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.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.     

Differentiation between human and ovine isolates of Bordetellaparapertussis using pulsed-field gel electrophoresis

Abstract The genetic relatedness of 18 human and 29 ovine isolates of Bordetella parapertussis was examined by macrorestriction digestion of DNA with the rarely cutting enzyme Xba I and resolution by pulsed-field gel electrophoresis. There was clear separation of human and ovine isolates and variation within host types. The human isolates were separated into three types as were the 24 Scottish ovine isolates. Species-specific bands were observed with the human isolates at 114, 134, 166, 213, 346 and 372 kb. No species-specific bands were found in the B. parapertussis ovine isolates. Isolates of B. parapertussis recovered from sheep in New Zealand gave a further two DNA banding patterns which were clearly different from the Scottish ovine and the human isolates. These results indicate that human and ovine isolates of B. parapertussis are genetically distinct and that variation exists within isolates from the same host species. Pulsed-field gel electrbphoresis therefore appears to be a powerful discriminatory tool for the classification of B. parapertussis .     

A comparison of Salmonella serotypes isolated from New Zealand sea lions and feral pigs on the Auckland Islands by pulsed-field gel electrophoresis

The Salmonella serotypes S. Cerro and S. Newport were isolated from New Zealand sea lions (Phocarctos hookeri) and feral pigs on the Auckland Islands in the New Zealand subantarctic region. The isolates were typed by pulsed-field gel electrophoresis using Xba1 as the restriction enzyme. The isolates were indistinguishable, which suggests that Salmonella infection cycles between sea lions and pigs in this environment. Apart from a previous isolation from a single New Zealand fur seal (Arctocephalus forsteri), S. Newport has not been recorded in any animals from New Zealand, but it is associated with gastroenteritis in humans. Contamination of the marine environment by human waste is a possible source of infection for marine mammals and warrants further investigation.     

Heterogeneity of genome sizes among natural isolates of Escherichia coli.

Comparisons of the genetic maps of Escherichia coli K-12 and Salmonella typhimurium LT2 suggest that the size and organization of bacterial chromosomes are highly conserved. Employing pulsed-field gel electrophoresis, we have estimated the extent of variation in genome size among 14 natural isolates of E. coli. The BlnI and NotI restriction fragment patterns were highly variable among isolates, and genome sizes ranged from 4,660 to 5,300 kb, which is several hundred kilobases larger than the variation detected between enteric species. Genome size differences increase with the evolutionary genetic distance between lineages of E. coli, and there are differences in genome size among the major subgroups of E. coli. In general, the genomes of natural isolates are larger than those of laboratory strains, largely because of the fact that laboratory strains were derived from the subgroup of E. coli with the smallest genomes.     

Karyotyping by PFGE of clinical isolates of Sporothrix schenckii

Abstract From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.     

Genome organization and localization of the pufLM genes of the photosynthesis reaction center in phylogenetically diverse marine Alphaproteobacteria

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.     

Analysis of genetic relationships and antimicrobial susceptibility of Escherichia coli isolated from Clethrionomys glareolus

Eleven strains of Escherichia coli were isolated from 54 bank voles living in the LomZa Landscape Park of the Narew River Valley, indicating that E. coli is not common in the alimentary tract of these mammals. On the basis of pulsed-field gel electrophoresis and computer-assisted analysis, the isolates were grouped into six genotypes at similarities of 39%. Chromosome length of E. coli under study differed by as much as 900 kb, ranging 2.7-3.6 Mb. All strains were susceptible to amikacin and ciprofloxacin, whereas, for tetracycline, streptomycin, ampicillin, and cefonicid, different results were noted. No differences were detected among the plasmid complements of eight strains (73%), for which plasmid profiles revealed the presence of two plasmidic bands. One, three and four plasmids were observed in a plasmid pattern of single isolates. The observation from the study indicated the high genetic polymorphism among the isolates recovered from the animals of one species living in the same environment.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Detection of a large linear plasmid in Borrelia spielmanii isolate

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.     

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.     

Transformation of Chlorobium limicola by a plasmid that confers the ability to utilize thiosulfate.

A modified transformation method for Chlorobium limicola confirms the role of an endogenous plasmid in thiosulfate metabolism. The plasmid was present in two forma specialis thiosulfatophilum (Tio+) strains and absent in one non-thiosulfate-utilizing (Tio-) strain. The plasmid (size, 14 kb) was transferred by transformation from Tio- to Tio+. The chromosomal restriction patterns, analyzed by pulsed-field gel electrophoresis, were used to distinguish between the different strains.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

Determination of genome sizes of Rickettsia spp. within the spotted fever group, using pulsed-field gel electrophoresis.

The chromosome lengths of six spotted fever group Rickettsia species (Rickettsia rickettsii, R. conorii, R. rhipicephali, R. sibirica, R. australis, and R. akari) were estimated by pulsed-field gel electrophoresis. The genome size of R. rickettsii was about 2,100 kb, but the chromosome lengths of the five other species were, surprisingly, much lower and ranged between 1,200 and 1,300 kb.     

Genome conservation in isolates of Leptospira interrogans.

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.     

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.