Dynamics of the reversible transition of Vibrio cholerae into the uncultivable state in the presence of organic and inorganic microcosmic components

The dynamics of the transition of V. cholerae into the uncultivable state in distilled, river and tap water, containing organic and inorganic components added, was studied. As additives, potassium nitrate, potassium phosphate, magnesium sulfate, ammonium chloride, lysine, alpha-ketoglutarate, succinic acid, catalase were used. The study of the influence of biotic factors on transition into the uncultivable state was carried out in the presence of one-celled green algae Scenedesmus quadricauda or infusoria Paramecium caudatum. The linear dependence of speed of transition into the uncultivable form on the concentration of cells was noted. The composition of the microcosmic medium was also found to have some influence on the speed of transition into the uncultivable form and on the reversibility of this process. The presence of organic substances, such as peptone solution or destroyed cells of phyto- and zooplankton, in the microcosmic medium prolonged the time of transition into the uncultivable form and produced a positive effect on the capacity of the population to reversion. In respect of live biotic components, no such dependence was found. Inorganic additives prolonged the time of transition into the uncultivable state, but did not promote reversion.     

Transition of Vibrio cholerae into the non-culturable state under the conditions of low temperature, different mineralization and illumination of artificial culture medium

The importance of the combined influence of temperature, mineralization and illumination of the medium on the time of the transition of V. cholerae into the uncultivable state has been shown. The reversion of 5- to 60-day variants of uncultivable forms after the elevation of temperature to 20-22 degrees C has been obtained.     

Experimental non-culturable Vibrio cholerae eltor and its biological properties

In experiments with the cultivation of V. cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained. The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V. cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones. The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle. The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.     

Vibrion variability under the action of antibiotics]

Tetracycline, streptomycin and monomycin resistant variants of the cholera and NAG-vibrios were obtained by means of repeated passages on nutrient media with increasing concentrations of the antibiotics (114 variants V. cholerae asiaticae, 1337 variants of V. cholerae eltor and 299 variants of NAG-vibrios of the 1st Heiberg group). The highest number of the antibiotic resistant variants was obtained under the effect of streptomycin and the resistance level to it was much higher (up to 8000 gamma/ml) than that to tetracycline or monomycin (160 or 320 gamma/ml respectively). The study of the differential-diagnostic properties of the above vibrios showed that 14.8 per cent of the strains of V. cholerae asiaticae and 4.6 per cent of the strains of V. cholerae eltor became non-typical with respect to the colony morphology and fermentative properties. Their agglutinability with the species or type specific cholera sera partially decreased. Nine per cent out of 299 antibiotic resistant NAG-vibrios had changes only in the colony structure. None of them changed their fermentative properties or acquired even minor capacity for agglutination with cholera sera or lysing with specific bacteriophages. The described changes in the properties of the antibiotic resistant cholera and NAG-vibrios were not stable and disappeared after 2- or 3-fold passages on media containing no antibiotics.     

Morphology and enzyme activity of nonculturable forms of Vibrio cholerae

The transition of V. cholerae into the uncultivable state under experimental conditions was accompanied by gradual changes in their morphology, motility and metabolic activity. The vibrios took the oval form, lost their flagellum, motility and enzyme activity on diagnostic media. Dehydrogenase activity tested by reduction of triphenyl tetrazolium chloride, increased at the initial stages and dropped to the initial level or even lower by the end of the observation period (10 months). Similar dynamics was noted when the cytoplasmatic marker enzyme--ATPase activity was studied. Glucose catabolism in the uncultivable forms shifted towards glycolysis. During 1-2 months ctx and tcp genes could be detected in these forms by the PCR. The dynamics of the biological properties under study made it possible to find out the existence of 3 functionally different stages in the development of an uncultivable population.     

Clonal structure of Yersinia pestis populations in experimental soil ecosystems

Two basic tendencies--formation of latent (uncultivable) form (LF) and hemin storage variability--has been revealed during study of clonal structure dynamics of Y. pestis populations in artificial soil ecosystems in long-term incubation conditions. Y. pestis populations disappeared within 3 - 6 months at 18 - 22 degrees C, whereas at 4 - 8 degrees C a subsequent replacement of vegetative cells on LF, which are capable to prolonged survival (up to 22 months) in soil with ability to reversion in the presence of abundance of nutrients, has been observed. Bacteria of virulent strain retained all determinants of pathogenicity when reverted to LF, whereas bacteria of avirulent strain (defective on plasmid of Ca-dependence), on the contrary, undergo further degradation that resulted in loss of a pgm locus and gradual disappearance of population. LF revertants of highly virulent strain restored properties of initial population and were highly virulent.     

