Analysis Of Electrophoretic Protein Patterns Research Articles

Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands.

In order to gain insight into the epidemiology of Campylobacter lari infection in The Netherlands due to the consumption of raw mussels and oysters, batches of these shellfish were screened for the presence of Campylobacter spp. during a 6 month period in 1993-1994. Apparently, 41 out of 59 batches of mussels and 11 out of 41 batches of oysters were colonized with Campylobacter spp. A subset of the isolates was further characterized by additional phenotypic tests, numerical analysis of electrophoretic protein patterns, and genotyping by random amplification of polymorphic DNA (RAPD). Protein electrophoretic analysis of 39 Campylobacter spp. cultured from 24 batches of mussels and oysters, revealed that all isolates, except two, were C. lari. Two strains with an aberrant protein pattern were identified as C. coli and C. hyointestinalis, respectively. Nalidixic acid susceptible campylobacters (NASC) and urease-positive thermophylic campylobacters (UPTC) did not form separate clusters and should be considered biovars only. Several strains were both urease positive and nalidixic acid susceptible, which represents a new biovar within C. lari. The results of RAPD demonstrated the presence of 3 distinct genetic variants, implying that even within a single batch of shell fish, relatively extensive DNA polymorphisms can be found. It is therefore apparent that this complex group of C. lari is characterized by a high degree of genetic diversity, implying the presence of a heterogenous population of C. lari in crustacean organisms living in marine waters in a restricted area in The Netherlands.

Identification and typing of Proteus penneri and Proteus vulgaris biogroups 2 and 3, from clinical sources, by computerized analysis of electrophoretic protein patterns

Seventy-six strains of the Proteus vulgaris complex (Pr. penneri and Pr. vulgaris biogroups 2 and 3) were characterized by one-dimensional SDS-PAGE of cellular proteins. The protein patterns were highly reproducible. The strains came from various countries and were mainly of human origin: urine (28), respiratory tract (13), wounds (8), faeces (7), blood (3), miscellaneous sources (6) and unknown sources (11). The patterns of these strains, together with those of the type strains of seven Morganella, Proteus and Providencia species were subjected to two numerical analyses. In the first, in which the principal protein bands (in the 35.0-42.0 kDa range) were excluded, the strains of the Pr. vulgaris complex formed four clusters at the 83% similarity level. These corresponded to Pr. penneri, Pr. vulgaris biogroup 2, and two clusters (3a and 3b) represented biogroup 3. Each of these clusters was distinct from the Morganella, Proteus and Providencia reference strains. In the second analysis, which included all the protein bands, the 41 Pr. penneri strains showed little heterogeneity but 17 subphenons could be recognized among the 35 strains of Pr. vulgaris biogroups 2 and 3. These results support the division of biogroup 3 strains into at least two separate taxa. Other results indicate that biogroup 3 is heterogeneous and may contain further genomic groups. The method also provides a basis for typing clinical strains of Pr. vulgaris biogroups 2 and 3.

Typing of Proteus mirabilis from clinical sources by computerized analysis of electrophoretic protein patterns

Seventy-five strains of Proteus mirabilis (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 24 were from urine, 17 from faeces (including one from a dog and one from a monkey), 12 from the respiratory tract (including nine from sputum), four from a cerebral abscess, 12 from miscellaneous sources and six from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of the 75 P. mirabilis cultures plus those of the type strains of six Proteus and Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the P. mirabilis cultures formed nine clusters at the 85% S level. In the second analysis, in which the principal protein bands (in the 34.0-44.6 kDa range) were excluded, 74 of the 75 cultures of P. mirabilis formed a single cluster at the 77% S level distinct from the six Proteus and Providencia reference strains. The P. mirabilis strain which failed to cluster with the others had a background band pattern typical of the species in the lower molecular weight region but appeared to be less typical in the heavier bands. It is concluded that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of P. mirabilis. Reference strains of each of the nine PAGE types identified are available from NCTC for inclusion in future studies.

