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Journal of Clinical Microbiology, July 2006, p. 2327-2332, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.00052-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

Beata Krawczyk,¹ Alfred Samet,² Justyna Leibner,¹ Anna ledziska,² and Józef Kur^1*

Gdask University of Technology, Department of Microbiology, Gdask, Poland,¹ Department of Clinical Microbiology, Clinical Hospital No. 1, Medical University of Gdask, Gdask, Poland²

Received 10 January 2006/ Returned for modification 31 March 2006/ Accepted 23 April 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdask, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsed-field gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over the past 20 years, a significant number of DNA-based techniques have been introduced into the field of bacterial characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are presently available (1-7, 11-13), most of which use PCR for detection of fragments. The choice of fingerprinting technique depends on the type of application. Ideally, a fingerprinting technique should require no prior investments in terms of sequence analysis, primer synthesis, or characterization of DNA probes. A number of fingerprinting methods which meet these requirements have been developed. The fingerprints are obtained by visualizing many parts of the genome. Differences in these fingerprints between individuals are interpreted as genetic distances. Obviously, the differences should reflect variations in DNA rather than artifacts due to a nonrobust method. Furthermore, the method should provide the appropriate level of discriminatory power, and it should be relatively rapid and cheap, especially in large-scale population genetic studies. Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the "gold standard" for molecular typing (10). In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3, 12, 13), and amplification of DNA fragments surrounding rare restriction sites (5, 6, 8). These techniques are now applied more and more because they involve less time, comparably low costs, and only standard equipment. In general, these methods work quite well compared to already established methods, such as REA-PFGE or ribotyping. However, there is still a need for a new method which may appear to be less complex, cheaper, and have a power of discrimination at least similar to that of REA-PFGE.

Here, we show application of a modified PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR), developed by Masny and Pucienniczak (9), for epidemiological studies. The proposed method allows gradual amplification of the genomic DNA differing in the thermal stability starting from the less stable DNA fragments amplified at lower denaturation temperature (T_d) values to more stable ones amplified at higher T_d values. We have implemented this method using a set of clinical Escherichia coli strains isolated from patients of the Clinical Hospital in Gdask (Poland). The utility of the PCR MP fingerprinting method was evaluated with data obtained using the pulsed-field gel electrophoresis (PFGE) method.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolates and patients. From October 2004 to May 2005, blood was collected from patients of Public Hospital No. 1 in Gdask (a 1,260-bed teaching hospital) with suspected bloodstream infection. This hospital provides medical and surgical care for children and adults. Blood was initially characterized as part of routine diagnostic testing using the BacT/Alert blood culturing system (bioMérieux, Hazelwood, Mo.). Bottles flagged by the instrument as positive were removed, and a portion of the blood broth mixture was used for gram staining. The remainder was subcultured onto solid plate medium according to the results of the gram staining on Columbia II agar base with 5% sheep blood, chocolate agar, and MacConkey agar (bioMérieux) for gram-negative microorganisms.

From patients with bloodstream infection, the isolates were recovered also from various kinds of specimens, including mainly stool and urine. Species identification and biochemical characterization were performed with the Vitek system (bioMérieux, Hazelwood, Mo.) with the GNI+ card for gram-negative microorganisms. The antimicrobial susceptibility test was performed according to the standardized disc diffusion Kirby-Bauer method according to CLSI (formerly NCCLS) recommendations. Each of the isolates was screened for susceptibility to ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefuroxime, ceftriaxone, cefotaxime, cefepime, ceftazidime, imipenem, meropenem, amikacin, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole.

We included in the present study 37 patients who had growth in blood culture of one or more isolates of E. coli. All patients gave informed consent to participate in the study, which was approved by the Local Ethics Committee. Seventy isolates were chosen for examination by molecular typing methods (one or several isolates from each patient). E. coli isolates were isolated from blood (45 samples), urine (9 samples), stool (14 samples), drainage (1 sample), and throat (1 sample). Initially, all isolates were tested for epidemiological relationships using REA-PFGE.

