aldrich, Poole, Dorset, UK) at ambient temperature for 15 min before DNA isolation. For restriction endonuclease analysis, plasmid DNA was dissolved in 10 µL of the appropriate buffer. Ten units of HindIII or PstI (Invitrogen, Paisley, Scotland) were then added and the reaction incubated at 37°C for 2 h. DNA fragments were separated electrophoretically in a 1% agarose gel.

PCR amplification

PCR amplification was performed as previously described.3

Determination of MICs

The MICs of cefotaxime and ceftazidime were determined by a macro broth dilution technique, according to BSAC guidelines.4 Each antibiotic was tested in two-fold dilutions in the range 0.25–128 mg/L. Escherichia coli C600 (R1010), a bacterium producing SHV-1, was used as a control strain containing blaSHV-1; UB1637 was included as a negative control.

Bacterial conjugation

Plate matings were performed by cross-streaking each donor and E. coli UB1637 onto heated blood agar and incubating them in air overnight at 37°C. Transconjugants were recovered by plating bacteria from the intersection onto selective media and by resuspending the bacteria in a small volume of LB broth. One hundred microlitres of the suspension was plated out on IsoSensitest agar containing ceftazidime 2 mg/L (AAH Pharmaceuticals, Leeds, UK) and streptomycin 100 mg/L (Sigma-Aldrich). Transconjugants were recovered by incubation overnight at 37°C. Controls in which each parental strain was incubated on the selective medium were checked for the absence of mutation to resistance.

Nucleotide sequence determination

Nucleotide sequence determination was carried out by Lark Technologies (Saffron Walden, Essex, UK) on PCR products generated from an E. coli UB1637 transconjugant. The nucleotide sequences of three amplimers were determined, each amplimer having been generated independently.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 18 isolates examined for this study were obtained from 13 children, some of whom had suffered treatment failure with an extended-spectrum cephalosporin. With the exception of one K. pneumoniae isolate that produced a colicin, as demonstrated by its inhibition of the growth of the E. coli recipient strain in plate-mating experiments, all isolates that contained target DNA amplifiable with PCR primers targeted at blaSHV were able to transfer an ESBL phenotype to E. coli strain UB1637. The MICs of ceftazidime and cefotaxime for four representative transconjugants were determined, and compared with E. coli harbouring SHV-1 and with UB1637 (Table 1). These results demonstrate that the plasmid encodes an ESBL phenotype.5


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Table 1. MICs of ceftazidime and cefotaxime for representative transconjugants producing SHV-34, compared with the MICs for E. coli C600 (R1010), a bacterium that produces SHV-1
 
The ESBL phenotype transferred to a recipient E. coli in plate-mating experiments. Transferable resistance in every case was associated with the movement of a single, large plasmid, larger than 100 kb. When digested with restriction endonucleases HindIII and PstI, alone and in combination, the digestion profiles were indistinguishable, demonstrating the presence of an epidemic plasmid in bacteria from this hospital, responsible for the dissemination of resistance to extended-spectrum cephalosporins (data not shown).

Nucleotide sequence determination of blaSHV in a representative bacterium was undertaken. This showed that the variant responsible for resistance has not been described previously. It was denominated SHV-34. Nucleotide sequence analysis shows that there is a unique mutation in blaSHV-34, GAA->GGA, affecting the codon at amino acid position 64 using the numbering system of Ambler et al.6 This mutation changes the glutamic acid currently seen in all other SHV ß-lactamases in this position to glycine.

Table 2 shows a summary of the amino acid changes and the associated codons found in SHV-34 and its neighbours compared with SHV-1. It should be noted that blaSHV-14, the gene most closely related to blaSHV-34, is separated by three mutations. In addition to the unique GAA->GGA mutation that changes the glutamic acid at position 64 in all but SHV-34 to glycine in that enzyme, there is a second mutation affecting amino acid 238 where AGC in SHV-34 encodes a serine, whereas a glycine residue is found in that position in SHV-14, encoded by GGC. There is, additionally, a silent mutation differentiating blaSHV-14 and blaSHV-34, where the CGC seen in blaSHV-14 becomes CGG in blaSHV-34 in the codon found at position 259.


