Application of different chromosomal DNA restriction digest fingerprints to specific and subspecific identification of Campylobacter isolates.

Eleven strains comprising representatives of different subspecies, biotypes, and serotypes of Campylobacter jejuni and reference strains of C. coli, C. fetus subsp. fetus, C. hyointestinalis, and C. sputorum subsp. sputorum were studied to assess the utility of different DNA profiles for measuring fine differences between allied bacteria. Strains were compared by analyses of HaeIII and XhoI digest patterns of chromosomal DNA and Southern blot hybridization patterns of XhoI digests obtained with an Escherichia coli 16S + 23S rRNA gene probe. Visual comparisons and numerical analyses of the HaeIII and XhoI digest patterns both revealed clear differences between the five Campylobacter species and between representatives of C. jejuni subspecies and biotypes. Only strains with the same Penner serotype gave identical total digest polymorphisms. The advantages of XhoI total digests and Southern blot hybridization patterns were that they were less complex than the HaeIII patterns and easier to compare visually. However, numerical analysis of XhoI data resulted in reduced discrimination. We conclude that DNA fingerprinting using either HaeIII or XhoI fragment polymorphisms has considerable potential as a generally applicable method for identification of Campylobacter isolates, especially at the infrasubspecific level.     

Biochemical Characteristics of Typhus Group Rickettsiae with Special Attention to the Rickettsia prowazekii Strains Isolated from Flying Squirrels

Six strains of Rickettsia prowazekii, two derived from human infections and four isolated from flying squirrels, two strains of R. typhi, and the single available strain of R. canada, were characterized by several biochemical procedures. The electrophoretic patterns on polyacrylamide gels of rickettsial proteins solubilized by sodium dodecyl sulfate revealed several species differences, but strains of the same species appeared to have identical patterns. Cytoplasmic fractions of the rickettsiae were examined for enzymatic activities and for polyacrylamide gel isoelectric focusing patterns. Some species differences were encountered in the activities or ratios of activities of glutamate-oxaloacetate transaminase, glutamate dehydrogenase, and malate dehydrogenase. When polyacrylamide gels were stained for malate dehydrogenase after electrophoresis, a single band became apparent with single extracts or mixtures of two strains of R. prowazekii, but two bands were seen with mixtures of a strain of R. prowazekii and one of R. typhi. The isoelectric focusing patterns of the soluble proteins revealed numerous species differences, especially between R. canada and the other two species, and a few differences among the strains of R. prowazekii. The patterns of the two human strains, Breinl and E(R), differed in at least one location, and both differed from the flying squirrel strains in the displacement of one band. One of the flying squirrel strains, GvF-16, contained a protein band not seen in the other five strains. Despite these minor differences, a striking similarity was revealed by all the biochemical tests performed between the R. prowazekii strains of human and flying squirrel origin.     

Chromosomal DNA, iron-transport systems, outer membrane proteins, and enterotoxin (heat labile) production in Salmonella typhi strains.

We examined a representative collection of Salmonella typhi strains from Chile, Peru, Mexico, India, and England for the presence of several properties. All strains had a conserved pattern of outer membrane proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoresis profiles of chromosomal DNA digested with EcoRI and PstI restriction enzymes were similar for all the strains. A conserved pattern of hybridization was observed when digested chromosomal DNA was hybridized with DNA probes for the 36-kilodalton porin, enterobactin synthesis, and enterobactin receptor genes. All the strains produced enterobactin but not aerobactin in bioassays. None of the strains produced heat-labile toxin, as measured by an enzyme-linked immunosorbent assay. Colony and Southern hybridizations with DNA probes for aerobactin synthesis and its receptor and heat-labile toxin genes were negative. These results indicate that S. typhi strains from different origins have similar phenotypic and genetic properties and, as has been suggested, constitute a clone.     

