Purification and some properties of beta-lactamases from Proteus rettgeri and Proteus inconstans

Two beta-lactamases were isolated from strains of Proteus species and purified, one from a strain of P. rettgeri and the other from a strain of P. inconstans. Each enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. Molecular weights of P. rettgeri and P. inconstans enzymes were found to be 42,000 and 43,000, and their isoelectric points pH 8.7 and 8.6, respectively. The two enzymes presented typical cephalosporinase profiles. Cefmetazole (CS-1170) and cefoxitin, both cephamycin antibiotics, not only resisted hydrolysis by both of the enzymes, but also inhibited their activities competitively. Rabbit antiserum against purified P. rettgeri enzyme inhibited the activity of both purified and crude enzyme preparations from other strains of P. rettgeri so far tested. None of the beta-lactamases produced by other species of Proteus including P. inconstans was inhibited by the antiserum, thus showing that the purified cephalosporinase was of the species-specific types. The enzymological properties of the preparations were compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

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.     

Antibiotic sensitivity of Proteus, Pseudomonas pyocyanea and staphyloccoci isolated from scleroma and ozena patients]

Antibiotic sensitivity of 292 strains of Proteus, 60 strains of Ps, aeruginosa, 309 strains of S. aureus and 88 strains of S. epidermidis isolated from the upper respiratory tract of patients with scleroma and ozena was studied. The cultures of Pr. mirabilis were sensitive to aminoglucosides (54.9-96.2 per cent) and Pr. morganii were sensitive to levomycetin (81.5 per cent) and neomycin (92.6 per cnet). Sensitivity of Pr. vulgaris and Pr. morganii was reliably higher (p less than 0.001) than that of Pr. mirabilis. The strains of Pr. morganii were less sensitive to monomycin (P less than 0.001) and streptomycin (p less than 0.01) as compared to the cultures of other Proteus species tested. The strains of Ps. aeruginosa were sensitive only to gentamicin (90 per cent) and neomycin (81.1 per cent). Most of the strains of S. aureus (85.4-100 per cent) were sensitive to oleadomycin, erythromycin, olemorphocycline, tetraolean, oxacillin, methicillin ceporin, lincomycin, ristomycin, kanamycin, monomycin and gentamicin. Benzylpenicillin (90.8 per cent of the sensitive strains), ampicillin (67.1 per cent), tetracycline (66.7 per cent), levomycetin (68.6 per cent) and streptomycin (38.1 per cent) were less effective. Antibacterial therapy in cases with scleroma and ozena should be directed not only against causative agents of the diseases but also against the microbes developing due to disbacteriosis. Combination of parenteral and local use of the antibiotics in the treatment of chronic clebsiellesis decreased the isolation rate of Proteus and Ps. aeruginosa in the patients.     

Selected properties of lactose-fermenting and non-fermenting Salmonella agona strains isolated from specimens from hospitalized infants

The aim of this study was to compare some of the properties of 28 lactose-positive and 28 lactose-negative Salmonella agona strains isolated from faeces of infants hospitalized in the same hospital. Some of biochemical properties, sensitivity to 14 antibiotics and chemotherapeutic agents and sensitivity to bacteriophages used for typing of this Salmonella genus were tested. Results of biochemical examinations revealed that lactose-fermenting strains retain the remaining of Salmonella of subspecies I. Two biochemical features are of particular importance: the ability to ferment lactose on all lactose containing media and a lack of the ability to produce H2S on Kligler medium. These two features differentiate lactose-fermenting strains of Salmonella from non-lactose fermenting ones. Antibiotic sensitivity pattern differed between lactose-positive and lactose-negative strains. Lactose-positive strains showed higher degree of resistance than lactose-negative strains. The differences in resistance were seen in the case of chloramphenicol, doxycycline, gentamicin and tetracycline. Both lactose-positive and lactose-negative strains were sensitive to colistin, neomycin, nitrofurantoin and nalidixic acid. They were resistant to ampicillin, cloxacillin, rifampicin, streptomycin, sulfatiazol and biseptol. Bacteriophage typing revealed that all lactose-negative strains isolated in this study from clinical samples belonged to the same phage pattern V. Lactose-positive strains belonged to two phage types VB and XI. Type VB prevailed.     

