Pulsed-field Gel Electrophoresis Procedure Research Articles

An improved procedure for the pulsed-field gel electrophoresis analysis of Vibrio vulnificus

Vibrio vulnificus is a marine bacterium that causes septicemia with high mortality via wound infection or seafood ingestion. Subspecies typing of V. vulnificus can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 12% of strains. This study presents a modified PFGE procedure. The outer membrane of V. vulnificus cells was lysed utilizing a sucrose-EDTA method and components in the periplasmic space were removed prior to making plugs. Cytoplasmic digestive enzymes were inactivated by an extended proteinase K reaction. Experimental results indicated that five out of eight strains exhibiting smeared DNA in a previous study (Appl. Environ. Microbiol. 70: 5153-5158. 2004) achieved clear banding patterns. This modified PFGE procedure can be applied to improve PFGE typing of untypeable strains of V. Vulnificus.

Rapid and simultaneous detection of linear chromosome and large plasmids in Proteobacteria

Bacterial genomic architectures are complex and play important roles in genome evolution. While most bacterial lineage genomes contain a single chromosome often accompanied by plasmids, more and more genomes are described as harboring a linear or a second chromosome. Thus, the development of bacterial genomics leads to the study of bacterial genome architectures. In order to avoid laborious techniques combining several methods, we developed an original plug-free pulsed field gel electrophoresis procedure, that enabled us to co-characterize reproducibly the presence of linear chromosomes and plasmids ranging between 30 kb and 2000 kb in various proteobacterial lineages.

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates

To develop a modified pulsed-field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates. A modified PFGE procedure was designed using CpoI to generate restriction maps of H. parasuis genomic DNA. This approach was used to characterize 47 H. parasuis clinical isolates and 15 reference strains. All strains could be typed by this method, and the procedure was completed in 36 h. A total of 39 different PFGE patterns were identified among 47 epidemiologically unrelated clinical isolates. The modified PGFE described in this report efficiently characterized H. parasuis isolates. This method can be adopted for studying the epidemiology of Glässer's disease outbreaks in addition to differentiating and classifying previously untypeable H. parasuis isolates. The modified PFGE method described is a novel means of characterizing H. parasuis isolates. It is also a highly discriminatory molecular typing method (discriminatory index of 0.98) that can overcome the limitations of serotyping.

The Optimization of a Rapid Pulsed-Field Gel Electrophoresis Protocol for the Typing of <i>Acinetobacter baumannii</i>, <i>Escherichia coli</i> and <i>Klebsiella</i> spp.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.

First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters (UPTC), and small restriction fragment sequencing

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a ρ-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible ρ-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

Molecular Fingerprinting of Clostridium difficile Isolates: Pulsed-Field Gel Electrophoresis versus Amplified Fragment Length Polymorphism

Two molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), were used to investigate the epidemiological relatedness among Clostridium difficile isolates from suspected outbreaks in three general hospitals. Analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. Although this degradation could be prevented to a certain extent by the inclusion of thiourea in the electrophoresis buffer, the weak DNA banding patterns obtained in this way were still far from optimal. AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform. AFLP analysis yielded high resolution and highly reproducible DNA fingerprinting patterns from which the epidemiological relatedness among the isolates could easily be determined. AFLP results could be readily obtained within 24 h, whereas 3 to 4 days were routinely required to complete the lengthy PFGE protocol. AFLP clearly proved to be a much more fail-safe fingerprinting method for C. difficile isolates, especially for those isolates for which a standard PFGE procedure yielded inconclusive results due to DNA degradation.

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis.

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Typing of nosocomial Strains of Serratia marcescens: Comparison of restriction enzyme cleaved genomic DNA fragment (PFGE) analysis with bacteriocin typing, biochemical profiles and serotyping

Eighty-eight selected clinical isolates of Serratia marcescens, representing 27 putative outbreaks of nosocomial cross-infection encountered during 1980-1995, were tested comparatively by bacteriocin typing, carbon source assimilation tests, serotyping (O and H antigens), and restriction pattern (RFLP) analysis of restriction cleaved (SpeI, XbaI) genomic DNA fragments after pulsed-field gel electrophoresis (PFGE). Serotyping served as the "gold standard" of the phenotypic methods. One pseudo-outbreak (bacteriocin typing incriminated type 26) was uncovered through serotyping as well as the biochemical profile and confirmed by PFGE analysis of genomic DNA. Bacteriocin typing and determination of biochemical profiles disclosed several instances of phenotypic variation; serotyping revealed two episodes of shifts from motility (H12) to nonmotility. Resolution of restricted genomic DNA fragments with the PFGE procedure permitted detection of 27 PFGE patterns (A-M, N-1-N-3, O-1, O-2, P-1-P-3, Q-1-Q-3, R-1-R-3, S-1, and T). Based on the analysis of PFGE patterns against the background of epidemiological data, the number of nosocomically significant strains of S. marcescens could be reduced to 16 (PFGE patterns A-M, N-2, O-1, P-2, and T). It was concluded that PFGE analysis of restricted genomic DNA of S. marcescens was superior to the three phenotypic methods.