Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.     

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.     

Molecular epidemiological characteristics of pneumococcal meningitis in children

The molecular epidemiological characteristics of pneumococcal meningitis in children were studied. Pneumococcal isolates were characterized by serotyping and two genotyping methods, BOX fingerprinting and restriction fragment end labeling, to evaluate whether clonal strains were present within the serotypes or serogroups. During a 17-year period, 68 children admitted to the Sophia Children's Hospital were diagnosed with meningitis due toStreptococcus pneumoniae. Pneumococcal isolates from 44 patients were still available for analysis. All strains were susceptible to penicillin. Serotypes/ serogroups 14,19, 6, and 18 represented 56% of all isolates. The results of the molecular typing methods demonstrate the absence of clonal relatedness between isolates from patients with pneumococcal meningitis.     

Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.     

Comparison of four genotyping assays for epidemiological study of methicillin-resistantStaphylococcus aureus

Twenty-six methicillin-resistantStaphylococcus aureus strains were genetically differentiated by interrepeat PCR and the results compared with those of ribotyping, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA analysis obtained in a previous study for the same strains. The comparison showed that the PCR-mediated assays were as discriminatory as PFGE, whereas ribotyping was the least powerful genotyping method. Due to the ease of performance, PCR fingerprinting may become the method of choice for establishing clonal relationships amongStaphylococcus aureus isolates.     

Comparison of Molecular Methods for Typing Vibrio parahaemolyticus

An outbreak of Vibrio parahaemolyticus gastroenteritis on Canada's west coast in 1997 emphasized the need to develop molecular methods for differentiation and typing of these organisms. Isolates were analyzed by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR, detection of restriction fragment length polymorphisms (RFLP) in rRNA genes (ribotyping), pulsed-field gel electrophoresis (PFGE), and RFLP analysis of the genetic locus encoding the polar flagellum (Fla locus RFLP analysis). ERIC PCR and ribotyping were the most informative typing methods, especially when used together, while Fla locus RFLP analysis was the least discriminatory. PFGE exhibited good discrimination but suffered from a high incidence of DNA degradation. ERIC PCR and ribotyping will be useful for the evaluation of genetic and epidemiological relationships among V. parahaemolyticus strains.     

Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.     

Evaluation of the discriminatory power of pulsed-field gel electrophoresis and PCR fingerprinting for epidemiologic typing of Candida species

Objective: To evaluate the discriminatory power of genotyping methods (PCR fingerprinting and pulsed-field gel electrophoresis) validated for Candida albicans in other Candida species.
Methods: Molecular typing methods are increasingly being applied for studies where the interpretation of data essentially relies on the typing results rather than epidemiologic data. In this situation, the discriminatory power (ability to identify differences among epidemiologically unrelated strains) of the typing method is important in allowing one to draw valid conclusions. By applying PCR fingerprinting, electrophoretic karyotyping, and restriction fragment endo-nuclease analysis using standard restriction enzymes and primers proven to be useful in previous studies, we evaluated whether the use of multiple genotyping methods is sufficient to delineate known unrelated strains among seven Candida species.
Results: All three methods identified individual genotypes for each of the seven Candida species studied. However, optimal strain delineation required the combined use of all three typing methods and was observed only within the small number of C. albicans and C. tropicalis isolates tested in this study.
Conclusion: Typing assays that are able to delineate a certain Candida species may not be used blindly for other species of that genus. Regarding the limited number of strains tested, further validation of the discriminative power of genotyping methods (including in C. tropicalis ) should be done.     

Epidemiological fingerprinting of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments.

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.     

