Adherence of Ureaplasma urealyticum to human epithelial cells.

Adherence of Ureaplasma urealyticum cells to eukaryotic cell monolayers was quantified using the Bertholet assay to monitor ammonia produced from urea by ureaplasma urease. Adherence was abolished by pre-treatment of ureaplasmas with HeLa cell extracts and inhibited to varying degrees by pretreatment of the ureaplasmas with N-acetylneuraminic acid, specific antisera and monoclonal antibodies. The data suggest the presence of several ureaplasma adhesins, some of which are species- or serotype-specific and some of which are proteinaceous and antigenic. The serotype-8-specific 96 kDa surface-expressed antigen may be one adhesin. Pre-treatment of HeLa cell monolayers with neuraminidase significantly reduced ureaplasma adherence and, using a novel 'immunoblot adherence assay', ureaplasmas were shown to bind to a number of HeLa cell components, three of which appear to terminate in sialic acid.

Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis.

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.

Urea-hydrolysis-dependent citrulline synthesis by Ureaplasma urealyticum.

Some of the ammonia produced by hydrolysis of urea by Ureaplasma urealyticum is channelled into an anabolic pathway with resultant 'de novo' synthesis of citrulline. The organism appears to possess ornithine carbamoyltransferase and carbamoyl phosphate synthetase or some modified form of these enzymes.

Characterization of the immunoglobulin A protease of Ureaplasma urealyticum.

Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1.

Isolation and detection of urease genes in Ureaplasma urealyticum.

Urease from ureaplasmas was purified by immunoaffinity chromatography, and the N-terminal amino acid sequence was determined for two of the three subunits. These sequences were used to design primers for a polymerase chain reaction (PCR) that amplified most of the gene coding for one of the subunits. By using a novel "PCR walking" technique, we synthesized almost the complete locus on two overlapping PCR products. We present here a partial nucleotide sequence of the urease locus from Ureaplasma urealyticum (serotype 8), which agrees with our N-terminal amino acid data but differs slightly from the sequence previously reported (A. Blanchard, Mol. Microbiol. 4:669-676, 1990). Also described are PCR primers, intended for diagnostic use, that amplify a sequence from all Ureaplasma strains tested but not from any other mycoplasmas or urease-positive bacteria.

Palmitoylated proteins in Ureaplasma urealyticum.

After incubation of Ureaplasma urealyticum serotype 8 in the presence of 3H-labeled palmitic acid, about 25 acylated proteins were detected by electrophoresis and fluorography. Of these, at least six were shown to be antigenic by immunoprecipitation of solubilized palmitate-labeled cells with a homologous polyclonal serum. These six included the serotype 8-specific surface-expressed 96-kDa antigen. After phase partition of palmitate-labeled cells with Triton X-114, all but six acylated proteins partitioned entirely into the detergent phase. The others, including the 96-kDa antigen, partitioned preferentially into the detergent phase and were apparently amphipathic. These results are consistent with the acylated proteins being mainly membrane associated.

Cross-reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal origin, shown by ELISA and immunoblotting with reference antisera.

Using enzyme-linked immunosorbent assays with Mycoplasma ovipneumoniae as antigen, the cross-reactivity of antigens between this species and 22 other mycoplasma species was examined using reference polyclonal antisera. Significant cross-reactivity with M. ovipneumoniae was demonstrated by five species, only, viz. M. bovoculi, M. dispar, M. flocculare, M. hyopneumoniae and M. hyorhinis. Using one-dimensional SDS-PAGE and immunoblotting techniques with homologous and heterologous antisera, cross-reacting antigens of M. dispar, M. flocculare, M. hyopneumoniae and M. ovipneumoniae were further investigated. Cross-reacting antigens with apparent molecular weights of 64, 44 and 32 kDa were common to all and a 184 kDa cross-reacting antigen occurred in all except M. ovipneumoniae. Further cross-reacting antigens (one-way and two-way) between two of the four species are reported. Four monoclonal antibodies against different antigens of M. ovipneumoniae did not recognise any antigen in the other three species examined.

The humoral immune response of lambs experimentally infected with Mycoplasma ovipneumoniae.