Comparison of morbillivirus proteins by limited proteolysis

Proteins of a number of measles and SSPE virus strains have been compared by limited proteolysis and they appear to be largely conserved amongst the various strains. Viruses derived from SSPE cannot be distinguished from other measles viruses by this technique. Small differences in the digest patterns of the M proteins have been observed between the Edmonston and other measles virus strains. Furthermore, in some strains where the M proteins migrate slower in SDS-PAGE the limited proteolysis patterns are slightly different from those in other MV and SSPE virus strains. The limited proteolysis pattern of some canine distemper virus (CDV) proteins have been determined and nucleocapsid breakdown products have been identified in infected cells. Comparisons of proteins of four strains of CDV have shown that these, too, are largely conserved, although the digest of proteins of CDV appear to show more pronounced differences than those present in the MV and SSPE virus group. Limited proteolysis can be used readily to distinguish MV from CDV isolates.     

Atrophic rhinitis in swine: correlation of Pasteurella multocida pathogenicity with membrane protein and lipopolysaccharide patterns

Cell envelope proteins and lipopolysaccharides (LPS) of Pasteurella multocida strains associated with atrophic rhinitis in swine were compared by using sodium dodecyl sulfate gel electrophoresis. Among 34 strains, three different types of cell envelope protein patterns, named I (16 strains), II (3 strains), and III (15 strains), could be distinguished. These differences were based on the electrophoretic mobility of the major protein, designated as protein H. Comparison of cell envelope protein type and pathogenicity of the strain, the latter property predicted by the guinea pig skin test, revealed that all type I strains, 6 of 15 type III strains, and none of the type II strains were pathogenic. Although pathogenicity has been correlated with extracellular toxin activity, no protein could be detected in either the cell envelopes or in the extracellular fluid that absolutely correlated with pathogenic strains. Electrophoretic analysis of the LPS revealed that all strains possessed low-molecular-weight LPS, which is inconsistent with the presence of a classical O antigen. The method allowed the detection of at least six types of LPS, which often coincided with a certain cell envelope protein type and with the presence or absence of the pathogenic character of the strain. These results strongly suggest that the sampled swine carry a limited number of P. multocida clones, in each of which the patterns of cell envelope proteins and LPS, as well as the presence or absence of the ability to produce extracellular toxin, are well conserved. Therefore, the possibility is discussed that sodium dodecyl sulfate gel electrophoresis of cell envelope proteins and LPS may be used for the prediction of the pathogenic character of part of the strains. Finally, the typing of strains based on cell envelope protein patterns might contribute to the development of vaccines containing outer membrane proteins as protective antigens.     

Comparison of protein and lipopolysaccharide patterns of several Coxiella burnetii strains in phase I

Electrophoresis in polyacrylamide gel gradients in the presence of sodium dodecyl sulphate revealed at least 18 identical protein bands in eight strains of Coxiella burnetii. By staining with Coomassie Brilliant Blue R-250 it was possible to visualize clearly at least 40 proteins. The protein pattern showed the greatest variability in the region from 27 to 18.5 kD. Strains S and Priscilla differed from the other strains also in proteins smaller than 18.5 kD. The lipopolysaccharide pattern obtained by the same technique consisted of from 6 to 12 various bands in the region from 23.5 to 11.8 kD. The protein and lipopolysaccharide patterns of four strains isolated from ticks did not significantly differ from those of strains Henzerling and L-35 isolated from men.     

Outer membrane protein profiles of Haemophilus pleuropneumoniae.

Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000- to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein profiles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States.     

Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.     

Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus

Monoclonal antibodies were produced to JHMV-DL, a neurotropic member of the mouse hepatitis virus (MHV) or murine coronavirus group. Of 23 antibodies isolated, 10 were specific for the major envelope glycoprotein, gp180/90, 10 for the nucleocapsid protein, pp60, and 3 for the minor envelope glycoprotein, gp25. Eleven different MHV isolates were used in antibody binding assays to study antigenic relationships among the viruses. Each MHV isolate tested had a unique pattern of antibody binding, indicating that each is a distinct strain. Conservation of JHMV-DL antigenic determinants varied among the three proteins, with pp60 showing intermediate conservation, gp180/90 little conservation, and gp25 marked conservation in the different MHV strains. Monoclonal antibodies to pp60 proved most useful in delineating antigenic relationships among MHV strains. These antigenic groups correlated with pathogenic types, indicating that pp60 may be one of the gene products which mediates the distinct disease patterns manifested by different murine coronaviruses.     

Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever.

Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.     

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.     

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.     

Two clones of methicillin-resistant Staphylococcus aureus in Poland

Objective: To evaluate relatedness among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Poland.
Method: Ninety-three MRSA hospital isolates were collected from different regions in Poland from 1990 to 1992. Strains were analyzed with respect to heterogeneity of methicillin resistance, phage types, resistance patterns, crystal violet staining, chromosomal DNA Sma I restriction patterns by PFGE, ERIC1 and ERIC2 AP-PCR types and DNA repeat polymorphism within the protein A gene. Resistance to methicillin was confirmed by the detection of the mecA gene by PCR.
Results: The combined results of typing methods demonstrate that all MRSA strains analyzed could be easily divided into two distinct clones (clonally related strains). The first consisted of strains with clear heterogeneous expression of resistance to methicillin (34 isolates) and the second showed more homogeneous resistance (59 isolates). In this study the best method for epidemiologic analysis of MRSA was found to be PFGE. A good correlation between the epidemic behavior of MRSA and a high number of repetitive DNA units within the protein A gene was observed.
Conclusions: Results show that in Poland two distinct clones of epidemic MRSA have circulated in the past, easily discriminated by pheno and genotyping methods, and both could be found together in a single hospital.     

Use of protein profiles to identify Acinetobacter calcoaceticus in a respiratory care unit.

The presence of acinetobacters in a respiratory care unit was prospectively studied because of an increase in the number of isolations of Acinetobacter calcoaceticus. Cell envelope protein electrophoresis was used to distinguish strains. Eleven protein patterns were observed in isolates from patients and their environment. One pattern (pattern 1) was seen in several patients and environmental samples. Another pattern (pattern 2) was identified repeatedly in samples from skin and mucous membranes of patients in the same ward. After thorough cleaning was undertaken throughout the unit, the pattern 1 strain was no longer cultivated from clinical samples. It is concluded that cell envelope protein electrophoresis is a useful method for tracing epidemic strains of A calcoaceticus.     

Antigen analysis of several pathogenic strains of Trichomonas vaginalis.

Analysis of several human strains of Trichomonas vaginalis and one bovine strain of Tritrichomonas foetus was accomplished with standard sodium dodecyl sulfate-gel electrophoresis and fluorography technology. Highly motile, live trichomonads were radiolabeled, and total trichloroacetic acid-precipitated proteins were electrophoresed. Complex protein profiles of the various human strains of T. vaginalis were obtained with proteins ranging in molecular weight from 20,000 to greater than 200,000. The parasite biosynthesis of the Coomassie brilliant blue-stained protein bands was demonstrated by efficient radiolabeling of trichomonads with [35S]methionine or a 3H-amino acid digest before electrophoresis and fluorography. Immunogenic trichomonal proteins were then identified by a radioimmunoprecipitation method. A detergent extract of [35S] methionine-labeled T. vaginalis proteins was mixed with serum from an immunized rabbit or pooled sera from subcutaneously infected mice and soluble antibody-antigen complexes isolated by adsorption to protein A-bearing Staphylococcus aureus. The radiolabeled protein antigens were then identified by gel electrophoresis and fluorography. Immunized rabbit serum and pooled sera from challenged mice contained high-titered antibody which reacted with numerous high- and low-molecular-weight proteins. Individual subcutaneously infected mice were found to possess identical antibody responses to these immunogenic trichomonal proteins. A high degree of serological cross-reactivity among the various trichomonads was demonstrated. No differences in the composition of immunogenic proteins were observed among cultures grown in vitro for various lengths of time under the experimental conditions employed. Finally, electrophoretic analysis of cloned colonies of T. vaginalis organisms revealed no differences in their protein composition. The biological relevance of these observations is discussed.     

Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(-)). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.     

IS6110 based amplityping assay and RFLP fingerprinting of clinical isolates of Mycobacterium tuberculosis.