Performance criteria of DNA fingerprinting methods for typing of Helicobacter pylori isolates: experimental results and meta-analysis

Typing systems are used to discriminate between isolates of Helicobacter pylori for epidemiological and clinical purposes. Discriminatory power and typeability are important performance criteria of typing systems. Discriminatory power refers to the ability to differentiate among unrelated isolates; it is quantitatively expressed by the discriminatory index (DI). Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed; it is quantitatively expressed by the percentage of typeable isolates. We evaluated the discriminatory power and the typeability of the most currently used DNA fingerprinting methods for the typing of H. pylori isolates: ribotyping, PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, and random amplified polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance criteria. A meta-analysis of typeability and discriminatory power was conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis demonstrated the optimal typeability (100%) and the excellent discriminatory powers of PCR-based typing methods: RAPD analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis, DIs, 0.70 to 0.97). Chromosome restriction-based typing methods (ribotyping and PFGE) are limited by a low typeability (12.5 to 75%) that strongly decreases their discriminatory powers: ribotyping, DI, 0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of ribotyping and PFGE for the typing of H. pylori isolates. We recommend the use of PCR-based methods.     

Comparison of PCR-based methods for typing Staphylococcus aureus isolates

In this study, we compared the potentials of (i) a multiplex PCR-based multilocus variable-number tandem repeat (VNTR) assay; (ii) a triplex PCR coamplifying fragments of spa, coa, and the hypervariable region adjacent to the mecA gene; (iii) restriction profile analysis of the STAR repetitive element; (iv) randomly amplified polymorphic DNA analysis; (v) inter-IS256 PCR; and (vi) rep-MP3 PCR. Multilocus VNTR typing and triplex PCR (coa, spa, and hypervariable region) approaches showed excellent reproducibility and high discriminatory power; however, only multilocus VNTR typing could distinguish all pulsed-field gel electrophoresis and spa types. Multilocus VNTR typing appears to be the most useful PCR-based method for the rapid genotyping of Staphylococcus aureus strains.     

DNA polymorphism in strains of Listeria monocytogenes.

DNA polymorphism in 35 Listeria monocytogenes strains belonging to serovars 1/2a, 1/2b, 1/2c, and 4b was studied by genomic DNA digestion. The restriction endonucleases ApaI and NotI, which cleave DNA at rare sequences, were used, and DNA fragments were analyzed by pulsed-field gel electrophoresis. Restriction fragment length polymorphism varied among different serovars and was used for epidemiological studies, but serovar 1/2c isolates could not be analyzed because their restriction patterns were indistinguishable. The genome sizes were calculated by addition of the sizes of the ApaI fragments and were found to be about 2,660 kb for serovar 1/2a strains, 2,640 kb for serovar 1/2b strains, and 2,710 kb for serovar 4b strains but only 2,340 kb for serovar 1/2c strains. This last group therefore appears to differ from the other serovar strains by the absence of restriction fragment length polymorphism and a chromosome that is 15% shorter, suggesting that strains of serovar 1/2c have quite recently emerged.     

Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods.

Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.     

Differentiation of pathogenic Bartonella species by infrequent restriction site PCR

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.     

Genotyping of 44 isolates of Fusarium solani, the main agent of fungal keratitis in Brazil

In a retrospective study performed over 6 years in Brazil, Fusarium solani was found to be the most common species causing mycotic keratitis. The genetic diversity of 44 isolates from 39 patients was assessed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) fingerprinting. ERIC-PCR was more discriminatory than PCR-RFLP for differentiating the strains. By combining of the results of both techniques, we identified 40 genotypes. Molecular typing revealed a high genomic heterogeneity of the strains of F. solani studied.     

Genomic DNA fingerprinting by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus.

In this study, we have compared genomic DNA fingerprintings among isolates of methicillin-resistant Staphylococcus aureus (MRSA) by using pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with SmaI were most suitable for the PFGE separation. SmaI cut genomic DNA into 15 to 20 fragments whose sizes ranged from about 30 to 1,500 kb. Thirty-one distinctive fragment patterns were identified in 111 infecting and colonizing MRSA isolates from six different hospitals in Japan. On the basis of the genomic typing by PFGE, we performed an epidemiological investigation of an outbreak of nosocomial MRSA infections among inpatients in Nagoya University Hospital. Ten types of chromosomal digestion were identified in the 20 strains isolated from 18 infected patients and 1 from colonized hospital personnel. According to the restriction patterns, we found that four types of these strains had caused epidemic infections among 13 patients in the outbreak. Two types (types 1 and 4) of the strains were involved in the death of five patients. The other infections were sporadic. The clarity and polymorphism of the chromosomal digestion patterns enabled us to discriminate between isolates which could not be differentiated by antibiogram or plasmid analysis. Classification of the genomic DNA fingerprinting patterns by PFGE is therefore proposed as a useful method for investigating the source, transmission, and spread of nosocomial MRSA infections.     

