The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.     

Comparative study of nutrient media for isolation of the agents of pertussis and parapertussis]

Taking into consideration literature data pointing to a possibility of a marked improvement of the quality of the medium for the isolation of the causative agents of pertussis and parapertussis, an experimental study was made of the sensitivity of the nutrient media produced by the Dagestan Institute of Nutrient Media, a medium made from dry and also of a number of media modified by the authors prepared on its basis. Pure freshly-isolated and museum B. pertussis cultures were used in the observations. Analysis of the results of a comparative study of the indices (detectability of the strains, the appearance of colonies and periods of their formation) allomed a recipee of the most sensitive medium to be elaborated. At the same time there were found considerable differences in the indices of the appearance of B. pertussis colonies and detectability of the museum cultures used for testing the industrial batches and of the freshly-isolated strains.     

Construction of the genetically attenuated bacteria Bordetella pertussis devoid of dermonecrotic toxin activity and producing modified nontoxic pertussis toxin form

The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.     

Isolation and characterization of sulphur-oxidizing Thiomonas sp. and its potential application in biological deodorization

AIMS: To isolate and characterize a sulphur-oxidizing bacterial strain from activated sludge and to evaluate its potential application in biological deodorization. METHODS AND RESULTS: A dominant sulphur-oxidizing bacterial strain, designated as strain SS, was isolated from an enrichment culture using thiosulphate as a sole energy source and CO2 as a sole carbon source. The cells of this organism were aerobic, rod-shaped, Gram-negative and motile. Strain SS could grow autotrophically, heterotrophically as well as mixotrophically. Autotrophic growth was observed at pH values ranging from 2.3 to 9.0. Phylogenetic analyses revealed that strain SS belonged to Group 1 of the genus Thiomonas, closely related to Thiomonas perometabolis and Thiomonas intermedia. The thiosulphate oxidation rates of strain SS at different pH values were evaluated in terms of oxygen uptake using a Micro-Oxymax respirometer. The results showed that the maximum oxidation rate of 5.65 mg l(-1) h(-1) occurred at 56 h of growth and pH 6.0. Continuous H2S removal study demonstrated that strain SS could remove more than 99% of H2S when the inlet concentration was below 58.6 ppm. Further increase of the inlet concentration to 118 ppm gave rise to a decline in the removal efficiency to ca 90%. CONCLUSIONS: The strong acidification of the culture medium during the later period could result in the deterioration of the growth activity and the metabolism activity of strain SS. In practical application, the problems caused by the end-product inhibition and the acidification can be alleviated by periodical replacement of culture medium with fresh medium. Given the physiological flexibility and the ability to remove H2S rapidly and efficiently, strain SS could be a good 'deodorizing' candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that Thiomonas species has been reported for biological deodorization application.     

Isolation of Shigellae: V. Comparison of Enrichment Broths with Stools

Many enteric media are more efficient for the detection of salmonellae than of shigellae. Comparisons of three enrichment broths and three plating media were made during analysis of 1,405 stool specimens to choose a combination of media which would enhance detection of shigellae as well. Gram-Negative (GN), Selenite, and Silliker's Broths were streaked to E M B, Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) Agars. The enrichment broths produced a twofold increase in isolations of both salmonellae and shigellae over direct streaking. All three broths performed equally well for Salmonella detection, but GN and Silliker's produced twice as many Shigella isolates as did Selenite. Comparison of the plating media showed that XLD was markedly more efficient than either E M B or SS Agar for the recovery of both genera. SS Agar was superior to E M B for isolation of salmonellae after enrichment, whereas E M B was better for isolation of shigellae by direct streaking. Both E M B and SS were more effective when used after GN and Silliker's than after Selenite. GN Broth and XLD Agar were the most efficient combination of media. During these analyses, 158 salmonellae and 49 shigellae isolates were obtained.     

A low-sodium-salt formulation for the fermentation of salinosporamides by <Emphasis Type="Italic">Salinispora tropica</Emphasis> strain NPS21184

In this paper, we described the development of a potassium-chloride-based-salt formulation containing low sodium concentrations (5.0 to 11 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). The sodium present in the media was essentially derived from the complex nitrogen sources Hy Soy, yeast extract, and peptone used in the media. We demonstrated that good growth rate and yield of S. tropica strain NPS21184 were detected in both agar and liquid media containing the potassium-chloride-based-salt formulation with sodium concentration as low as 5.0 mM, significantly less than the critical seawater-growth requirement concentration of 50 mM sodium for a marine microorganism. We also observed good production of NPI-0052 (176 to 243 mg/l) by S. tropica strain NPS21184 grown in production media containing the potassium chloride-based-salt formulation. The production of deschloro analog, salinosporamide B (NPI-0047), was significantly lower in the low-sodium-salt-formulation medium than in the high-sodium-salt-formulation media. We demonstrated that while S. tropica strain NPS21184 is a novel marine actinomycete that requires high salt content for growth, it does not require sodium-chloride-based seawater-type media for growth and production of NPI-0052.     