Using sera from lambs experimentally infected with Mycoplasma ovipneumoniae and Pasteurella haemolytica, the development of a good humoral immune response to M. ovipneumoniae was detected by ELISA. The antibody titres peaked 41 days post-infection and good antibody titres were maintained over the 16-week experimental period. Immunoblotting revealed that antibodies to specific antigens appeared in the sera in a sequential manner, some being seen shortly after infection and others developing only after a substantial time lag. Antibodies were raised against almost all the major antigens detected in one laboratory strain (956/2) and against all antigens previously shown to be conserved in 22 Scottish field isolates of M. ovipneumoniae.

A comparison of four major antigens in five human and several animal strains of ureaplasmas.

A comparison of the antigens of single representatives of five serotypes of Ureaplasma urealyticum, of three strains of U. diversum and of single ureaplasmal strains from four other animal hosts was performed by immunoblotting with monoclonal antibodies and a urease 'enzyme-catch test'. The U. urealyticum serotype 8-specific, surface-expressed, 96-Kda antigen was not found in any of the strains of non-human origin. Differences in the distribution of 16- and 17-Kda antigens were also seen, not only between seroclusters A and B of U. urealyticum, but also with respect to animal strains. Five distinct epitopes were expressed on the urease from U. urealyticum and from chimpanzee ureaplasmal strains, but between one and three of these epitopes were either poorly expressed or not detected on the urease from the other animal strains. Apart from lacking the 96-Kda antigen of U. urealyticum serotype 8, chimpanzee strains gave results similar to those obtained with serocluster A of U. urealyticum. The results with the marmoset strain differed from those of all other non-human strains.

Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and immunoblotting.

Comparison of the polypeptide patterns of 22 isolates of M. ovipneumoniae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a marked degree of heterogeneity with only limited groupings identifiable. Of the 50 major polypeptides identified in one strain (956/2), 35 were shown to be antigenic using immunoblotting with a homologous polyclonal serum. Radioimmune precipitation of 125I-surface-labelled proteins and phase partition using Triton X-114 detergent indicated that these were membrane associated. Cross-reactivity between the isolates was examined by immunoblotting using one polyclonal serum and four monoclonal antibodies (MAbs), all raised against strain 956/2. The polyclonal serum revealed considerable antigenic heterogeneity, but at least nine major antigens were conserved across all isolates. Two MAbs cross-reacted with all 22 strains, but the other two MAbs allowed some differentiation of the strains. One (MO/3) divided the isolates into groups of 16 and 6 based on the presence of absence of a 26-kDa antigen. All strains isolated from sheep with pulmonary adenomatosis fell into the smaller group and did not possess the 26-kDa antigen.

Serotype 8- and serocluster-specific surface-expressed antigens of Ureaplasma urealyticum.

The polypeptides of all 14 serotypes of Ureaplasma urealyticum were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. The electrophoretic patterns did not allow ready discrimination of individual serotypes or seroclusters. The analysis of the antigens of serotype 8 was reported previously (B. L. Precious, D. Thirkell, and W. C. Russell, J. Gen. Microbiol. 133:2659-2676, 1987). In this study, three of the surface-expressed membrane antigens of 16, 17, and 96 kilodaltons were further investigated, and monoclonal antibodies were raised against these three polypeptides. The major 96-kilodalton polypeptide was serotype 8 specific, and the 16-kilodalton polypeptide was present only in the larger serocluster. We describe monoclonal antibody probes that unequivocally differentiate serotype 8 from the other serotypes and that separate the two seroclusters of the organism.

The urease of Ureaplasma urealyticum.

The urease from Ureaplasma urealyticum (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6.9 and pI values were determined. Storage of the purified enzyme at -70 degrees C followed by thawing to 20 degrees C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes of the enzyme, all distinct from its active site.

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

The survival of anaerobes during sewage treatment employing biological filter beds.