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Table 2. Summary of the amino acid changes and the associated codons found in SHV-34 and its neighbours compared with SHV-1. Nucleotide changes are underlined; those causing changes in amino acids are shown in bold text. The amino acid number refers to the Ambler scheme6
 
Four mutations differentiate blaSHV-34 from blaSHV-18 and from blaSHV-7.

The SHV-7 ß-lactamase differs by two amino acids from SHV-34. The unique GAA->GGA mutation at position 64 is accompanied by a mutation affecting the amino acid at position 240, where GAA encoding glutamic acid in blaSHV-34 is changed to AAA encoding lysine in blaSHV-7. Additionally, two silent mutations separate blaSHV-34 from blaSHV-7. The first silent mutation is also shared with blaSHV-14: the CGC seen in blaSHV-7 and blaSHV-14 becomes CGG in blaSHV-34 at position 259. Additionally, there is a change between ACG in blaSHV-34 encoding threonine at position 268 and ACC in blaSHV-7.

In the case of blaSHV-18, all the mutations that differentiate it from blaSHV-34 cause amino acid changes. The first mutation is the unique GAA->GGA mutation that changes the glutamic acid typically found at position 64 to the glycine found in SHV-34. In blaSHV-18, the codon for amino acid 238 suffers a double mutation. The AGC codon in blaSHV-34 at amino acid 238 specifies serine at that position; in blaSHV-18, the codon for amino acid 238 is GCC, which codes for alanine. The fourth mutation differentiating blaSHV-34 and blaSHV-18 is independent of blaSHV-14. This mutation affects the amino acid at position 240 where AAA encoding lysine in blaSHV-18 is mutated to GAA encoding glutamic acid in blaSHV-34.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dissemination of SHV ESBLs may be largely limited to plasmid or clonal spread. In this study, we have identified a newly described ESBL, SHV-34, seen in 16 of the 18 K. pneumoniae isolates collected for this study. The remaining bacteria that produce SHV-34 identified in this study comprised one isolate of E. coli and one of Citrobacter koseri.

It has recently been asked why some bacterial strains achieve epidemic spread whereas others, that are equally resistant, do not persist. Whereas this question remains unanswered, it is suggested that its focus needs to be narrowed, at least with respect to resistance to ESBLs. Here, it is the persistence of the replicon that encodes the resistance determinant that has persisted and spread, not just the bacteria that express this resistance.


    Acknowledgements
 
We would like to thank Professor George Jacoby for helpful discussions on the analysis of the nucleotide sequence data presented in this paper. We also thank the staff of the Clinical Microbiology Laboratory at the American hospital for their assistance in the identification, preparation and storage of the isolates described in this study.


    Footnotes
 
* Corresponding author. Tel: +44-113-343-5592; Fax: +44-113-343-5638; E-mail: j.heritage{at}leeds.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Yan, J. J., Wu, S. M., Tsai, S. H. et al. (2000). Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum ß-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan. Antimicrobial Agents and Chemotherapy 44, 1438–42.[Abstract/Free Full Text]

2 . Bennett, P. M., Heritage, J. & Hawkey, P. M. (1986). An ultra-rapid method for the study of antibiotic resistance plasmids. Journal of Antimicrobial Chemotherapy 18, 421–4.[Abstract]

3 . Chanawong, A., M’Zali, F. H., Heritage, J. et al. (2001). Discrimination of SHV ß-lactamase genes by restriction site insertion-PCR. Antimicrobial Agents and Chemotherapy 45, 2110–14.[Abstract/Free Full Text]

4 . Holt, A. & Brown, D. (1989). Antimicrobial susceptibility testing. In Medical Bacteriology (Hawkey, P. M. & Lewis, D. A., Eds), pp. 176–96. IRL Press, Oxford, UK.

5 . Livermore, D. M. & Brown, D. F. J. (2001). Detection of ß-lactamase- mediated resistance. Journal of Antimicrobial Chemotherapy 48, Suppl. 1, 59–64.[Abstract/Free Full Text]

6 . Ambler, R. P., Coulson, A. F. W., Frere, J. M. et al. (1991). A standard numbering scheme for the Class A ß-lactamases. Biochemical Journal 276, 269–70.[ISI][Medline]