Bacteriophage Typing of Proteus mirabilis, Proteus vulgaris, and Proteus morganii

A bacteriphage typing scheme for differentiating Proteus isolated from clinical specimens was developed. Twenty-one distinct patterns of lysis were seen when 15 bacteriophages isolated on 8 Proteus mirabilis, 1 P. vulgaris, and 1 P. morganii were used to type 162 of 189 (85.7%) P. mirabilis and P. vulgaris isolates. Seven phages isolated on 3 P. morganii were used to type 13 of 19 (68.4%) P. morganii isolates. Overall, 84.1% of the 208 isolates were lysed by at least 1 phage at routine test dilution (RTD) or 1,000 x RTD. Fifty isolates, retyped several weeks after the initial testing, showed no changes in lytic patterns. The phages retained their titers after storage at 4 C for several months. A computer analysis of the data showed that there was no relationship between the source of the isolate and bacteriophage type. This bacteriophage typing system may provide epidemiological information on strains involved in human infections.     

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.     

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli.

Secreted hemolysins were extremely common among clinical isolates of Proteus mirabilis, Proteus vulgaris, and Morganella morganii, and hemolytic activity was either cell associated or cell free. Southern hybridization of total DNA from hemolytic isolates to cloned regions of the Escherichia coli alpha-hemolysin (hly) determinant showed clear but incomplete homology between genes encoding production of hemolysins in the four species. One of the two E. coli secretion genes, hlyD, hybridized only with DNA from P. vulgaris and M. morganii, which produced cell-free hemolysis, but not with that from P. mirabilis, which showed only cell-associated activity. Molecular cloning of the genetic determinants of cell-free hemolytic activity from P. vulgaris and M. morganii chromosomal DNA allowed their functional analysis via inactivation with the transposons Tn1000 and Tn5. Both hemolysin determinants were about 7.5 kilobase pairs and comprised contiguous regions directing regulation, synthesis, and specific secretion out of the cell. Transposon mutations which eliminated secretion of the Proteus and Morganella hemolysins could be complemented specifically by the E. coli hemolysin secretion genes hlyB or hlyD. Alignment of the physically and functionally defined hly determinants from P. vulgaris and M. morganii with that of the E. coli alpha-hemolysin confirmed a close genetic relationship but also indicated extensive evolutionary divergence.     

Penicillin-binding proteins in Proteus species.

Penicillin-binding proteins in three species of Proteus, Proteus mirabilis, P. morganii, and P. rettgeri, were investigated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Penicillin-binding proteins in these Proteus species were compared with those in Escherichia coli K-12. An approximate correlation between penicillin-binding proteins in E. coli and those in Proteus species was shown by several criteria: electrophoretic mobilities; affinities of several beta-lactam antibiotics which show characteristic patterns of binding to penicillin-binding proteins in E. coli; relation between affinities of antibiotics to the proteins and effects on morphological changes in Proteus species; location of beta-lactamase activity among penicillin-binding proteins; and thermostability. The electrophoretic mobilities and several other characteristics of penicillin-binding proteins among the Proteus species examined were found to be similar from species to species and differed only slightly from those of E. coli.     

Comparative activity of tobramycin and gentamicin against Pseudomonas,Proteus and Providencia species.

Tobramycin is a new antibiotic resembling gentamicin. We measured the minimal inhibitory concentrations of these two antibiotics against five bacterial species that cause hospital-acquired infections and are resistant to many presently available antibiotics. The organisms tested were 500 strains of Pseudomones aeruginosa, 100 strains of each of Proteus rettgeri and Pr. morganii, 50 strains of Pr. vulgaris and 250 strains of Providencia stuartii. Tobramycin was 2 to 4 times more active than gentamicin against Ps. aeruginosa; all except 6 of 70 strains resistant to 4 mug/ml of gentamicin were sensitive to 4 mug/ml of tobramycin. The two antibiotics showed a similar degree of activity against the other four species. Tobramycin promises to be of particular value in the treatment of Pseudomonas infections.     

Characterization of Indole-Positive Proteus mirabilis

Thirteen indole-producing, swarming strains of Proteus were identified by additional biochemical testing as being Proteus mirabilis. These strains were characterized by 40 biochemical tests and by susceptibility testing to 11 antibiotics. All produced ornithine decarboxylase and were susceptible to members of the penicillin-cephalosporin groups of antibiotics. These indole-positive strains are similar to indole-negative P. mirabilis and are distinctly different from P. vulgaris. For greatest accuracy and to insure greatest clinical relevancy, P. mirabilis and P. vulgaris should be distinguished from one another in the laboratory by performing both the indole and ornithine decarboxylase tests.     