Comparison of Automated Ribotyping to Pulsed-Field Gel Electrophoresis for Genetic Fingerprinting of Streptococcus pneumoniae

Fifty-two isolates of Streptococcus pneumoniae were characterized by pulsed-field gel electrophoresis (PFGE) and automated ribotyping by using HindIII and PvuII. HindIII ribotypes correlated well with PFGE. PvuII produced fewer bands and was less discriminatory. Automated ribotyping with HindIII is an accurate method for genetic fingerprinting of S. pneumoniae and can complement PFGE.     

Risk factors for colonization and infection in a hospital outbreak caused by a strain of Klebsiella pneumoniae with reduced susceptibility to expanded-spectrum cephalosporins

Between February 2001 and January 2002, an increase in the number of Klebsiella pneumoniae isolates with reduced susceptibility to expanded-spectrum cephalosporins (RSKp) was detected in the neonatal unit of the Juan Canalejo Hospital, and 21 patients were either colonized or infected by the bacterial isolates. The current "gold standard" method for typing K. pneumoniae isolates is pulsed-field gel electrophoresis. However, this technique is expensive and time-consuming. In a search for faster and accurate alternatives to this method, we investigated PCR-based fingerprinting techniques (enterobacterial repetitive intergenic consensus sequence PCR [ERIC-PCR], repetitive extragenic palindromic sequence-based PCR [REP-PCR], and RAPD [randomly amplified polymorphic DNA]) for their ability to characterize K. pneumoniae isolates. The causal agent of the nosocomial outbreak was characterized by these techniques and was found to be a single epidemic strain (RSKp). A multiple regression logistic model was developed to identify potential independent factors associated with colonization and/or infection by RSKp. Logistic regression analysis was applied to all significant variables (P < 0.05) in the univariate analysis, and it was revealed that intubation (odds ratio [OR], 27.0; 95% confidence interval [95%CI], 5.39 to 135.14) and prematurity (OR, 4.4; 95%CI, 0.89 to 21.89) were such independent factors. Moreover, oxime cephalosporins did not appear to be statistically significant. Overall, the results showed that PCR-based techniques are expeditious and useful methods for typing K. pneumoniae isolates. Of the techniques studied, ERIC-PCR showed the highest discriminatory index (D = 0.828), followed by RAPD (D = 0.826) and REP-PCR (D = 0.773)     

Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds.

One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.     

Comparison of pulsed-field gel electrophoresis and amplified fragment length polymorphism techniques for investigating outbreaks of enteritis due to campylobacters

Campylobacters are the most commonly reported cause of acute bacterial enteritis in the United Kingdom and United States, with poultry, milk, and water implicated as sources or vehicles of infection. The majority of campylobacter infections are sporadic, although outbreaks may occur, and these provide an opportunity to evaluate genotypic fingerprinting techniques. In this study, pulsed-field gel electrophoresis (PFGE) was compared with single-enzyme-amplified fragment length polymorphism (SAFLP). The results for the three separate episodes indicated that SAFLP and PFGE both clustered the strains from the first incident as 100% homologous. The strains from the second and third incidents clustered as distinct from both the first incident and from each other. PFGE is well recognized as a discriminatory fingerprinting technique for campylobacters; however, SAFLP has proven to be equally discriminatory, but far less labor intensive and with the added advantages of less "hands-on" time and inexpensive equipment, it is an excellent alternative to PFGE for investigation of outbreaks.