Classification of Streptomyces and Streptoverticillium Species by Numerical Analysis of Electrophoretic Protein Patterns

Summary Cellular proteins of 32 strains of Streptomyces and 5 of Streptoverticillium were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The protein profiles obtained were analysed by computer-assisted techniques and the results compared with those of a previous numerical taxonomic study of phenotypic characteristics. Valuable taxonomic correlations were found between the protein electrophoretic and phenotypic data though it was evident that certain numerically circumscribed taxa, notably Streptoverticillium griseocarneum, were electrophoretically heterogeneous. Species specific profiles were observed for Streptomyces albidoflavus, Streptomyces anulatus and Streptomyces cyaneus. It can be concluded that numerical analysis of SDS-PAGE patterns of cellular proteins provides a rapid way of clarifying and refining subgeneric relationships established within the genera Streptomyces and Streptoverticillium.

Numerical analysis of electrophoretic protein patterns of Morganella morganii strains from faeces, wound, urine and other clinical sources

Sixty-five strains of Morganella morganii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 13 were from stools (including one from a toucan), 13 from wounds, 11 from urine, five from blood (including one from a snake), five from the respiratory tract (four sputum, one lung), 12 from miscellaneous sources and six from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 67 M. morganii cultures plus those of the type strains of seven Proteus and Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the M. morganii strains formed 21 clusters at the 91% S level. In the second analysis, in which the principal protein bands (in the 31.6-43.2 kDa range) were excluded, the 67 M. morganii cultures formed a single cluster at the 80% S level distinct from the seven Proteus and Providencia reference strains. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of M. morganii. Reference strains of each of the 21 PAGE types identified are available from NCTC for inclusion in future studies.

Identification of catalase negative/weakCampylobacter jejunifrom human blood and faecal cultures by numerical analysis of electrophoretic protein patterns

Identification of catalase negative/weak<i>Campylobacter jejuni</i>from human blood and faecal cultures by numerical analysis of electrophoretic protein patterns

Identification of catalase negative/weak Campylobacter jejuni from human blood and faecal cultures by numerical analysis of electrophoretic protein patterns

Twenty-eight isolates of catalase-negative/weak (CNW) thermophilic campylobacters from human blood and faecal cultures were characterized by one-dimensional (1-D) high-resolution SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. Partial protein patterns were used as the basis for numerical analysis, which showed that all of the hippurate-positive strains had a high similarity to C. jejuni. Two subclusters were formed within C. jejuni corresponding to C. jejuni subsp. doylei (15 strains) and C. jejuni subsp. jejuni (4 strains). Most of the paediatric strains from South Africa were members of C. jejuni subsp. doylei. Hippurate-negative CNW thermophilic strains were identified as “ C. upsaliensis”. The analysis demonstrated that the catalase-negative C. jejuni strains were quite distinct from “ C. upsaliensis” and that electrophoretic protein patterns provide an excellent criterion for the identification of subspecies within C. jejuni.

Numerical analysis of electrophoretic protein patterns of ‘Achromobacter’group B, E and F strains from human blood

Thirty-two clinical strains representing 'Achromobacter' groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins. All the strains were isolated from blood samples from hospital patients in the United Kingdom. The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands. The 32 'Achromobacter' strains formed two clusters at the 77% S level. The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F. The strains in each phenon were characterized by a clearly distinct pattern of protein bands. Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD. Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria. Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies.

Numerical analysis of electrophoretic protein patterns of Providencia stuartii strains from urine, wound and other clinical sources

Eighty-six strains of Providencia stuartii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 52 were from urine, 11 from wounds, five from blood (one of these also from urine), four from ear infections, two each from faeces and sputum, one from 'alimentation' and nine from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 46 Prov. stuartii strains (selected to represent the full range of protein pattern diversity) plus those of the type strains of the four other Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the Prov. stuartii strains formed 13 clusters at the 88% S level. In the second analysis, in which the principal protein bands (in the 33.8-40.7 kDa range) were excluded, 45 of the 46 Prov. stuartii strains formed a single cluster at the 82% S level, whilst the four Providencia reference strains remained unclustered. The 40 strains of Prov. stuartii not included in the cluster analysis were assigned to a protein type by calculating their similarity with the strains in the database used for the cluster analysis. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. stuartii. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.

