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.     

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.     

Sequence diversity of intervening sequences (IVSs) in the 23S ribosomal RNA in Salmonella spp

Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.     

Characterization of enterobacteria by esterase specific-activity profiles

The spectrum of specific activities and the electrophoretic mobilities of esterases produced by 550 strains of Enterobacteriaceae belonging to 36 species and subclassified into six groups (group 1, Escherichia coli, Shigella and Escherichia hermanii; group 2, genus Salmonella and genus Citrobacter; group 3, genus Klebsiella and genus Enterobacter; group 4, genus Serratia and Serratia fonticola; group 5, genus Proteus, genus Providencia and genus Morganella; and group 6, genus Yersinia) were analysed by acrylamide/agarose gel electrophoresis using standardized methods for staining and mobility comparisons. Nineteen types of esterase were defined by their respective esterase specific-activity profile (ESAP). A multiple correspondence analysis (MCA) of the ESAP data enabled 82% of the strains in the 36 species to be correctly classified. In each group, the species were clearly delineated after MCA on both ESAP and electrophoretic mobility data. In addition, the smallest number of characters providing species identification of Yersinia strains by esterase polymorphism was identified by means of a binary segmentation tree technique.     

A novel glutamate-dependent acid resistance among strains belonging to the Proteeae tribe of Enterobacteriaceae

Morganella, Providencia and Proteus strains were capable of surviving pH 2.0 for 1 h if glutamate was present. These strains did not have glutamic acid decarboxylase activity and the gadAB genes were not detected in any of these bacteria. When exposed to pH 2.0 acid shocks, the survival rate of these bacteria was significantly increased with glutamate concentrations as low as 0.3 mM in the acid media. Escherichia coli cells incubated at pH 3.4 consumed four times more glutamate and produced at least 7-fold more gamma-amino butyric acid than Morganella, Providencia and Proteus strains. These results indicate that strains belonging to the Proteeae tribe might have novel glutamate dependent acid-resistance mechanisms.     

Fragmentation of 23S rRNA in strains of Proteus and Providencia results from intervening sequences in the rrn (rRNA) genes

Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.     

利用培养组学技术分离鉴定体外降解胆固醇肠道细菌及其功能评价

高胆固醇是诱发心脑血管疾病的重要因素之一。目前国内外降低胆固醇的主要方式是药物治疗,但其存在费用高及副作用多的弊端。研究表明,肠道细菌对机体的胆固醇代谢有重要影响,但降胆固醇肠道细菌的筛选方式及功能评价却少有报道。本研究通过培养组学方法,使用牛胆汁酸或人工混合胆汁酸作为筛选条件,从健康人体肠道筛选出36种耐胆汁酸细菌。以鼠李糖乳杆菌GG株 (Lactobacillus rhamnosus GG,LGG) 作为阳性对照,设置0 g/L、0.3 g/L、3 g/L三种胆汁酸浓度组,对耐胆汁酸细菌进行体外降胆固醇能力评估,确定奇异变形菌、斯氏普罗威登斯菌、普通变形菌等10种细菌为降胆固醇优势菌。随后对其中6种降胆固醇优势菌——奇异变形菌、斯氏普罗威登斯菌、普通变形菌、潘氏变形菌、污蝇解克杆菌、雷氏普罗威登斯菌进行体外降甘油三酯能力评估以及人工胃液耐受能力评估。结果显示,上述6株细菌体外降甘油三酯能力均优于LGG。伴随人工胃液pH值的下降和作用时间的延长,6株细菌的生存率均下降。上述筛选实验及功能评价为进一步开发潜在的降胆固醇细菌制品提供研究基础。     

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.     

Bacteriophage D: an IncD group plasmid-specific phage

The existence of the plasmid incompatibility group D was reaffirmed as a result of compatibility experiments done on plasmids R687, R711b, R778b and R840 which were previously tentatively accepted as constituting the group. The group was further delineated by the isolation of a phage, phage D, which adsorbed specifically to IncD plasmid-encoded pili produced by Escherichia coli K12 strains and strains of Salmonella typhimurium, Proteus morganii and Klebsiella oxytoca harbouring one of these plasmids. Plaque formation, like that of phage pilH alpha, was temperature sensitive in that plaques formed at 26 degrees C but not at 37 degrees C. Plaques were fairly clear, regular in outline and varied from pinpoint to about 1.5 mm in diameter on E. coli hosts where plaques were detected, but on the other hosts the plaques were more turbid and often irregular in outline. The phage did not plate (or propagate) on IncD plasmid-carrying strains of Providencia alcalifaciens, Providencia stuartii or Serratia marcescens. The phage had an isometric hexagonal outline with a diameter of about 27 nm. It contained RNA and resembled two other RNA-containing phages, M and pilH alpha, by being sensitive to chloroform. It adsorbed to the sides of the very distal ends of the shafts of IncD plasmid-coded pili.     

Intervening Sequences in rrl Genes and Fragmentation of 23S rRNA in Genera of the Family Enterobacteriaceae

Intervening sequences (IVSs) in the rrl genes for 23S rRNA are transcribed but later removed by RNase III without religation during RNA processing, leading to fragmented rRNA. We examined about 240 strains of the family Enterobacteriaceae for presence of IVSs using PCR. No IVSs were detected in strains belonging to Escherichia, Shigella, Enterobacter, Erwinia, Ewingella, Hafnia, Kluyvera, Morganella, Pantoea, or Serratia. Previously unreported IVSs were detected in Klebsiella oxytoca, Citrobacter amalonaticus, and Providencia stuartii; previously reported IVSs are in species of Salmonella, Proteus, Providencia, and Yersinia. The sporadic distribution of IVSs indicates lateral genetic transfer of IVSs.     

