SHV-27, a novel cefotaxime-hydrolysing ß-lactamase, identified in Klebsiella pneumoniae isolates from a Brazilian hospital

John E. Corkilla,*, Luis E. Cuevasb, Ricardo Q. Gurgelc, Julie Greensilla and C. Anthony Harta

a Royal Liverpool University Hospital, Department of Medical Microbiology, Liverpool L7 8XP; b Liverpool School of Tropical Medicine, University of Liverpool, Liverpool L3 5QA, UK; c Department of Paediatrics, Federal University of Sergipe, Aracaju, Brazil


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From a collection of cefotaxime-resistant Klebsiella pneumoniae isolated from neonatal blood culture specimens in a maternity hospital in Aracaju, Brazil, two isolates (strains KPBRZ-842 and -843, indistinguishable by pulsed-field gel electrophoresis) were found to produce ß-lactamases with isoelectric points (pI) of 5.4 and 8.2, respectively. Using a gel overlay method, cefotaxime hydrolysis was shown to be associated with the pI 8.2 protein. Nucleotide sequencing of the gene encoding the pI 8.2 ß-lactamase revealed a blaSHV-ESBL-type gene differing from the gene encoding SHV-1 by three silent point mutations, and a fourth that resulted in an amino acid substitution, aspartate for glycine, at position 156. This novel SHV-type extended-spectrum ß-lactamase is designated SHV-27.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following their recognition in Germany in 1983, extended-spectrum ß-lactamases (ESBLs) were reported predominately in Europe, but are now found worldwide in various members of the family Enterobacteriaceae. Stepwise mutations in SHV parental genes, causing between one and seven amino acid substitutions, have resulted in >20 different enzymes showing various activities towards third generation cephalosporins and reduced susceptibility to mechanism-based inhibitors.1 During a recent study of multi-antibiotic-resistant Klebsiella pneumoniae from a hospital in Brazil, two isolates were shown to express cefotaxime-hydrolysing ß-lactamases that appeared novel compared with published data. We have characterized this ESBL, SHV-27, which confers resistance to cefotaxime, ceftazidime and aztreonam.


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

K. pneumoniae KPBRZ-842 and -843 were isolated in 1999 from blood cultures and identified using the API-20E system (bioMérieux, Marcy l'Etoile, France). Escherichia coli J62-1 (nalidixic acid-R) was used as a conjugation recipient, and E. coli 39R861 (NCTC 50192) and V417 (NCTC 50193) as standards for plasmid analysis. E. coli DP38 and DP42, encoding SHV-1 and TEM-1 ß-lactamases, respectively (from Dr D. Payne, SmithKline Beecham Pharmaceuticals) were used as controls for iso-electric focusing (IEF). E. coli (NCTC 10418) was used to detect cefotaxime hydrolysis by gel overlay after analytical IEF.

Susceptibility testing

Disc susceptibility testing was performed according to BSAC guidelines on Iso-Sensitest agar (Oxoid Ltd, Basingstoke, UK).2 Detection of ESBLs was achieved with a combination of the Etest method (AB Biodisk, Solna, Sweden) and Mast DD test (Mast Laboratories Ltd, Bootle, Merseyside, UK) employing both cefotaxime and ceftazidime.

Plasmid analysis and conjugation experiments

Plasmid DNA was prepared by standard alkaline lysis and analysed by gel electrophoresis. Conjugal transfer of resistance determinants was performed in broth cultures, with K. pneumoniae KPBRZ-842 and -843 as donors, and E. coli J62-1 as recipient. After 3 and 24 h incubation, mating mixtures were plated on to agar containing nalidixic acid (30 mg/L) plus ampicillin (10 mg/L) and nalidixic acid (30 mg/L) plus cefotaxime (5 mg/L).

Analytical IEF

ß-Lactamase extraction and IEF were performed as described previously3 on polyacrylamide gels containing ampholines in the pH range 3.5–9.5 (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). Detection of ß-lactam hydrolysis by proteins separated postelectrophoresis was achieved by layering the ampholine gel with agar containing cefotaxime (0.4 mg/L). After incubation for 2 h at 37°C, the agar was flooded with E. coli (NCTC 10418) at McFarland 0.5 and re-incubated overnight.

Genomic analysis

Pulsed-field gel electrophoresis (PFGE) was performed with a CHEF DR 111 system (Bio-Rad, Hemel Hempstead, UK) as described previously.4 DNA insert blocks were digested overnight with XbaI.

