Numerical analysis of electrophoretic protein patterns of Providencia rustigianii strains from human diarrhoea and other sources

Twenty strains of Providencia rustigianii (including the type strain of Prov. friedericiana ) have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 12 strains (almost exclusively associated with the intestinal tract) from humans, plus eight largely from the intestinal tract of pig, penguin and environmental sources. 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 20 Prov. rustigianii strains formed six clusters at the 88% S level. One of these clusters included the type strains of both Prov. friedericiana and Prov. rustigianii , thereby confirming the synonymy of these two species. In the second analysis, the principal protein bands were excluded. At the 86% S level the 20 Prov. rustigianii strains formed a single cluster, whilst a field strain of Morganella morganii and the respective type strains of three other Providencia species remained unclustered. The total protein pattern of the type strain of Prov. alcalifaciens was very similar to that of Prov. rustigianii phenon 3 and the M. morganii field strain, which indicates that careful biochemical characterization may be necessary to ascribe strains to a species before typing by the PAGE technique. Alternatively, a selective analysis of the protein bands may be used to confirm the identity of the strains, as shown in this study.     

Numerical analysis of electrophoretic protein patterns of Providencia alcalifaciens strains from human faeces and veterinary specimens

Twenty-five strains of Providencia alcalifaciens from various countries have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 15 from human faeces, one from duck faeces, one from a guinea-pig eye and eight from unknown sources. Also included, for reference purposes, were the type strains of three 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, in which the principal protein bands (in the 33-40 kD range) were excluded, the 25 Prov. alcalifaciens strains formed, at the 83% S level, a single cluster whilst the three Providencia reference strains remained unclustered. In the second, which included all the protein bands, the 25 Prov. alcalifaciens strains formed 10 clusters at the 85% S level. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. alcalifaciens. Reference strains of each of the 10 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia alcalifaciens strains from human faeces and veterinary specimens

Twenty-five strains of Providencia alcalifaciens from various countries have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 15 from human faeces, one from duck faeces, one from a guinea-pig eye and eight from unknown sources. Also included, for reference purposes, were the type strains of three 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, in which the principal protein bands (in the 33–40 kD range) were excluded, the 25 Prov. alcalifaciens strains formed, at the 83% S level, a single cluster whilst the three Providencia reference strains remained unclustered. In the second, which included all the protein bands, the 25 Prov. alcalifaciens strains formed 10 clusters at the 85% S level. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov, alcalifaciens . Reference strains of each of the 10 PAGE types identified are available from NCTC for inclusion in future studies.     

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.     

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 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.     

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.     

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.     

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

B. HOLMES, M. COSTAS AND A.C. WOOD. 1991. 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.     

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.     

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.     

A method for rapid extraction of Providencia chromosomal DNA and restriction enzyme digestion in agarose pellets

A simple and fast (2–3 d) method is described for the extraction and restriction endonuclease digestion of bacterial chromosomal DNA in low melting point agarose pellets. The technique minimized the random mechanical shearing of DNA caused by conventional preparative methods and improved the resolution of electrophoretic band patterns, particularly for the smaller (< 10 kb) fragments. The method was tested on 14 strains of Providencia stuartii and Prov. rustigianii .     

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 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.     

A Numerical Taxonomic Study of Proteus-Providence Bacteria

One hundred and six strains from the Proteus-Providence group and 27 other strains from the rest of the Enterobacteriaceae were subjected to 178 morphological. physiological and biochemical tests and the results analysed by computer. Most of the Proteus-Providence strains grouped into six main clusters; (1) Proteus mirabilis , (2) Proteus vulgaris , (3) Proteus morganii , (4) Providencia alcalifaciens, Shigella dysenteriae , (5) Proteus rettgeri , (6) Providencia stuartii . On the basis of these groupings a scheme has been drawn up for distinguishing between the different taxa in the Proteus-Providence group.     

Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.     

Inducible xylitol dehydrogenases in enteric bacteria.

Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp. strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium. The xylitol dehydrogenases were partially purified from the four strains, and those from M. morganii ATCC 25829, P. stuartii ATCC 25827, and S. marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose. These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecular weights ranging from 130,000 to 155,000. In contrast, the xylitol dehydrogenase from Erwinia sp. strain 4D2P oxidized xylitol at the C-4 position to produce L-xylulose, had a Km for xylitol of 72 mM, and had a molecular weight of 102,000.     

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.