AIMS--To evaluate the usefulness of two IS6110 based typing methods, an amplityping assay and restriction fragment length polymorphism (RFLP) analysis, for fingerprinting respiratory isolates of Mycobacterium tuberculosis. METHODS--For amplityping, a pair of primers which amplify the intervening sequence between the repetitive insertion sequence IS6110 was used to generate a banding pattern which was confirmed by hybridisation. This assay was compared with conventional chromosomal DNA RFLP typing in the evaluation of 110 epidemiologically diverse isolates. RESULTS--Polymerase chain reaction (PCR) amplityping generated a single pattern in Hong Kong Chinese strains, but two and four diverse patterns in Filipino and Vietnamese strains, respectively, and could be completed within four days. When compared with chromosomal DNA RFLP typing, which took three weeks to complete, four different RFLP patterns could be seen among the Chinese strains, while seven patterns were found in the Filipino and Vietnamese strains. No change in amplityping or RFLP patterns was found in 36 sequential isolates from the same patients after anti-tuberculosis treatment for up to 12 months, despite the emergence of resistance in three of these strains. No specific amplityping or RFLP pattern could be related to different patterns of drug susceptibility. CONCLUSION--PCR amplityping could be used initially as a rapid typing method to distinguish strains originating from different localities. This could be important for investigation of outbreaks of tuberculosis--for example, in refugee camps.     

Biochemical and immunological characterization of cell surface proteins of Pasteurella multocida strains causing atrophic rhinitis in swine

In a previous paper (B. Lugtenberg, R. van Boxtel, and M. de Jong, Infect. Immun., 46:48-54, 1984) we showed that among 34 isolates from swine the membrane protein and lipopolysaccharide (LPS) patterns, as analyzed by sodium dodecyl sulfate-gel electrophoresis, could be classified into three and six patterns, respectively. In all cases a certain LPS pattern was correlated with a certain protein pattern. Certain combinations of types of cell surface proteins and LPSs were correlated with pathogenicity, the latter property being judged by the guinea pig skin test. In the present paper the immunological and biochemical properties of cell surface constituents were analyzed. The reaction between electrophoretically separated cell surface constituents with guinea pig and sow antisera showed that LPS as well as several proteins were immunogenic. Among these is protein H, whose electrophoretic mobility is the main criterium for typing of cell envelope protein patterns. Protein H was the most heavily labeled component when whole cells were iodinated by the Iodo-Gen procedure showing its accessibility at the cell surface. These properties of protein H make it an attractive vaccine candidate. Further biochemical analyses revealed that protein H shares many properties with pore proteins of members of the family Enterobacteriaceae. One of these properties, association between pore proteins and peptidoglycan, was used as the basis for a simple procedure developed to partially purify protein H.     

Non-toxigenic pattern II and III Bacteroides fragilis strains: coexistence in the same host

Diarrhoeic stool samples from 334 0-5-year-old children were analysed with respect to the incidence of Bacteroides fragilis as well as other enteropathogens. B. fragilis was recovered in 9.3% (31/334) of the samples, and 79 strains were examined for the presence of the bft gene or the BfPAI flanking region using polymerase chain reaction assays. No enterotoxigenic B. fragilis strains were detected. In 29% (9/31) of the samples the coexistence of both II and III non-toxigenic B. fragilis (NTBF) patterns could be seen. In 51.6% (16/31) of the samples there existed a pattern II NTBF only, and in 19.4% (6/31) only pattern III could be detected. Strains from the same patient representing different patterns were submitted to pulsed-field gel electrophoresis assays. Fingerprints obtained by this technique showed that there was strong heterogeneity among strains from different individuals. However, different patterns from the same individual shared 100% similarity.     

Polyacrylamide gel electrophoresis of cellular proteins from parasitic flagellates of the order Trichomonadida

Some Trichomonas hominis and Dientamoeba fragilis strains were compared by electrophoretic patterns of total proteins from trophozoites. Easily distinguished patterns divided T. hominis in different types. Significant differences were also seen comparing T. hominis and D. fragilis whilst among strains of D. fragilis electrophoretic protein patterns were mostly found to be accordant.