Evaluation of Genetic Relatedness of Bacteroides fragilis Strains Isolated from Different Sources by AP-PCR and Pulsed-Field Gel Electrophoresis Assays

The genetic relatedness of 71 Bacteroides fragilis strains isolated from different sources (human intestinal and non-intestinal infections and animal intestinal infections, human and animal intestinal microflora and polluted aquatic environment) was evaluated by arbitrarily primed-polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE). The presence of the enterotoxin gene (bft) and β-lactamase genes (cep A, cfi A) was also determined by PCR. The amplification with the arbitrary primer AP12h produced electrophoretic profiles and the use of a biostatistical program (NTSYS) provided a dendrogram that revealed nine amplitypes, clustered in two groups, AI and AII, at a genetic distance of 0.30. Eight strains harbouring cfi A gene presented homogeneous profiles and could be clustered (amplitype A8) as well as 82.4% of the strains isolated from non-intestinal infections (amplitype A4). EnterotoxigenicB. fragilis strains (ETBF) were clustered in group AI as well as non-enterotoxigenic B. fragilis strains (NTBF). PFGE was used to analyse strains representative of each ampli-type formed. DNA restriction with Not I generated 25 PFGE profiles and only two pairs of strains presented more than 90% of similarity when Dice's coefficient and UPGMA clustering were applied. Although our data suggest a relevant relatedness among cfi A positive strains and among strains isolated from non-intestinal infections using AP-PCR, the use of a method with a greater discriminatory power revealed the wide diversity. These data reinforce the idea of infinite heterogeneity among B. fragilis strains.     

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.     

Genetic Fingerprinting of Brevibacterium linens by Pulsed-Field Gel Electrophoresis and Ribotyping

Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied. Received: 13 January 2000 / Accepted: 9 February 2000     

A Comparison of Non-Typhoidal Salmonella from Humans and Food Animals Using Pulsed-Field Gel Electrophoresis and Antimicrobial Susceptibility Patterns

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella, differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT), in 92% of human and 80% of food animal Salmonella isolates. The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize their impact on prevention.     

Genetic Diversity in the Microsporidian Encephalitozoon hellem Demonstrated by Pulsed-Field Gel Electrophoresis

Encephalitozoon hellem is a microsporidian species responsible for opportunistic infections in AIDS patients. Use of a novel chitinase-based method allowed unsheared chromosomal DNA to be recovered from eleven E. hellem isolates derived from three geographic regions. All isolates were typed by 18S rDNA sequencing, which showed that they belonged to intemal transcribed spacer type 1. After ethidium bromide staining, pulsed-field gel electrophoresis (PFGE) analysis discriminated two new karyotypes comprising 7 and 8 chromosomal bands respectively, ranging in size from 205- to 272-kb pairs. Genomic size was estimated to be 2.39 Mb. Our data indicate PFGE is useful for typing E. hellem and confirms genetic diversity among E. hellem genotypes.     

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AGAH00000000.1","term_id":"364889226","term_text":"AGAH00000000.1"}}AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell''s Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.     

Genetic Characterization of Atypical Enteropathogenic Escherichia coli Isolates from Ewes' Milk,Sheep Farm Environments,and Humans by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes'' milk and suggest a transmission route for this emerging pathogen through contaminated water.     

DNA Fingerprinting of Dairy Propionibacteria Strainsby Pulsed-Field Gel Electrophoresis

Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and industrial collections; endonucleases XbaI and SspI gave satisfactory restriction patterns. However, whereas XbaI can be used for Propionibacterium freudenreichii, SspI seems more suitable for the three other species: P. acidipropionici, P. thoenii, and P. jensenii. It is a convenient method to differentiate the dairy propionibacteria from closely related bacteria and from others usually present in dairy products. We observed a considerable restriction fragment length polymorphism among the Propionibacterium chromosomes and especially for P. freudenreichii: among 48 strains we detected 40 different patterns. This species is the most commonly encountered in the Swiss-type cheeses and is the only Propionibacterium species used as a cheese starter. Conversely, the species P. acidipropionici is not very diverse: among nine strains we observed only four different patterns, two of which were closely related. This is probably because this species is not used as a starter in cheese manufacture and consequently is poorly represented in collections. When strains come from geographical different isolates, their patterns are always different with very few common bands. The presence of numerous identical strains was due to the fact that they were present at the same time in the national collections, research laboratory collections, and in the industrial ones.     