The occurrence of the trehalose fermenting, tetracycline and polymyxin resistant phenotype among the Enterobacteriaceae

In Proteus morganii, P. mirabilis, and Providencia stuartii the ability to ferment trehalose and resistance to tetracycline were associated in 90%-97% of the strains. The same was true of at least 78% of the strains of Serratia marcescens. Proteus vulgaris showed a more quantitative association of the two traits. As the characters occur independently in 3-10% of the strains, the association is considered to be due to simultaneous selection in some natural niche. The trehalose fermenting, tetracycline and polymyxin resistant species ferment few other carbohydrates, fewer than the remainder of the Serratia species.     

Chlorhexidine resistance and the lipids of Providencia stuartii

The lipid composition of Providencia stuartii has been shown to resemble closely that of Proteus mirabilis. The ability of some Pv, stuartii strains to survive exposure to high concentrations of the antiseptic chlorhexidine could not be explained in terms of differences in lipid content between sensitive and resistant strains. In addition, resistance could not be attributed to either reduced adsorption of the antiseptic or to its gross enzymic degradation.     

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.     

Phage X: a plasmid-dependent, broad host range, filamentous bacterial virus

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R- strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.     

Pyrazinamidase activity of bacteria from Enterobacteriaceae family as characteristic for taxonomy

Pyrazinamidase activity in 330 strains of bacteria from Enterobacteriaceae family (14 genus, 27 species) has been assessed. Pyrazinamidase activity detected in species from following genuses: Citrobacter, Escherichia, Klebsiella, Kluyvera, Morganella, Providencia, Raourtella, Salmonella, Shigella, and also in Proteus mirabilis, and nonpathogenic serovars of Yersinia enterocolitica, Y. frederiksenii. Pirasinamidase was absent in Serratia (S. marcescens, S. liguefaciens), Hafnia alvei, P. vulgaris, P. penneri, Y. pseudotuberculosis and pathogenic serovars of Y. enterocolitica. Absence of pyrazinamidase activity in bacteria from Hafnia and Serratia genus is a key taxonomic characteristic for identification of enterobacteria with microvolume assay technology.     

A review of Providencia bacteremia in a general hospital,with a comment on patterns of antimicrobial sensitivity and use.

Six cases of Providencia bacteremia occurring between 1969 and 1978 were reviewed. These cases represented 3% of the gram-negative bacteremias occurring at one hospital. All six cases of Providencia bacteremia were secondary to urinary tract infection with P. stuartii, and in most the infection developed only shortly before the onset of bacteremia; in three cases the bacteremia developed immediately following manipulation of the urinary tract. Patients with long-standing Providencia infections did not acquire bacteremia. The signs and symptoms of Providencia bacteremia were typical of those of septicemia except that vascular collapse was not a prominent feature, occurring in only one patient. The mortality was 33%. All the Providencia strains cultured from the bloodstream were susceptible to gentamicin, although the frequency of gentamicin resistance increased from roughly 10% to 50% during the period studied; the increase in gentamicin use over this period was more gradual. Also noted was a decrease in resistance to ampicillin that paralleled a decrease in ampicillin use. All the Providencia strains were susceptible to amikacin.     

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.     

Release of surface enzymes in Enterobacteriaceae by osmotic shock

The process of osmotic shock, which has been used to release degradative enzymes from Escherichia coli, can be applied successfully to other members of the Enterobacteriaceae. Cyclic phosphodiesterase (3'-nucleotidase), 5'-nucleotidase (diphosphate sugar hydrolase), acid hexose phosphatase, and acid phenyl phosphatase are released from Shigella, Enterobacter, Citrobacter, and Serratia strains. Some strains of Salmonella also release these enzymes. Members of Proteus and Providencia groups fail to release enzymes when subjected to osmotic shock and do not show a lag in regrowth, although they do release their acid-soluble nucleotide pools. In contrast to E. coli, release of enzymes from other members of the Enterobacteriaceae studied is affected by growth conditions and strain of organism. None of the organisms was as stable to osmotic shock in exponential phase of growth as was E. coli. Exponential-phase cells of Shigella, Enterobacter, and Citrobacter could be shocked only with 0.5 mm MgCl(2) to prevent irreparable damage to the cells. These observations suggest that this group of degradative enzymes is probably loosely bound to the cytoplasmic membrane through the mediation of divalent cations.     

Extent of peptidoglycan O acetylation in the tribe Proteeae.

The degree of peptidoglycan O acetylation in 18 strains of the different genera of the tribe Proteeae (Proteus, Providencia, and Morganella) has been determined by high-performance liquid chromatography-based organic acid analysis of mild-base-released acetic acid and quantitation of peptidoglycan concentrations by simultaneous amino sugar-amino acid analysis using high-performance anion-exchange chromatography with pulsed amperometric detection. The N,O-diacetylmuramyl content of all isolated and purified peptidoglycans was greater than 29% and ranged up to 57% relative to total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by hen egg white lysozyme.