Synergistic Antibacterial Activity of Ampicillin-Cloxacillin Mixtures Against Proteus morganii

Synergistic antibacterial effects of mixtures of ampicillin and cloxacillin and induced penicillinases were investigated in 48 strains of Proteus. The serial tube dilution method was used to determine the minimal inhibitory concentration of ampicillin, cloxacillin, and 2:1 and 1:1 mixtures of ampicillin and cloxacillin. Production of penicillinases was determined by the cellulose acetate membrane method, with ampicillin, cloxacillin, mixtures of ampicillin and cloxacillin, penicillin G, and cephalothin as inducing agents and as substrates for penicillinase. Synergism occurred against P. morganii, but against no other species. The 1:1 ampicillin-cloxacillin combination was synergistic against 13 of 17 P. morganii strains; the 2:1 combination was synergistic against only 9 strains. Penicillinases, demonstrated in all species except P. mirabilis, hydrolyzed penicillin G and cephalothin. Although only P. vulgaris hydrolyzed ampicillin, no species of Proteus hydrolyzed cloxacillin or the ampicillin-cloxacillin mixtures. Penicillinases were, however, induced by ampicillin, cloxacillin, and the mixtures. There was no relationship between production of penicillinase and synergism with mixtures of ampicillin and cloxacillin.     

Diagnostic value of a novel nutrient medium for isolation and cultivation of pathogens causing enteric yersiniosis and pseudotuberculosis

A new nutrient medium for isolation and cultivation of the causative agents of enteric yersiniosis and pseudotuberculosis was found to have advantages over Endo medium in its differentiating and inhibiting properties. This medium permitted the easy differentiation of Yersinia pseudotuberculosis from Y. enterocolitica, as well as from Escherichia coli, Shigella flexneri, Klebsiella pneumoniae, K. rhinoscleromatis, Hafnia, Enterobacter and Citrobacter by color; from Proteus inconstans by swarming. In addition, weakly swarming of P. vulgaris differed by their light bluish color and Pseudomonas aeruginosa, by the brilliance and size of colonies. Endo medium could be used only for differentiation of E. coli from lactose-negative Yersinia colonies, Klebsiella (by mucous growth) and, to a certain extent, all Proteus species (by swarming). The medium under test and the control medium inhibited the growth of Staphylococcus aureus. In contrast to Endo medium, the medium under test partially inhibited the growth of K. rhinoscleromatis and the swarming of P. inconstans. The new medium is now introduced into practice.     

Colistin-induced surface changes in the cells of Proteus spp.

The effects of colistin on some strains of Proteus mirabilis, P. morganii and P. vulgaris are described. Two strains of P. mirabilis , NCTC 60 and NCTC 4199, took up greater amounts of colistin from solution than did the other strains. Pretreatment of strains 60 and 4199 resulted in osmotic instability and in increased susceptibility to Tris and deoxycholate. Colistin-induced lysis in Tris could be overcome by means of 0–16 mol/l sodium chloride or 0–33 mol/l sucrose. Pre-treated, but not control, cells of these two strains exposed to Tris (0–05 mol/l, pH 9) developed surface protuberances (blebs).     

Some biological features of Proteus bacilli. 2. Haemolytic activities of Proteus mirabilis and Proteus vulgaris strains

The haemolytic activities of Proteus mirabilis and P. vulgaris strains were studied under different conditions. No filterable alpha haemolysin could be detected in P. mirabilis uropathogens provided from patients with urinary tract infections. Together with the results presented in the accompanying paper, in which three clinical isolates with temporary ability to produce a soluble haemolysin were described, the occurrence of alpha haemolytic P. mirabilis isolates did not exceed 3%. Cell bound beta haemolysin is present in nearly 35% of P. mirabilis urinary strains. Another kind of haemolytic activity was observed when P. mirabilis and P. vulgaris strains were grown in liquid media supplemented with erythrocytes. During the logarithmic growth phase nearly 100% of P. mirabilis and P. vulgaris strains of various origin haemolyzed 100-50% of erythrocytes. Except for Serratia, the other representatives of Enterobacteriaceae did not demonstrate such activity in the same conditions. The preliminary characteristics of this phenomenon is given.     

Proteus virulence: involvement of the pore forming alpha-hemolysin (a short review)

The genus Proteus belongs to the tribe of Proteae in the family of Enterobacteriaceae, and consists of five species: P. mirabilis, P. vulgaris, P. morganii, P. penneri and P. myxofaciens. They are distinguished from the rest of Enterobacteriaceae by their ability to deaminate phenylalanine and tryptophane. They hydrolyze urea and gelatin and fail to ferment lactose, mannose, dulcitol and malonate; and do not form lysine and arginine decarboxylase or beta-galactosidase [1]. Colonies produce distinct "burned chocolate" odor and frequently show the characteristics of swarming motility on solid media. P. mirabilis, P. vulgaris and P. morganii are widely recognized human pathogens. They have been isolated from urinary tract infections, wounds, ear, and nosocomial bacteremic infections, often in immuncompromised patients [2-6]. P. myxofaciens has no clinical interest to this time. P. penneri as species nova was nominated by the recommendation of Hickman and co-workers [7]. Formerly it was recognized as P. vulgaris biogroup 1 or indole negative P. vulgaris [8, 9]. Although it has been less commonly isolated from clinical samples than the other three human pathogenic Proteus species, it has nevertheless been connected with infections of the urinary tract, wounds and has been isolated from the feces of both healthy and diarrheic individuals [10-12]. Potential virulence factors responsible for virulence of Proteae are: IgA protease, urease, type3 fimbriae associated with MR/K haemagglutinins of at least two antigenic types, endotoxin, swarming motility and HlyA and/or HpmA type hemolysins [for review see ref. 13]. In the followings we give a survey of accumulated concepts about the position and characteristics of HlyA type alpha-hemolysins both in general and with emphasis on virulence functions in the tribe of Proteae.     