During the passage of sewage through a typical treatment plant employing biological filter beds and operating under dry weather flow conditions, about 7% of the input of anaerobic organisms survive to be released in the effluent. The greatest fall in numbers occurs in the first of two primary settling tanks operating in series and during passage through the filter beds. The predominant organisms were Bacteroides species, gram-positive cocci and clostridial species. There is no significant difference in the rate of survival of any of the genera through the different stages of treatment.

A screen for Clostridium difficile in the vagina: an out-patient study using and comparing selective media.

Cycloserine-Cefoxitin-Fructose Agar (CCFA) gives good presumptive identification of Clostridium difficile after 1- or 2-day incubation whereas Reinforced Clostridial Medium (RCM)/p-cresol is not very selective for the organism from the vagina. The identification of 91.5% of the isolates from an initial screen subjected to biochemically based tests was achieved. Conventional screening of vaginal swabs failed to confirm any significant occurrence of Cl. difficile in the vagina of pregnant or non-pregnant women. The incorporation of an enrichment stage in the isolation procedure, however, did reveal a significant presence of the organism in the vagina of both pregnant and non-pregnant women.

Enumeration and speciation of group D streptococci from above and below a sewer outfall, their susceptibilities to six antibiotics and a comparison with clinical isolates.

Isolates of group D streptococci from above and below a sewer outfall and a series of clinical isolates have been speciated to sub-species level. From below the sewer outfall, Streptococcus faecalis var. faecalis predominates whereas above the outfall, S. faecium var. casseliflavus predominates. S. faecalis var. faecalis, S. faecalis var. liquefaciens and S. faecalis var. zymogenes were the predominant sub-species in the clinical isolates where S. faecium var. casseliflavus was virtually absent. S. faecalis var. liquefaciens and S. faecalis var. zymogenes were uncommon in the environmental isolates. S. faecium and S. durans were more abundant in the environmental than in the clinical isolates. The use of group D streptococci as indicators of faecal pollution would seem more suited to higher, rather than lower, levels of pollution. A statistically significant increase in resistance to antibiotics (ampicillin, penicillin, streptomycin, gentamicin, erythromycin and tetracycline) was seen in isolates from below the outfall compared with those from above and a further significant increase was seen in the clinical isolates compared with the former. Resistance to tetracycline was most common and ampicillin was the only antibiotic tested to which no resistance was detected. Multiple antibiotic resistance was rare in the environmental isolates. Even in moderately polluted water, there is not a large pool of antibiotic resistance.

The speciation of coliform genera from above and below a sewer outfall and their susceptibilities to antimicrobial agents.

The occurrence of coliforms in a small water course was shown to increase by a factor of thirty six below the outfall of a sewage treatment plant. Speciation of the bacteria from above and below the sewer outfall showed that Escherichia coli and Enterobacter species predominated. Drug resistance levels were significant in microorganisms from both sampling sites and the occurrence of a significant number of multiple-resistant microorganisms, particularly E. coli, is reported. Both E. coli and Enterobacter species from below the sewer outfall show a statistically significant increase in resistance to ampicillin and E. coli from below the outfall also shows a statistically significant increase in resistance to sulpha-methoxazole as compared with isolates from above the outfall.

Variation in pigment production by Planococcus citreus Migula with cultural age and with sea salt concentration in the medium.

The biosynthesis of carotenoids and the type of carotenoids produced by Planococcus citreus Migula appear to be influenced by the concentration of sea salt in the medium and by the age of culture. Quantitatively, pigmentation peaks at a time corresponding to the presence of maximum cell numbers and then undergoes a rapid decline.

The effect of varying sea salt concentration in the growth medium on the chemical composition of a purified membrane fraction from Planococcus citreus Migula.

Membrane preparations were prepared from cells of Planococcus citreus grown in the presence of three final concentrations of sea salt in a basic growth medium. The concentration of salt in the medium affects the amount of membrane in the cell. The three preparations were subjected to chemical analysis and no significant changes in chemical composition were seen as the salt concentration in the medium was increased. Values for the various components generally were within normal ranges and were similar to those of non-halophiles rather than extreme halophiles. The protein levels were slightly higher and it it cellular ion balance. Atomic absorption analysis of the major cations associated with the membranes showed that divalent ions were present in a 2:1 ratio with ...