REA-PFGE. PFGE was performed according to the method described in the Instruction Manual and Application Guide for the FIGE Mapper Electrophoresis System (Bio-Rad). Restriction digestion of the chromosomal DNA was performed with 20 U of XbaI (Sigma) for 1/3 of a plug at 37°C. Restriction fragments were resolved in a 1% agarose gel (Bio-Rad) with the FIGE Mapper Electrophoresis System (Bio-Rad) in 0.5x TBE buffer (45 mM Tris, pH 8.0, 45 mM boric acid, 1 mM EDTA). Conditions for FIGE included a voltage linear gradient of 10 V/cm forward and 6.7 V/cm reversed, an initial forward pulse of 0.1 s, a final forward pulse of 8.5 s, and a total run time of 31 h. Electrophoresis was performed in a cold condition at 10°C. The optimal resolution range was from 0.1 to 150 kbp. Chromosomal bands were visualized by UV transillumination after ethidium bromide staining and were analyzed using a Versa Doc Imaging System version 1000 (Bio-Rad).

PCR MP procedure. DNA isolations (from 1.5 ml of culture) were carried out with the Genomic DNA Prep Plus (A&A Biotechnology, Poland) according to the manufacturer's procedure. The DNA concentration range was about 100 to 500 ng per microliter. Genomic DNA was digested with HindIII (10 U/µl; Fermentas, Lithuania). Digestion reactions were performed under uniform conditions; approximately 1 µg of DNA sample was added to 2.5 µl of buffer R (10x concentrated; 10 mM Tris-HCl, pH 8.5, 10 mM MgCl₂, 100 mM KCl, 0.1 mg/ml bovine serum albumin; Fermentas, Lithuania), and 0.5 µl (5 U) of the endonuclease (25-µl total volume). Following a 30-min incubation at 37°C, ligation mix comprising 2 µl of two oligonucleotides forming an adaptor (POW, 5'-CTCACTCTCACCAACGTCGAC-3'; HINDLIG, 5'-AGCTGTCGACGTTGG-3'; 20 pmol each, complementary sequences are underlined), 2.5 µl of ligation buffer (330 mM Tris-acetate, pH 7.8, 660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM dithiothreitol; Epicentre, Madison, WI), 0.5 µl of 25 mM ATP (Epicenter), and 0.5 µl of T4 DNA ligase (5 U; Epicentre, Madison, WI) was added, and the samples were incubated for 1 h at room temperature. Next, the mixture was heated in a thermoblock for 10 min at 70°C and cooled for 10 min at room temperature. The PCR was carried out in a 50-µl reaction mixture containing 1 µl ligation solution, 5 µl 10x PCR buffer (100 mM Tris-HCl, pH 8.5, 500 mM KCl, 1% Triton X-100), 2 µl 50 mM MgCl₂, 5 µl of a deoxynucleoside triphosphate mixture (concentration of each deoxynucleoside triphosphate, 2.5 mM), 1 µl (2 U) of Pwo polymerase (DNA Gdask II, Poland), and 50 pM of POW-AGCTT primer (5'-CTCACTCTCACCAACGTCGACAGCTT-3'). In a Biometra Tgradient thermal cycler, PCRs were performed as follows: 7 min at 72°C to release unligated oligonucleotides HINDLIG and to fill in the single-stranded ends and create amplicons, followed by initial denaturation at 87.2°C for 90 s and 22 cycles of denaturation at 87.2°C for 1 min, and annealing and elongation at 72°C for 2 min. After the last cycle, samples were incubated for 5 min at 72°C. The denaturation temperature was calculated during the optimization experiments for several E. coli isolates using a gradient thermal cycler (Biometra Tgradient Engine) with a gradient range of 85 to 89°C for the denaturation step in 200-µl thin-walled PCR tubes with PCR mixtures as described above. The reproducibility of the technique was examined by performing three PCR MP fingerprinting runs for each isolate with three separate DNA extractions and by using two different thermal cyclers (Biometra Tgradient cycler or Perkin-Elmer GENEAMP PCR System 2400).