Numerical analysis of electrophoretic protein patterns of Group EF-4 bacteria, predominantly from dog-bite wounds of humans

Thirty-seven strains of Group EF-4 bacteria (from various countries) were characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 21 from dog-bite wounds of humans, three from cat-bite wounds of humans and five from human limb wounds which may have been inflicted by dogs or cats; there was also one each from a pet monkey, a tiger lung (fatal), a dog tonsil, a mouse, a cat liver, a wallaby mandible, a human vagina and one from a human limb wound which was apparently not inflicted by an animal. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible and were used as the basis for three numerical analyses. In the first, in which the principal protein bands (in the 34.8 to 41.3 kD range) were excluded, the 37 Group EF-4 strains formed, at the 62% S level, two major clusters corresponding to strains producing a dihydrolase for arginine and those not doing so. In the second analysis, which included all the protein bands and which was performed only on the 22 arginine-positive strains, two phenons formed (one of which could be further divided into two sub-phenons) at the 56% S level. The third analysis, also based on all the protein bands, divided the 15 arginine-negative strains into three clusters at the 56% S level. We conclude that high resolution PAGE combined with computerised analysis of protein patterns correlates exactly with the separation of Group EF-4 into two biovars (also with the distinction of the biovars on the basis of G + C content). Reference strains of each of the PAGE types identified are available from the NCTC for inclusion in future studies.

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens.

Thirty-one strains of Providencia rettgeri (mainly from humans) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries and comprised 14 from urine, eight from faeces, two from bile (plus one from the liver of a sheep), two from sputum, one from an insect pupa and three the sources of which were unknown. Also included, for reference purposes, were the type strains of the four other Providencia species. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 31 Prov. rettgeri strains formed 13 clusters at the 84% S level. In the second analysis, in which the principal protein bands (in the 33.3-41.3 kD range) were excluded, 29 of the 31 Prov. rettgeri strains formed a single cluster at the 81% S level, whilst the four Providencia reference strains remained unclustered. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. rettgeri. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.

Identification of 'Campylobacter upsaliensis' and other catalase-negative campylobacters from paediatric blood cultures by numerical analysis of electrophoretic protein patterns.

Twenty-one strains of catalase-negative campylobacters from paediatric blood cultures were characterized by one-dimensional SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. The partial protein patterns were used as the basis of a numerical analysis, which showed that 17 hippurate-negative strains had a high similarity to 'C. upsaliensis' (r greater than or equal to 0.82) irrespective of their geographical location, and that three hippurate-positive isolates had a high similarity (r greater than or equal to 0.87) to C. jejuni subsp. jejuni. One hippurate-positive CNW strain was not identified. The analysis of SDS-PAGE protein patterns proved an excellent method of characterizing these thermophilic campylobacter as they were difficult to identify by traditional methods.

Identification of ‘Campylobacter upsaliensis’ and other catalase-negative campylobacters from paediatric blood cultures by numerical analysis of electrophoretic protein patterns

Twenty-one strains of catalase-negative campylobacters from paediatric blood cultures were characterized by one-dimensional SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. The partial protein patterns were used as the basis of a numerical analysis, which showed that 17 hippurate-negative strains had a high similarity to 'C. upsaliensis' (r greater than or equal to 0.82) irrespective of their geographical location, and that three hippurate-positive isolates had a high similarity (r greater than or equal to 0.87) to C. jejuni subsp. jejuni. One hippurate-positive CNW strain was not identified. The analysis of SDS-PAGE protein patterns proved an excellent method of characterizing these thermophilic campylobacter as they were difficult to identify by traditional methods.