PCR, restriction analysis and sequencing

Total DNA for PCR was extracted by suspending bacteria in 5% Chelex-100 resin slurry (Bio-Rad) in injection grade water followed by boiling for 10 min. Samples were centrifuged (10 min at 13 400g) and used immediately or stored at –20°C. PCR amplification of blaSHV genes (1017 bp amplicon), followed by restriction analysis with NheI to detect the glycine to serine substitution at position 238 (numbering in accordance with Ambler et al.5) was performed according to the method of Nüesch-Inderbinen et al.6 The PCR was optimized by titration to a magnesium concentration of 1.0 mM and annealing temperature of 60°C. Further characterization of the blaSHV gene was performed by a second PCR–restriction fragment length polymorphism method (PCR–RFLP)7 using NheI, DdeI, TaqI and BglI. In addition, the concentration of magnesium chloride was reduced from 1.87 to 1.0 mM. After digestion, products were analysed by gel electrophoresis using 3% low-melting agarose (Metaphore; FMC Bio-Products, Flowgen, Staffordshire, UK). Sequence determination of the SHV gene was performed on both strands of the 1017 bp amplicon with a dideoxynucleotide-chain termination method using an automated DNA sequencer ABI PRISM 377 (Perkin Elmer, Warrington, UK). Sequence analysis was performed using commercial software (Lasergene, DNAStar Inc., Madison, WI, USA). Nucleotide sequence structure was compared with a consensus SHV-1 (strain KPZU-8, DDBJ/EMBL/GenBank accession no. X98100).

Nucleotide sequence accession number

The nucleotide sequence data reported here will appear in the DDBJ/EMBL/GenBank database under accession no. AF293345.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates KPBRZ-842 and -843 were found to be indistiguishable by both PFGE and phenotypic susceptibility tests. The other multi-antibiotic-resistant klebsiellae (not reported here) from the same hospital were distinguishable from KPBRZ-842 and -843 by PFGE, and were found to carry both SHV-1 and SHV-2 ß-lactamases or SHV-2 alone.

By disc susceptibility testing, KPBRZ-842 and -843 were sensitive to ciprofloxacin, imipenem, cefoxitin and amikacin, but resistant to amoxycillin, gentamicin, cefotaxime, ceftazidime, aztreonam and trimethoprim. Susceptibility to piperacillin–tazobactam and co-amoxiclav was reduced. By Etest, the addition of clavulanate to cefotaxime and ceftazidime reduced MICs from 96.0 to 0.1 mg/L and 8.0 to 0.5 mg/L, respectively. The presence of ESBLs was also confirmed by the use of the Mast DD test (ratio of inhibition zones >1.5) for cefotaxime but not for ceftazidime (ratio of inhibition zones 1.4). Reversal of aztreonam resistance could be demonstrated by the presence of closely placed clavulanate-containing discs.

Flooding of ampholine IEF plates with nitrocefin showed strong ß-lactamase activity at pI 8.2, with a weaker band at pI 5.4. By a gel overlay technique, with E. coli as indicator, cefotaxime hydrolysis was only associated with the basic protein.

Both clinical isolates contained two small plasmids of 4.0 and 5.0 kb, but conjugative transfer of resistance determinants to E. coli J62-1 was unsuccessful.

Nucleotide sequencing was performed on both strands of the 1017 bp amplicon and the coding region (861 bp) used to predict the amino acid sequence. This sequence was compared with strain KPZU-8 (DDBJ/EMBL/GenBank accession no. X98100) for nucleotide sequence homology and predicted amino acid sequence. The following silent point changes were found at positions His-112 (CAC to CAT), Cys-123 (TGC to TGT) and Leu-138 (CTA to CTG). A single amino acid substitution was found at position 156, resulting in a glycine (GGC) to aspartate (GAC) change. The alteration at amino acid position 156 created a new TaqI recognition site, absent in SHV-1, but to the deletion of a BglI recognition site. As this substitution is not shared by other known SHV ß-lactamases, the pI 8.2 enzyme from KPBRZ-842 and -843 appears to be a novel ESBL and has been designated SHV-27.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two isolates of K. pneumoniae, indistinguishable by PFGE and plasmid profiles, and showing resistance by disc sensitivity testing to cefotaxime and ceftazidime and other classes of antimicrobials, were investigated. We identified blaSHV-ESBL genes by the PCR–NheI method6 and confirmed the result by both nucleotide sequencing and a second PCR–RFLP7 method.