Gel electrophoresis of PCR products. PCR products, 15 µl of 50 µl, were electrophoresed on 6% polyacrylamide gels (29:1 acrylamide-N,N-methylene bis-acrylamide) with TBE buffer and stained in ethidium bromide (0.5 mg/liter aqueous solution) for 10 to 15 min. Images of the gels were analyzed using a Versa Doc Imaging System version 1000 (Bio-Rad).

Top Abstract Introduction Materials and Methods Results Discussion References

 
PCR MP procedure. We applied the newly discovered PCR MP technique based on using low denaturation temperatures during LM PCR for fingerprinting of bacterial strains (9). The method allows specific gradual amplification of the genomic DNA differing in the thermal stability starting from the less stable DNA fragments amplified at lower T_d values to more stable ones amplified at higher T_d values. As suggested, this technique may be used for strain typing because it gives very specific fingerprints. Here, we show for the first time the usefulness of that technique for epidemiological examination. The outline of that method applied in our experiments is shown in Fig. 1. A genomic total DNA is digested completely with restriction enzyme (HindIII). The mixture of restriction fragments is ligated with a synthetic adaptor. All 5' ends of DNA fragments produced by digestion are modified by joining the same synthetic oligonucleotide. Melting temperatures of genomic DNA fragments obtained by digestion of restriction nuclease depend on their GC content and length. It means that restriction fragments, which arise as a result of the digestion, have different thermal stabilities. This feature may be used to obtain sets of electrophoretic patterns of DNA fragments amplified during LM PCR performed at various denaturation temperatures—PCR MPs. Lowering of T_d applied during LM PCR should decrease the number of amplified fragments because only single-stranded DNA molecules may serve as a template during LM PCR. A low T_d during LM PCR leads to limited and specific amplification of a small number of the less stable DNA fragments. The electrophoretic patterns of DNA fragments obtained after such amplifications are characteristic for the bacterial strain taken for DNA isolation.

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FIG. 1. Diagram illustrating the PCR MP fingerprinting technique used in the study.

The modified protocol of the PCR MP technique shown in this study differs from the original technique (9) in several points, giving a method which appears to be less complex, fast, and reproducible. A main difference was the combination of the restriction digestion and ligation reaction steps. In the original method, genomic DNA is digested with restriction enzyme in two steps: first 2 h of digestion and after that another 2 h of digestion with a new portion of enzyme. Next, DNA is extracted twice with chloroform/isoamyl alcohol and ethanol precipitated before ligation reaction. In a modified procedure, DNA is digested completely after 30 min and directly used for the adaptor ligation process. Next, modification refers to PCR which is performed without "hot start." The modified and optimized method considerably shortens the time of the experiment from about 24 h to only 8 h.

E. coli isolate typing by PCR MP and REA-PFGE. The obtained PCR MP fingerprinting patterns for representative E. coli isolates are presented in Fig. 2A. Each pattern consists of approximately 15 to 25 fragments in the size range of 100 to 1,200 bp. Comparative pairwise analysis of PCR MP patterns was performed with the Dice band-based similarity coefficient. The patterns with the Dice coefficient of 0.85 were assigned to the same type. PCR MP analysis distinguished 36 types among all isolates studied (Table 1). One genotype was markedly predominant (H2), as this was represented by 10 isolates from 4 patients. This genotype probably represents an E. coli strain from a hospital infection. Seventeen other PCR MP types grouped from 2 to 5 isolates each and altogether included 41 isolates. Finally, the remaining 19 types were unique among the isolates studied.