Numerical analysis of electrophoretic protein patterns of Campylobacter laridis and allied thermophilic campylobacters from the natural environment.

Twenty-one strains comprising Campylobacter laridis (nine), nalidixic acid sensitive campylobacters (NASC) (four), and urease-positive thermophilic campylobacters (UPTC) (eight) were characterized by one-dimensional SDS-PAGE of cellular proteins. The UPTC and NASC strains included six from river water, two from mussels and four from sea water. The type strains of three other Campylobacter species were included for reference. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 21 strains formed nine clusters at the 80% similarity (S) level. The typical C. laridis strains were restricted to two phenons (2 and 5); the atypical strains being distributed among the remaining phenons. In the second analysis, which excluded the principal protein bands (40-48.5 kD range), the 21 strains formed five clusters at the 80% S level. The typical C. laridis strains were relatively homogeneous and fell into a single phenon (2) within which two subgroups were discernable. The atypical strains were more heterogeneous with respect to background protein pattern, with representatives appearing in all five phenons. An electropherotyping scheme comprising six electropherotypes, and based on both analyses is proposed. The high within-group S level and separation from reference strains of Campylobacter in the second analysis, suggested that UPTC and NASC strains belonged within C. laridis possibly as biovars.

Numerical analysis of electrophoretic protein patterns of Bacteroides ureolyticus clinical isolates.

A total of 37 Bacteroides ureolyticus clinical isolates (mainly from the United Kingdom) comprising 18 nongonococcal urethritis-associated strains and 19 other strains, including the type strain (NCTC 10941T) and strains from abscesses, ulcers, and various other soft tissue lesions, and five reference strains of other Bacteroides, Fusobacterium, Megamonas, and Sebaldella species were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible and were used as the basis for a numerical analysis. Four clusters comprising 31 strains of B. ureolyticus were obtained at the 70% similarity level, and one of these was further divided to give two subclusters at the 82% similarity level. The analysis showed that the clusters obtained corresponded with three previously identified polyacrylamide gel electrophoresis types (I, II, and III) within the species, although two slightly different patterns within type I were detected. Evidence is presented for a new protein pattern (type IV) which was characteristic of NCTC 10941T and seven isolates. There was no clear-cut correlation between the pattern type and the clinical source of the isolates, except for the type Ib and type IV strains, which were nongonococcal urethritis-associated and wound isolates, respectively.

Classification of Campylobacter sputorum and allied campylobacters based on numerical analysis of electrophoretic protein patterns

A total of 28 strains of Campylobacter comprising representatives of C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, C. sputorum subsp. mucosalis, C. concisus, “C. fecalis”, “C. hyointestinalis” and the CNW Campylobacter group have been characterized by SDS-PAGE of cellular proteins. The protein patterns, which contained 40 to 50 discrete bands, were highly reproducible and were used as the basis of a numerical taxonomic analysis. Eight clusters were obtained at the 55% similarity level and each corresponded to a recognized species or group. The analysis demonstrated unequivocally that C. sputorum subsp. mucosalis constituted a distinct species although the serogroup A strains differed slightly from the serogroup B and C strains. Also, it was shown that C. sputorum subsp. bubulus and “C. fecalis” were closely related and should be classified in the same species. As that taxon contained catalase-positive and catalase-negative strains, over-reliance on that test could lead to incorrect identification. Each of the species clusters had distinctive protein band patterns and the main bands provide useful diagnostic markers for the rapid identification of human and animal isolates.

Fingerprinting Acinetobacter strains from clinical sources by numerical analysis of electrophoretic protein patterns.