Optimization of Pulsed-Field Gel Electrophoresis for Legionella pneumophila Subtyping

A total of 32 strains of Legionella pneumophila were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of L. pneumophila. Twenty-six isolates of L. pneumophila with various origins and 11 isolates from five different water systems were used as the panels. For optimization of electrophoretic parameters (EPs) of SfiI PFGE, 26 isolates were analyzed with SfiI digestion, using four EPs yielding the same D value. The EP of a switch time of 5 to 50 s for 21 h had the smallest similarity coefficients and was declared the optimal EP for SfiI PFGE of L. pneumophila. By software analysis and pilot study, AscI was chosen as another PFGE enzyme. AscI PFGE could cluster the isolates from each water system into the same or very similar patterns and had a high degree of typing concordance with other molecular methods. In evaluating the discriminatory power of AscI with the panel of 26 isolates, AscI PFGE gave one single pattern and a D value of 100%. AscI PFGE had a high discriminatory power and a high degree of consistency with epidemiological data and other molecular typing methods for L. pneumophila subtyping, and hence, AscI could be used as a restriction enzyme in PFGE subtyping of L. pneumophila.Legionella pneumophila is an environmental organism that can cause disease in humans and is increasingly recognized as an important pathogen causing nosocomial pneumonia. Potable water systems (14, 26), spa water (28), and cooling towers (7, 13) are among the sources implicated in outbreaks of Legionnaires’ disease. Transmission of bacteria from the environment to humans occurs via inhalation or aspiration of Legionella-containing aerosols (3, 5). Strain differentiation is necessary for the identification of sources of contamination and determination of routes of transmission; this could in turn enable us to more accurately detect outbreaks and limit the spread of L. pneumophila infections. A variety of subtyping techniques have been used to identify and characterize L. pneumophila strains, including monoclonal antibody (MAb) analysis (16, 19), ribotyping (4), amplified fragment length polymorphism (AFLP) analysis (9, 22), PCR-based methods (15, 24), sequence-based typing (SBT) (9, 16), and pulsed-field gel electrophoresis (PFGE) (1, 6).Preliminary reports demonstrated that PFGE is a highly discriminative epidemiological marker for subtyping of L. pneumophila (6, 11, 23, 25), and a number of L. pneumophila PFGE protocols have been described in the literature (1, 2, 4, 14); however, most laboratories that use PFGE to subtype L. pneumophila cannot compare their results because the protocols differ from each other in critical parameters, such as the restriction enzymes and electrophoresis conditions used to generate the DNA fingerprints. To enhance our ability to monitor this pathogen, there is an urgent need for a standardized L. pneumophila PFGE protocol which can readily be implemented in different laboratories for information interpretation.An optimal PFGE protocol produces a suitable number of restriction fragments and gives distinct patterns by agarose gel electrophoresis, with these determined by the restriction enzymes and the electrophoretic parameters (EPs) used. SfiI is the most frequently used enzyme in conventional PFGE protocols for L. pneumophila, and there are several different EPs for SfiI digestion used by investigators for characterization and epidemiological studies. For a certain restriction enzyme, selection of the EP with the smallest similarity coefficients will increase the discriminatory power of PFGE. As the first phase of this study, we compared the similarity coefficients obtained for four EPs with SfiI digestion and determined the one with the maximal discriminatory power.There were some problems found in practical applications of epidemiological investigation of L. pneumophila by PFGE with single SfiI digestion, such as having epidemiologically unrelated strains exhibit the same patterns (30) and the appearance of “ghost” or “phantom” bands. Combination use of two enzymes would give a higher discriminatory power and more accurate results (10, 29). Thus, as the second phase of this study, we selected another suitable enzyme and compared it with SfiI to evaluate the possibility of its use in characterization and epidemiological studies of L. pneumophila.     

In Vitro and In Vivo Invasiveness of Different Pulsed-Field Gel Electrophoresis Types of Listeria monocytogenes

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L. monocytogenes commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10⁹ L. monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.     

用凝胶电泳方法鉴定苏云金芽孢杆菌溶源性菌株

根据原噬菌体的可诱导性,将苏云金芽孢杆菌(Bacillus thuringiensis,简称B t)培养液用丝裂霉素C(MMC)诱导,诱导液经高速离心除菌和2.5×SDS-EDTA染料混合液处理后,琼脂糖凝胶电泳检测有无DNA带,以确定菌株的溶源性。实验证明,该DNA为溶源菌诱导出的噬菌体DNA,而非溶源菌以同样方法不能获得DNA。用此方法,可作为鉴定B t溶源性菌株的一个手段,有助于B t工业发酵中噬菌体污染的预防。     

Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ^sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Pulsed-Field Gel Electrophoresis Analysis of Vibrio vulnificus Strains Isolated from Taiwan and the United States

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.     

Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.     

Typing of Staphylococcus epidermidis Colonizing in Human Nares by Pulsed-Field Gel Electrophoresis

From the nares of 11 healthy adults, 253 strains of coagulase negative staphylococcus were isolated and 88% of them were identified as Staphylococcus epidermidis using the API STAPH system. Chromosomal DNA fingerprinting of the isolated strains revealed that each person carried multiple types of S. epidermidis in his or her nares. The colonization of the strains was not stable; the types of the isolates changed in the first and the second examinations 5 months apart. The results contrasted with previous findings in which only one strain of S. aureus colonized persistently in the nares of healthy adults.     

Molecular Characterization of Clostridium tetani Strains by Pulsed-Field Gel Electrophoresis and Colony PCR

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.     

Meta-Analysis of Pulsed-Field Gel Electrophoresis Fingerprints Based on a Constructed Salmonella Database

A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE) patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13−15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.