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FIG. 2. (A) PCR MP fingerprints for representative E. coli isolates from the whole hospital. (B) All 20 PCR MP types identified among the E. coli isolates from the Hematological Unit. (C) H2 genotype from 4 patients (10 isolates). Lanes M, DNA molecular size marker (1,008, 883, 615, 517, 466, and 396 bp). PCR MP fingerprinting types are given above each lane. The DNA amplicons were electrophoresed on 6% polyacrylamide gels by using TBE buffer at a field strength of 13 V cm–1.

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TABLE 1. E. coli genotyping by PCR MP

Twenty PCR MP types were identified among the E. coli isolates from the Hematological Unit (Fig. 2B), with the most prevalent type being H2, which included 10 isolates (Fig. 2C). Six other PCR MP types were identified from the Transplantology unit, three were identified from the Cardiology and Internal Medicine units, two were identified from the Pneumology, Intensive Care, and Urology and Gynecology units, and one was identified from the Surgery unit. Some of these were probably involved in hospital infection (5 genotypes: H2, H3, H19 = C3, T6 = C2 = IM2 and IC1) (Table 1) because isolates from different patients or different hospital units represented the same genotype.

Molecular typing by REA-PFGE also found 36 unique profiles (representative results are shown in Fig. 3). Each pattern consists of approximately 12 to 21 fragments. Clustering of REA-PFGE fingerprinting data exactly matched PCR MP data.

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FIG. 3. PFGE profiles for representative E. coli isolates from whole hospital (the same isolates as shown in Fig. 2A). Lane M, DNA molecular size marker (Pulse Marker, Sigma) (145.5, 97, 23.1, 9.4, 8.5, 6.6, 4.4, 2.3, 2.0, 0.6, and 0.1 kbp).

We also validated the reproducibility of the PCR MP fingerprinting method. The E. coli isolates were taken from pure culture for PCR MP fingerprinting on three separate occasions to assess reproducibility of the method. The resulting PCR MP fingerprints from each separate run produced identical profiles. There was small variation in intensity of the bands, but this did not result in the gain or loss of any information.

PCR MP in comparison to E. coli isolated from different sites within the same patient. PCR MP was next investigated for the similarities of the E. coli isolated from different sites of examined patients. As shown in Fig. 4 and Table 1, PCR MP fingerprinting analysis exhibited usefulness for monitoring spread of bacteria within patients. As expected, the epidemiologically related isolates show a high degree of similarity. Genotype similarity was documented in 10 of 37 patients with E. coli bacteremia for whom paired blood and fecal or urine isolates were available for genomic typing and was probably the major source of these bacteremias (Table 2) .

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FIG. 4. Representative results of monitoring spread of bacteria within patients by using the PCR MP technique (isolates from the first 6 patients shown in Table 2). Lanes marked B, S, and U contain strains isolated from blood, stool, and urine, respectively.

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TABLE 2. Comparison of E. coli isolated from different sites within the same patient

To ensure that the PCR MP technique has good reproducibility and satisfactory differentiation efficiency, isolates suspected of playing a role in the spread of bacteria within patients were tested with PCR MP at increasing denaturation temperatures. A steady increase in the number of amplified DNA fragments, which is dependent on T_d increase, was observed and still produced identical profiles for isolates belonging to the same genotype (Fig. 5). Thus, the order of appearance of DNA bands in PCR performed in subsequent increasing temperatures is invariable for a given genomic DNA (genotype).

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FIG. 5. Spread of bacteria within patients tested with PCR MP at increasing denaturation temperatures (85.1°C, 85.4°C, 86.2°C, and 88.6°C). A steady increase in the number of amplified DNA fragments, which is dependent on Td increase, was observed and still produced identical profiles for isolates belonging to the same genotype.