A total of 57 strains of Acinetobacter calcoaceticus was fingerprinted by SDS-PAGE of cellular protein. All strains were also examined by conventional, API and N/F-Tek methods, and antibiotic sensitivity patterns were determined. In general, using the API 20E and N/F-Tek methods, it was possible to assign isolates of A. calcoaceticus to the two accepted "biotypes" or "varieties", A.c. anitratus and A.c. lwoffi, but these methods did not offer useful subdivision of the biotypes. Gel electrophoresis permitted subdivision of the strains into clusters in a manner which suggests that the technique may be valuable in typing strains isolated during outbreaks of infection in hospital.

Klebsiella trevisanii: a New Species from Water and Soil

We determined the taxonomic position of 17 strains of bacteria that were previously classified as “Klebsiella-like” strains, corresponding to group K of Gavini et al. [Ann. Microbiol. (Paris) 128B:45–59, 1977]. These strains were isolated from sewage, surface waters, drinking waters, and unpolluted soils. Deoxyribonucleic acid (DNA)-DNA hybridizations and a numerical analysis of electrophoretic protein patterns were used in this study. At least 82% of the group K strains investigated formed a tight protein electrophoretic cluster. The DNA homology values for all of the selected group K strains were higher than 73% and indicated the genetic homogeneity of this group. Two strains, CUETM 77-177 and CUETM 78-134, which were classified as group L strains by Gavini et al., were identified as true members of group K by the above-mentioned methods. The protein gel electrophoretic technique permitted distinction of group K strains from other species of the genus Klebsiella. DNA-DNA hybridization experiments revealed relatedness values of 40 to 71% between the reference strain of group K and the species Klebsiella mobilis, Klebsiella pneumoniae, Klebsiella oxytoca, and Klebsiella terrigena. Phenotypic characteristics, protein electrophoretic patterns, and the results of DNA-DNA hybridizations supported the individuality of group K, and we propose the name Klebsiella trevisanii sp. nov. for the strains of this group. These strains were positive in the Voges-Proskauer, urease, and β-galactosidase tests, grew at 4 and 41°, utilized histamine as a carbon source, did not ferment melezitose, and did not use m-hydroxybenzoate; indole was produced by 42% of the isolates. Strain CIP 81-36 (= CUETM 78-120) was designated the type strain of this species.

Classification of Corynebacterium species from axillary skin by numerical analysis of electrophoretic protein patterns.

The genus Corynebacterium, defined by the presence of mesodiaminopimelic acid, arabinose and corynomycolic acids in the cell wall, contains a very diverse group of organisms according to the results of numerical analysis of protein patterns; 13 groups were distinguished in this study. Strains isolated from axillary skin and hair included representatives of several groups but many strains were placed in groups 1 and 6A. Neither of these groups contained any reference strains and may constitute hitherto undescribed species. The reference strains of C. diphtheriae formed a coherent group not closely related to any non-pathogenic Corynebacterium species.

Klebsiella terrigena, a New Species from Soil and Water

Deoxyribonucleic acid (DNA)-DNA hybridization and numerical analysis of electrophoretic protein patterns were carried out to determine the taxonomic position of 37 “Klebsiella pneumoniae-like” strains from nonclinical origin (soil and water) and previously studied by numerical taxonomy (group L of Gavini et al., Ann. Microbiol. [Inst. Pasteur] 128B: 45-59, 1977). The DNA interrelatedness for 20 selected strains of group L was at least 87%, except for two strains (63 and 69%). Protein electrophoretograms indicated likewise that at least 85% of the L strains constitute a genetically homogeneous group. The latter technique enabled the distinction of strains of group L from those of K. pneumoniae, K. oxytoca, and an unnamed Klebsiella cluster (group K) related to K. pneumoniae and K. oxytoca. On the basis of their phenotypic characters, protein electrophoretic patterns, and DNA-DNA relatedness, we propose for these strains the name Klebsiella terrigena sp. nov., with strain CIP 80-07 (= CUETM 77-176) as the type strain. The DNA of this strain was found to be 49 to 64% homologous to K. oxytoca DNA, 48 to 63% to K. pneumoniae DNA, and 51% to K. ozaenae DNA.