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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2005, p. 4716-4720, Vol. 49, No. 11
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.11.4716-4720.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary,1 Division of Infectious Diseases, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213,2 Department of Pathology, Division of Molecular Diagnostics, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213,3 Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 441064
Received 28 March 2005/ Returned for modification 12 June 2005/ Accepted 25 August 2005
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ABSTRACT |
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INTRODUCTION |
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There are a number of ways in which different ß-lactamase types can be differentiated within a single isolate (8). Analytical isoelectric focusing can provide a clue to the presence and isoelectric points (pIs) of multiple ß-lactamases produced by a single isolate. However, many different ß-lactamases share common isoelectric points, so this method lacks the ability to identify the ß-lactamase type. We have previously performed surveys in which blaSHV, blaTEM, or blaCTX-M genes were amplified, and the primary PCR product was identified by sequencing (16). Such a procedure is easily performed but may not identify multiple ß-lactamases of the same type: genes existing in a lower copy number may be less likely to be detected than those which predominate. The definitive method for detecting multiple ß-lactamases of the same type is extensive cloning and sequencing of the bla genes.
Herein, we report an Enterobacter cloacae isolate which simultaneously produced two different SHV-type ESBLs. We describe a real-time PCR method for detecting single-nucleotide polymorphisms (SNPs) that uses fluorescently labeled oligonucleotide hybridization probes (20). We demonstrate a rapid, sensitive, and specific method that can detect mutations of the blaSHV gene in a single reaction. As a result of this analysis, we identified a novel SHV variant, SHV-30, in a complex ß-lactamase background.
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MATERIALS AND METHODS |
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Plasmid isolation and transformation.
Plasmid DNA from the E. cloacae ES24 isolate was extracted and purified using the protocol and reagents of a commercial kit (Quantum Prep; Bio-Rad, Inc., Hercules, CA) The plasmids were analyzed by electrophoresis on a 0.8% (wt/vol) agarose gel and visualized by ethidium bromide staining under UV light. 
HindIII (Promega, Madison, WI) was used as a molecular weight marker. The plasmid DNA of the E. cloacae ES24 isolate was used to transform electrocompetent E. coli EP-MAX10B (Bio-Rad, Inc.). Transformants were selected on Luria-Bertani agar (Beckton Dickinson, Sparks, MD) containing 0.5 µg/ml cefotaxime (Sigma, St. Louis, MO).
Isoelectric focusing. Analytical isoelectric focusing (IEF) was performed to determine the pIs of ß-lactamases extracted from the E. cloacae ES24 isolate and the plasmid-transformed E. coli EP-MAX10B isolate (17). Electrophoresis was performed on precast polyacrylamide gels, pH 3 to pH 10 (Bio-Rad Inc.). Isoelectric points were determined by placing filter paper soaked in nitrocefin (500 µg/ml; Becton Dickinson) on top of the focused gel (17).
PCR and cloning. A single colony of each test isolate was resuspended in 400 µl water and boiled for 15 min. The resulting supernatant was used as the bacterial DNA template for PCR amplification. The primers are shown in Table 2. Reaction mixtures (30 µl) contained 50 mM KCl, 1.5 mM MgCl2, 0.5 µM of each primer, 1.5 mM of each deoxynucleotide triphosphate (Sigma), 1 U of RedTaq polymerase (Sigma), and 2 µl of the bacterial DNA template. Initially, direct sequencing of the products was performed.
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Real-time PCR SNP genotyping. To detect the single-nucleotide polymorphisms in blaSHV, we used the 5' nuclease assay developed by Sevall (20). We designed fluorogenic probes consisting of oligonucleotides that possess a reporter dye (6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein [HEX] or 6-carboxyfluorescein [FAM]) and a quencher dye (6-carboxytetramethylrhodamine [TAMRA]) at each end. The probe adopts a hairpin structure while free in solution. The close proximity of the reporter and the quencher in this hairpin suppresses reporter fluorescence (fluorescence resonance transfer [FRET]). During PCR, the dual-labeled probe annealed to the target of interest (blaSHV) between the forward and reverse primer sites. During extension, the probe was cleaved by the 5' nuclease activity of the Taq polymerase. This separated the reporter dye from the quencher dye, generating an increase in the reporter dye's fluorescence intensity (FRET does not occur). Thus, by using two probes containing only a single nucleotide difference combined with two different reporter dyes, single-nucleotide polymorphisms were detected.