Susceptibility testing. The results of susceptibility testing revealed a high degree of the susceptibilities of the isolates tested. However, six isolates (genotype H2) from one patient were interpreted to be putative extended-spectrum ß-lactamase producers with resistance to ampicillin, cefuroxime, and ceftriaxone. They were also resistant to amikacin, ciprofloxacin, trimethoprim-sulfamethoxazole, and gentamicin. Twenty-four isolates were susceptible to all antibacterial agents tested, and 15 isolates were susceptible to all, with the exception of ampicillin. Cefotaxime, cefepime, ceftazidime, imipenem, and meropenem were 100% active agents in the study. Ampicillin was the least active compound in the study (34% susceptible isolates). The same patterns of susceptibility to antibacterial agents were observed among the E. coli isolates involved in each case of spread of bacteria within patients, confirming the genotyping correctness.

Top Abstract Introduction Materials and Methods Results Discussion References

 
REA-PFGE, especially when combined with serotyping, is currently considered the gold standard for molecular typing of isolates recovered from patients and the environment in the course of investigation and control of nosocomial outbreaks. However, REA-PFGE is time-consuming and labor intensive and can be performed only in reference laboratories with skillful technicians. Due to these drawbacks, REA-PFGE is not an ideal typing method for health departments undertaking routine analysis of large numbers of isolates. Here, we show for the first time the evaluation of a modified PCR MP technique, based on using low denaturation temperatures during LM PCR, described by Masny and Pucienniczak (9) for epidemiological studies. The high differentiation power of the PCR MP fingerprinting method is shown on clinical strains of E. coli. There are several advantages of the PCR MP fingerprinting method: (i) the method does not require prior knowledge of an analyzed sequence, (ii) results can be easily analyzed on polyacrylamide gels stained with ethidium bromide, (iii) the same adaptor and enzymes can be applied to analyze DNA from diverse species of bacteria. We suggested, based on this study, that there is at least a similar power of discrimination between the present gold standard REA-PFGE and the PCR MP method. The PCR MP fingerprinting method described here (with the same restriction enzyme, adaptor, and primer) was successfully used also for epidemiological studies of Klebsiella pneumoniae and Candida albicans strain differentiation (unpublished data).

Extraintestinal E. coli infections are increasing in frequency and becoming more difficult to treat. Thus, they represent a growing problem that requires more attention than ever before. Commensal E. coli strains can participate in extraintestinal infections when an aggravating factor is present, such as a foreign body (e.g., urinary catheter), host compromise (e.g., local anatomical or functional abnormalities such as urinary or biliary tract obstruction or immunocompromise), or a high or mixed bacterial species inoculum (e.g., with fecal contamination of the peritoneal cavity). Besides, isolation of E. coli from a blood culture is almost always clinically significant and is typically accompanied by sepsis syndrome, severe sepsis (sepsis-induced dysfunction of at least one organ or system), or septic shock. The data presented in this paper illustrate only a fragment of the natural history of E. coli in the hospital studied in Gdask. The organism had often been observed in the hospital before the beginning of the study, and it continues to occur to date. Molecular typing, performed by PCR MP and REA-PFGE analysis, revealed a very high degree of clonal diversity of the E. coli isolates, reflected best by the 36 genotype patterns produced by all 70 isolates collected. The distribution frequency of particular genotypes revealed that the vast majority of the types represented patients ' endogenic strains. However, 10 isolates studied (from 4 patients) were categorized as one type (H2). It may be assumed that this strain has been endemic to this setting. Isolates recovered from the same patient at different times and from different specimens were of either the same or different PCR MP and REA-PFGE types, and the susceptibilities of the isolates matched the genotype differences. These data suggest that all possible forms of blood infection or colonization could have occurred in this group of patients, including hospital infection. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a single hospital and medical ward.

 
This work was supported by a grant from The State Committee for Scientific Research (Poland).

 
* Corresponding author. Mailing address: Gdask University of Technology, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdask, Poland. Phone and fax: 48 58 3471822. E-mail: kur{at}altis.chem.pg.gda.pl.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, July 2006, p. 2327-2332, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.00052-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Krawczyk, B. Articles by Kur, J. Search for Related Content PubMed PubMed Citation Articles by Krawczyk, B. Articles by Kur, J.