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.     

Transparent solid nutrient medium for studying the lytic spectrum of bacteriophages of microbes of the genus Bordetella

A solid, transparent culture medium for the study of the lytic spectrum of the phages, active against B. pertussis and B. bronchiseptica, in respect to homologous and heterologous bacteria of the genus Bordetella has been developed. The Cohen-Wheeler liquid medium with nicotinic acid and nicotinamide added, solidified with agar, is nicotinamide added, solidified with agar, is used as the base of the new medium. This base ensures the growth of B. parapertussis and B. bronchiseptica. To stimulate the growth of B. pertussis, the tissue stimulant of B. pertussis growth (a transparent substrate obtained from the tissue of the large intestine of a rabbit) has been used. With 10% of this stimulant added, B. pertussis cells have been found to preserve their typical morphological and immunobiological properties.     

Osmolarity affects Bvg-mediated virulence regulation by Bordetella pertussis

Bordetella pertussis dramatically alters its phenotype by sensing its environment via the BvgAS regulatory system. Increased concentrations of specific chemicals are used in vitro to induce modulation of the bacterium from the Bvg(+) virulent phenotype to a fully Bvg(-) phenotype. Varied expression of sets of Bvg(-)regulated molecules depends on the modulating capacity of the environment. We examined the effect of a number of chemicals on the modulating capacity of B. pertussis growth media, both alone and in combination with known modulators. It was demonstrated that under certain conditions the Bvg(-)intermediate protein, BipA, is coexpressed with the Bvg(-) antigen, VraA. This demonstrates that the patterns of molecules expressed in the different phenotypes of B. pertussis are more fluid than has previously been demonstrated. The in vitro modulator, sulfate, was found to be a relatively inefficient modulator of our Tohama I-derived B. pertussis strain. However, addition of nicotinic acid, MgCl2, or sucrose in combination with relatively low sulfate concentrations resulted in effective modulation. This suggests that multiple signals may affect modulation through the BvgAS system or possibly through other regulatory networks. In addition, the cooperative modulating effect of sucrose implicates osmolarity as an environmental stimulus that affects phenotypic modulation.     

Evolutionary relationships in the genus Bordetella

The nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Aricò and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base-pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica. Computer analysis of the sequence data allows the construction of an evolutionary 'tree' showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Type III secretion-dependent host defence elicitation and type III secretion-independent growth within leaves by Xanthomonas campestris pv. campestris

In many plant-bacterial interactions, loss of the type III secretion system (T3SS) severely reduces bacterial growth, symptom causation and suppression of defences in host plants. In the present study of Xanthomonas campestris pv. campestris (Xcc), Xcc strain B305 grew better than strain B186 in Arabidopsis thaliana after hydathode inoculation, and B305 strains mutated to the loss of T3SS (ΔhrcC and/or ΔhrpE; also ΔhrcCΔflgBC) grew similarly to wild-type B305 in Arabidopsis leaves. Unlike Xcc strain B186, wild-type B305 was relatively inefficient in secreting the exogenous T3S effector AvrBsT, but ΔhrcC and/or ΔhrpE attenuated the disease symptoms caused by Xcc B305, showing that the partially compromised T3SS of this strain still promotes necrotic leaf symptoms. In contrast with the T3SS-dependent defence suppression that has been observed for some other plant pathogenic bacteria, the Xcc B186 and B305 wild-type strains (which are virulent on Arabidopsis) caused greater elicitation of host PR-1 and PR-5 expression and callose deposition in comparison with their respective T3SS mutants. A defence-suppressing/virulence-enhancing activity of the Xcc T3SS effector suite was detectable when co-inoculation with wild-type Xcc B186 increased the growth of ΔhrcC Xcc, but this activity did not prevent the above defence elicitation. Experiments using T3SS mutants and Arabidopsis fls2 mutants suggested that FLS2 does not play a prominent role in restriction of the examined Xcc strains. However, ectopic overexpression of the Pseudomonas syringae effector AvrPto promoted in planta growth of wild-type and ΔhrcC Xcc. In summary, the T3SS components or effector suite from virulent Xcc strains elicit some host defence responses, but suppress other defences and stimulate more severe disease symptoms, AvrPto-disruptable elements other than FLS2 apparently contribute to the host restriction of Xcc, and in some virulent Xcc strains the T3SS is not absolutely required for wild-type levels of bacterial growth within the plant.     

Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.