Real-time PCR allelic discrimination assays (SNP genotyping) were designed using Primer Express software (Applied Biosystems, Foster City, CA). Primer and probe combinations designed using Primer Express are listed in Table 2. Genotyping was performed in 50-µl reaction mixtures that contained 5 µl of genomic DNA, 900 nM of each primer, 200 nM of each probe, and 25 µl of Taqman Universal PCR Master Mix (contains PCR buffer, passive reference dye ROX [carboxy-X-rhodamine], deoxynucleotides, uridine, uracil-N-glycosylase, and AmpliTaq Gold DNA polymerase; Perkin-Elmer Applied Biosystems). Amplification was performed using an ABI 7000 real-time thermal cycler. Cycling conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Real-time fluorescence detection was performed during the 60°C annealing/extension step of each cycle. ABI allelic discrimination software was used to plot and automatically call genotypes based on a two-parameter plot using fluorescence intensities of FAM and HEX at 40 cycles. This software uses autoscaling for the allelic discrimination plot.
Nucleotide sequence accession number. The DNA sequence and deduced amino acid sequence of SHV-30 has been deposited in GenBank and assigned accession number AY661885.
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RESULTS |
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0.8 kb. In an attempt to isolate plasmids harboring genes encoding ß-lactamases, transformation experiments were performed. It was only possible to transform the largest plasmid (9.4 kb) into E. coli EP-MAX10B. The antibiotic susceptibilities of both the E. cloacae ES24 and the plasmid-transformed E. coli EP-MAX10B isolates are shown in Table 3. ES24 was resistant to ceftazidime, aztreonam, and cefoxitin but was intermediate to cefotaxime and cefepime. The cefepime MIC decreased in the presence of clavulanic acid. The MICs of the plasmid-transformed E. coli EP-MAX10B isolate were much lower than those of E. cloacae ES24 (Table 3), with MICs for all tested antibiotics being in the susceptible range.
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9 were identified in the E. cloacae ES24 isolate. The plasmid-transformed E. coli EP-MAX10B isolate produced only one ß-lactamase (with a pI value of 6.7) (Fig. 1).
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The plasmid-transformed E. coli EP-MAX10B isolate was found to be negative for blaTEM by PCR amplification, but it was found to be positive for blaSHV. The sequence analysis showed that the plasmid-transformed E. coli EP-MAX10B isolate carried only the novel blaSHV-30 gene. This finding was further confirmed by IEF (Fig. 1).
Real-time PCR SNP genotyping. Real-time PCR SNP was performed on the E. cloacae ES24 isolate (SHV-7 and SHV-30) and plasmid-transformed E. coli EP-MAX10B (SHV-30) as well as the following control isolates: K. pneumoniae ATCC 700603 (SHV-18), KPC-1-producing K. pneumoniae isolate (SHV-29), an E. coli EP-MAX10B isolate, AmpC ß-lactamase-producing clinical E. cloacae isolate, and ESBL (SHV-5, SHV-7, and SHV-14)-producing and AmpC ß-lactamase-producing clinical E. cloacae isolates (Table 1).
In our study, probes were designed to identify mutations affecting amino acid position 240 alleles GAA (Glu) and AAA (Lys) (1). Table 4 lists the results of the isolates tested and a comparison of their nucleotide sequences at the positions of probes. If the probes hybridized, the increase in fluorescence signal of one dye over the other indicated homozygosity for that PCR allele (FAM fluorescence for the G allele, and HEX fluorescence for the A allele). An increase in both signals indicated heterozygosity. The amplification fluorescence intensities are compared in a graphical form (Fig. 3). Clusters of points on the graph correspond to the homozygous G genotype, the homozygous A genotype, the heterozygous GA genotype, or to a genotype with no amplification.
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The plasmid-transformed E. coli EP-MAX10B (SHV-30- producing) isolate and the SHV-14-producing clinical E. cloacae isolates showed increased FAM fluorescence and unchanged HEX fluorescence, indicating homozygosity for the FAM-specific G allele. Only the G at the 703 nucleotide position was present (Fig. 3). However, the SHV-14-producing E. cloacae isolate contained one nucleotide difference at position 700 relative to the probe (Table 4).
The two SHV-5-producing and the five SHV-7-producing clinical E. cloacae isolates showed increases in only HEX dye fluorescence, indicating the specific A allele at nucleotide position 703 (Fig. 3).