Of note in this study is the characterization of a novel ESBL, designated here as SHV-27. The amino acid sequence predicted from the nucleotide sequence of the gene encoding SHV-27 ß-lactamase is different from those of other SHV-type enzymes.1 Unlike the earliest described ESBLs (SHV-2, -3, -4, -5, -7, -9, -10 and -12), SHV-27 does not possess a serine for glycine substitution at position 238, thought initially to be necessary for the hydrolysis of cefotaxime, but neither is a change found at the other important position, 179, which is responsible for the ESBL phenotype in SHV-6, -8 and -24. In SHV-27, an aspartate for glycine substitution at position 156 closely precedes the conserved Box V, which resides on the opposite side of the active site cavity (Ser-70), and during hydrolysis the ß-lactam molecules are sandwiched between them.8 The sequenced SHV gene also contained three previously known silent point mutations within the open reading frames of other sequences. In SHV genes, including SHV-27, the most frequent silent point mutation is in the codon triplet for Leu-138 (CTA to CTG). In SHV-27, the switch for His-112 (CAC to CAT) has only been reported for SHV-11 (a non-ESBL).9 The introduction of gene technology has now reduced the reliance that researchers had on determination of pI values for newly described SHV-ß-lactamases.1 Evolutionary pathway relationships between the SHV family of ß-lactamases with recorded enzymes of pI 8.2 (SHV-5, -9, -10 and -12), like SHV-27, suggest that they derive from mutations occurring in SHV-5, which itself is derived from SHV-2 by a lysine for glutamic acid at position 240.10 The single coding mutation at amino acid 156 in SHV-27, however, suggests that it has evolved directly from SHV-1.

Lack of transfer of resistance markers in mating experiments implies localization of the gene on a non-conjugative plasmid or transposon on the chromosome of K. pneumoniae KPBRZ-842 and -843. Therefore, in the absence of transconjugants, the absolute contribution of ß-lactamase production to in vitro resistance in the host strains cannot be quantified absolutely.


    Acknowledgments
 
We would like to thank Dr J. Heritage, University of Leeds, for his advice on PCR–RFLP, and Drs Flavia Oliveira da Costa and Jorge Machado for providing the isolates.


    Notes
 
* Corresponding author. Tel: +44-151-706-4410 ext. 4421; Fax: +44-151-706-5849; E-mail: jecmm{at}liverpool.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Jacoby, G. & Bush, K. (2000). Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant betalactamases. [On-line.] http://www.lahey.org/studies/webt.htm [30 August 2000, date last accessed].

2 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1998). Antimicrobial Sensitivity Testing Guidelines. British Society for Antimicrobial Chemotherapy, Birmingham, UK.

3 . Corkill, J. E., Hart, C. A., McLennan, A. G. & Aspinall, S. (1991). Characterization of a beta-lactamase produced by Pseudomonas paucimobilis. Journal of General Microbiology 137, 1425–9.[ISI][Medline]

4 . Ledson, M. J., Gallagher, M. J., Corkill, J. E., Hart, C. A. & Walshaw, M. J. (1998). Cross infection between cystic fibrosis patients colonized with Burkholderia cepacia. Thorax 53, 432–6.[Abstract/Free Full Text]

5 . Ambler, R. P., Coulson, A. F., Frère, J. M., Ghuysen, J. M., Joris, B., Forsman, M. et al. (1991). A standard numbering scheme for the class A beta-lactamases. Biochemical Journal 276, 269–70.[ISI][Medline]

6 . Nüesch-Inderbinen, M. T., Hachler, H. & Kayser, F. H. (1996). Detection of genes coding for extended-spectrum SHV betalactamases in clinical isolates by a molecular genetic method, and comparison with the E test. European Journal of Clinical Microbiology and Infectious Diseases 15, 398–402.[ISI][Medline]

7 . Chanawong, A., M'Zali, F. H., Heritage, J., Lulitanond, A. & Hawkey, P. M. (2000). Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCRsingle strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP). FEMS Microbiology Letters 184, 85–9.[ISI][Medline]

8 . Collatz, E., Labia, R. & Gutmann, L. (1990). Molecular evolution of ubiquitous beta-lactamase towards extended-spectrum enzymes active against newer beta-lactam antibiotics. Molecular Microbiology 4, 1615–20.[ISI][Medline]

9 . Nüesch-Inderbinen, M. T., Kayser, F. H. & Hachler, H. (1997). Survey and molecular genetics of SHV beta-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrobial Agents and Chemotherapy 41, 943–9.[Abstract]

10 . Heritage, J., M'Zali, F. H., Gascoyne-Binzi, D. & Hawkey, P. M. (1999). Evolution and spread of SHV extended spectrum betalactamases in Gram-negative bacteria. Journal of Antimicrobial Chemotherapy 44, 309–18.[Abstract/Free Full Text]

Received 6 September 2000; returned 16 November 2000; revised 22 November 2000; accepted 15 December 2000