Effect of polymyxins on the lipopolysaccharide-defective mutants of Proteus mirabilis

The effects of polymyxins (Pmx) B and E on smooth and rough Proteus mirabilis strains were investigated. P. mirabilis mutant R4/028 which completely lacked 4-amino-4-deoxy-L-arabinose was sensitive towards both polymyxins, and the other P. mirabilis strains investigated were resistant. Lipopolysaccharide (LPS) from Pmx-sensitive R4/028 strain, binds 50% more Pmx B than LPS derived from resistant P. mirabilis strains. The presence of iodoacetamide, N-ethylmaleimide and chloramphenicol rendered the Pmx-resistant P. mirabilis strains sensitive towards both polymyxins.     

Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.     

Antibiotic sensitivity of the H- and O-forms of bacteria in the genus Proteus]

Proteus strains isolated from the gastro-intestinal tract of children not older than 1 year were characterized by resistance to oxytetracycline, chlortetracycline, benzylpenicillin and erythromycin. The strains were more sensitive to neomycin, monomycin and streptomycin. Antibiotic sensitivity of Pr. mirabilis and Pr. vulgaris strains increased on transfer from H- to O-form. Inverse dependence of the urease activity of the strains on their sensitivity to tetracyclines was noted.     

Nonspecific Toxicities in the Mouse Assay Test for Botulinum Toxin

In inoculated pack experiments on Clostridium botulinum type E, unirradiated and 0.1-Mrad irradiated haddock fillets often gave nonspecific toxicities by the mouse assay test for botulinum toxin. Samples given 0.2-Mrad radiation failed to produce nonspecific reactions. Nonspecific deaths sometimes occurred within 24 hr after injection, although deaths between 24 and 48 hr were more common. The symptoms and the pattern of these deaths suggested a septicemia. Heart-blood cultured from mice showing nonspecific symptoms indicated an infectious process. Among 23 isolates from the blood, eight were identified as Proteus vulgaris, two P. morganii, one P. rettgeri, one Providence subgroup B, two Aerobacter aerogenes, one Actinobacillus, three enterococci, one Alcaligenes marshalli, and four Erysipelothrix insidiosa. The E. insidiosa, Aerobacter, Providence group, and most of the Proteus isolates were infectious for mice when injected by the intraperitoneal route. But the enterococci, Alcaligenes, and Actinobacillus isolates were not infectious and probably represent secondary invaders. The cultural characteristics of the E. insidiosa isolates conform to those described in the literature, with the exception that the four strains grew in the temperature range 50 F (10 C) to 40 F (4.4 C). Nonspecific toxicities were avoided in assays for botulinum toxin by the protection of mice with chloramphenicol and oxytetracycline.     

Intestinal enterobacteria of the hibernating Apis mellifera mellifera L. bees

Dynamics of enterobacteria of normal intestinal microflora was studied in Apis mellifera mellifera L. bees hibernating under snow in the Western Urals. The cell numbers (N) of the predominant species Klebsiella oxytoca increased from 10-10(6) CFU/bee in November 2004 to 10(4)-10(7) CFU/bee in March 2005; its frequency of occurrence (P) increased from 92 to 100%. Increase of Providencia rettgeri (11.2004: N up to 10(6), P 25%; 03.2005: N 10(2)-10(6), P 80%) was accompanied by the substitution of Morganella morganii (11.2004: N up to 10(6), P 25%) with Proteus vulgaris (03.2005: N up to 10(5), P 8%). By spring, Hafnia alvei and Citrobacter sp., which are pathogenic to bees, disappeared (11.2004: N up to 10(5), P 13 and 10%, respectively). Endophytic species Pantoea agglomerans, Leclecria sp., and other representatives of the "Enterobacter agglomerans" group were present in November and after the first emergence in spring (N up to 10(5); November: P 15%; April: P 23%). In April, the number of enterobacteria decreased to 10(5), and P. rettgeri became the predominant species (P 54%) instead of K. oxytoca (P 43%).