The SHV-18-producing K. pneumoniae ATCC 700603 isolate and the SHV-29-producing KPC-1 isolate showed no change in the fluorescence intensity for either FAM or HEX (Fig. 3). Based on their sequencing results, the SHV-18-producing K. pneumoniae ATCC 700603 isolate had an A nucleotide at position 703, and the SHV-29-producing KPC-1 isolate had a G nucleotide at position 703; despite this, there was no increase in fluorescence signal for the HEX-specific A allele and the FAM-specific G allele. The reason is that both isolates have two differences at nucleotide positions 700 and 701 relative to the probes (Table 4), resulting in a lack of binding of the specific probe.
The nontransformed E. coli EP-MAX10B isolate and the chromosomal AmpC ß-lactamase-producing E. cloacae isolate also showed no change in fluorescence intensity for either FAM or HEX (Fig. 3) because they did not contain the blaSHV gene.
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DISCUSSION |
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Our E. cloacae ES24 isolate produced AmpC, TEM-1, and SHV-7 ß-lactamases and a novel ß-lactamase, SHV-30, with a pI of 6.7. This is the first isolation of SHV-30 from an Enterobacter isolate and the first report of an E. cloacae isolate producing two different SHV enzymes. The amino acid sequence of SHV-30 differed from the amino acid sequence of SHV-1 by three amino acid substitutions: isoleucine for phenylalanine at position 8, arginine for serine at position 43, and glycine for serine at position 238. An investigation of the effect of these mutations on the active site of the enzyme and on antibiotic hydrolysis is under further investigation.
Methods that can be used to rapidly and reliably characterize ß-lactamases and the genes that encode them are still being sought. To identify two different blaSHV genes within a single bacterial isolate is not simple. To assist with this, a combination of isoelectric focusing and restriction fragment length polymorphism (RFLP) analysis of SHV-specific PCR products has been developed (8). As noted previously, determination of the pI of the enzyme is insufficient to identify SHV-derived ß-lactamases due to the frequent similarity of pI values. The PCR-RFLP technique is a simple and rapid alternative, but it cannot identify all known mutations, such as those affecting amino acids at positions 8, 238, or 240. PCR single-strand conformational polymorphism analysis was developed to characterize ß-lactamases in the SHV family (13). PCR single-strand conformational polymorphism analysis has also been used to differentiate two different blaSHV gene types within a single bacterial isolate (14, 24). Restriction site insertion-PCR has been used to detect mutations of blaSHV genes that cannot be identified by PCR-RFLP. This technique uses primers with one to three base mismatches near the 3' end which modulate target restriction sites (5). All of these techniques, however, rely on the high specificity of restriction endonucleases to identify the restriction sites.
Real-time PCR and melting-curve analysis have been previously used for rapid detection of the blaSHV ESBLs (18). We have used a rapid method based on FRET real-time PCR to identify two different blaSHV genes in a single E. cloacae isolate. Real-time allelic discrimination is a fluorescence detection system that collects fluorescence measurements during the amplification. The DNA assay with amplification and detection in a single step allows the analysis of the amplification product without extensive postamplification processing. A mismatch between probe and target greatly reduces the efficiency of probe hybridization and its subsequent hydrolysis. In our case, if there were two additional nucleotide mismatches, the probes were unable to hybridize.
With this method, probes also can be designed to identify every target, even those that do not generate unique restriction endonuclease sites. With this method, more than two alleles could potentially be distinguished. The further advantage of this method is that it could be used for genes encoding other types of ß-lactamases, such as blaCTX-M or blaTEM.
In summary, the real-time PCR SNP assay proved useful in a situation where amplification from genome extracts, with sequencing of the primary PCR product, suggested that two ß-lactamases were present. We found the technique to be highly reproducible, and it could be performed without postamplification steps. The present assay was designed to conform to standard thermocycling conditions, thus allowing several SNPs to be identified simultaneously. This approach may be extremely useful when applied to large population survey studies that attempt to define the molecular epidemiology of ß-lactamase genes.
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ACKNOWLEDGMENTS |
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We are grateful to Fred Tenover for providing the K. pneumoniae isolate which produced KPC-1.
R.A.B. was supported by the Department of Veterans Affairs Merit Review Program and NIH grant R01AI063517-01.
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FOOTNOTES |
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REFERENCES |
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, SHV-30-producing, plasmid-transformed E. coli EP-MAX10B isolate and SHV-14-producing E. cloacae isolate; 
, E. cloacae ES24; •, two SHV-5-producing and five SHV-7 producing E. cloacae isolates; -, SHV-18-producing K. pneumoniae ATCC 700603, SHV-29-producing KPC-1 isolate, E. coli EP-MAX10B, and chromosomal AmpC ß-lactamase-producing E. cloacae isolate; 
, water.