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.     

Analysis of Legionella pneumophila serogroup 6 strains isolated from a hospital warm water supply over a three-year period by using genomic long-range mapping techniques and monoclonal antibodies.

Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown from warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NotI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 300-kb NotI fragment when a legiolysin (lly)-specific DNA probe was used. The NotI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six NotI cleavage types obtained by pulsed-field electrophoresis.     

Analysis of Legionella pneumophila serogroup 6 strains isolated from a hospital warm water supply over a three-year period by using genomic long-range mapping techniques and monoclonal antibodies.

Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown from warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NotI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 300-kb NotI fragment when a legiolysin (lly)-specific DNA probe was used. The NotI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six NotI cleavage types obtained by pulsed-field electrophoresis.     

Pulsed-field gel electrophoresis of the genomic restriction fragments of coagulase-negative staphylococci

Abstract The genomes of 47 coagulase-negative staphylococcal strains assigned to different species were analysed by pulsed-field electrophoresis. The strains were clustered on the basis of their similarity in the Sma I restriction patterns into various groups, each group consisting of the type strain and the strains whose Sma I restriction patterns were similar to that of the type strain. The Sma I restriction groups seem to correspond to the following species: Staphylococcus warneri, S. hominis, S. xylosus, S. lugdunensi, S. kloosii, S. haemolyticus, S. lentus, S. cohnii, S. equorum, S. chromogenes, S. saprophyticus, S. simulans, S. carnosus, S. capitis and S. auricularis . The species S. sciuri, S. caseolyticus, S. gallinarum, S. epidermidis and S. schleiferi were represented only by their type strains and showed no similarity in their Sma I restriction patterns neither to each other nor to all the species investigated here. Thus, the classification of coagulase-negative staphylococcal strains into the above species seems to be confirmed also by genome restriction analysis carried out by pulsed-field gel electrophoresis.     

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.     

DNA fingerprinting by pulsed-field gel electrophoresis to investigate a nosocomial pneumonia caused by Legionella bozemanii serogroup 1.

We typed 18 isolates of Legionella bozemanii obtained from clinical and environmental sources by pulsed-field gel electrophoresis. Each of the unrelated strains showed individual restriction patterns of the genomic DNA when either the SfiI or NotI restriction enzyme was used. One strain isolated from a patient with nosocomial legionellosis and two strains from the corresponding hospital water supply were indistinguishable, arguing for a transmission of L. bozemanii from the water supply to the patient. In conclusion, macrorestriction analysis is a valuable tool for studies of the molecular epidemiology of L. bozemanii.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

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.     

Analysis of the genome of the five Bifidobacterium breve strains: plasmid content, pulsed-field gel electrophoresis genome size estimation and rrn loci number

Abstract The genomes of the five Bifidobacterium breve strains available from culture collections were compared by restriction endonuclease analysis. Electrophoretic migration of undigested DNA allowed us to detect a 5.6-kb circular plasmid in two of these strains. A restriction map of this plasmid was constructed using 10 enzymes. With Dra I endonuclease, pulsed-field gel electrophoresis has allowed the determination of the five B. breve genome sizes to 2.1 Mb. This estimation was further confirmed for CIP 6469 (type strain) and ATCC 15698 using Xba I and Spe I enzymes. In addition, rRNA gene regions were used as probes for strain characterization and suggest that there are at least three rrn loci in B. breve .     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

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.     

Listeria monocytogenes in Faeces from Clinically Healthy Dairy Cows in Sweden

Faecal samples from 102 clinically healthy dairy cows, representing 34 farms in the Swedish province of Uppsala, were analysed for the presence of Listeria spp. using an enrichment procedure. Listeria monocytogenes was isolated from six (6%) and L. innocua from 2 (2%) cows. From each of the 6 samples positive for L. monocytogenes, 5 isolates were further characterised by restriction enzyme analysis using the 3 enzymes Apa I, Sma I, and Asc I, followed by pulsed-field gel electrophoresis. Three of the L. monocytogenes positive cows lived at the same farm, and they all harboured the same clonal type. One of these 3 cows also harboured a further clonal type of L. monocytogenes. The fact that one of the cows harboured 2 different clonal types of L. monocytogenes is important from an epidemiological point of view when routes of infection are to be investigated.     

Typing Listeria monocytogenes isolates from fish products and human listeriosis cases.

Seventy-two Listeria monocytogenes isolates originating from 10 different fish products of 12 producers and 47 isolates from human listeriosis cases were typed by serotyping and multilocus enzyme electrophoresis. Seventy-five of these isolates were further subtyped by restriction analysis of genomic DNA with the enzyme XhoI and by pulsed-field gel electrophoresis using the enzymes ApaI and SmaI. The results show that several L. monocytogenes clones identified by multilocus enzyme electrophoresis are frequently found in fish products of different origins. One of these clones is the same as another previously shown to be frequently associated with meat and meat products. The epidemic-associated electrophoretic type 1 was only rarely found in fish products. No association was found between any type of fish product and a particular lineage of L. monocytogenes. Both long-term persistence of a strain and simultaneous presence of several clearly distinct strains in the products of single producers were observed. The comparison of L. monocytogenes isolates from human clinical listeriosis cases in Switzerland and those from imported fish products by use of multilocus enzyme electrophoresis showed that they do not form two clearly distinct lineages but nevertheless belong to two separate populations. None of the 48 subtypes distinguished by the combination of all four typing methods could be found in both populations of human origin and those of fish origin.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.     

First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.     

Construction of physical and genetic maps of Chlamydia trachomatis serovar L2 by pulsed-field gel electrophoresis.

We constructed the physical map of Chlamydia trachomatis serovar L2 by using three restriction endonucleases, NotI (GC[GGCCGC), SgrAI (C(A/G)[CCGG(T/G)G), and Sse8387I (CCTGCA[GG), and we analyzed the fragments by pulsed-field gel electrophoresis. A total of 25 restriction endonuclease sites and 13 genes and/or operons were located on the map. The genome size was determined to be 1,045 kb. Neither highly transcribed chlamydia genes nor developmental cycle-specific genes were clustered on the genome.     

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     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.