Investigation of populations heterogeneous according to O-antigen in Vibrio cholerae cultures

The O-antigenic composition of 36 cultures of Vibrio cholerae agglutinating simultaneously with 01 cholera sera and 0 sera to NAG vibrios of the Sakazaki collection was investigated. It has been established experimentally that under the effect of medium and environmental conditions such cultures dissociate to subcultures differing in their affiliation to different serological groups according to 0 antigen. The passage of these cultures in the organism of susceptible animals promotes preservation of 01-group clones whereas the passage in peptone water or prolonged storage under unfavourable conditions result in the predominance of clones of different serological affiliation. The proposition has been put forward that the observed vibrio cultures are genotypically capable of producing, besides the 01 group, a number of 0 antigens. Phenotypical manifestation of the antigenic structure in the respective individuals of the population depends on the conditions of the environment.     

Formation of Streptococcus pneumoniae non-phase-variable colony variants is due to increased mutation frequency present under biofilm growth conditions

In this report, we show that biofilm formation by Streptococcus pneumoniae serotype 19 gives rise to variants (the small mucoid variant [SMV] and the acapsular small-colony variant [SCV]) differing in capsule production, attachment, and biofilm formation compared to wild-type strains. All biofilm-derived variants harbored SNPs in cps19F. SCVs reverted to SMV, but no reversion to the wild-type phenotype was noted, indicating that these variants were distinct from opaque- and transparent-phase variants. The SCV-SMV reversion frequency was dependent on growth conditions and treatment with tetracycline. Increased reversion rates were coincident with antibiotic treatment, implicating oxidative stress as a trigger for the SCV-SMV switch. We, therefore, evaluated the role played by hydrogen peroxide, the oxidizing chemical, in the reversion and emergence of variants. Biofilms of S. pneumoniae TIGR4-ΔspxB, defective in hydrogen peroxide production, showed a significant reduction in variant formation. Similarly, supplementing the medium with catalase or sodium thiosulfate yielded a significant reduction in variants formed by wild-type biofilms. Resistance to rifampin, an indicator for mutation frequency, was found to increase approximately 55-fold in biofilms compared to planktonic cells for each of the three wild-type strains examined. In contrast, TIGR4-ΔspxB grown as a biofilm showed no increase in rifampin resistance compared to the same cells grown planktonically. Furthermore, addition of 2.5 and 10 mM hydrogen peroxide to planktonic cells resulted in a 12- and 160-fold increase in mutation frequency, respectively, and gave rise to variants similar in appearance, biofilm-related phenotypes, and distribution of biofilm-derived variants. The results suggest that hydrogen peroxide and environmental conditions specific to biofilms are responsible for the development of non-phase-variable colony variants.     

Specificity of lectin receptors of cholera vibrios

Spectrum of carbohydrate specificity of lectin receptors of epidemically significant cholera vibrios (ctx(+) tcp(+) Hly(-)) as well as non epidemic hemolytic variants with or without tcp A gene (ctx(-) tcp(-) Hly(+), ctx(-) tcp(+) Hly(+)) was studied under the carbohydrates-mediated inhibition of hemagglutination between human erythrocytes of four blood groups and sheep erythrocytes. It was demonstrated that in toxigenic cultures lectin receptors specific for glucose, mannose, sacharose, lactose dominate whereas receptors specific for aminosugars are virtually absent. The latter are detected in hemolytic vibrios that can explain their ecologic flexibility.     

Detection of L forms of Vibrio cholerae in the water of open water reservoirs

L-forms of cholera vibrios were isolated from the river water for the first time. The presence of L-forms in water permitted to suppose that such variants served as one of the forms of cholera causative agent preservation in the external medium.     

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Factors Affecting the Resistance of Staphylococcus aureus to Hydrogen Peroxide Treatments in Milk

Staphylococcus aureus 196E was treated with 0.05% hydrogen peroxide in milk under varying conditions to determine the effects of treatment conditions and characteristics of the culture on bactericidal effectiveness of hydrogen peroxide. Time intervals required for 90 to 99.99% destruction of S. aureus decreased significantly as treatment temperatures increased from 37.8 to 57.2 C. Plots of survivor curves showed extended lags in destruction at 37.8 C, slight lags followed by logarithmic rates of destruction at 48.9 C, and logarithmic rates at 54.4 and 57.2 C except for trials in which there was very rapid initial destruction followed by logarithmic rates. S. aureus 196E was significantly more resistant to heat treatments at 54.4 C without added hydrogen peroxide than to treatment with 0.05% hydrogen peroxide at this temperature. Cultures grown at 37 C for 16 hr in milk were more resistant to hydrogen peroxide than were cultures grown at 35 C. Storage of cultures for 96 hr in milk at 4 C caused a decrease in the resistance of the culture. Numbers of staphylococci being treated had little effect on rates of destruction.     

Rapid Method of Determining Cholera Vibrio Biotype

The characteristic motility of cholera vibrios, as viewed through a dark-field microscope, and the adhesiveness of chicken cell-positive vibrios provide a means for rapidly identifying and biotyping cholera vibrios. Dilute suspensions of vibrios, such as one might find in a fresh rectal swab specimen from a cholera patient, when mixed with a 0.25% suspension of chicken erythrocytes in saline, can be used to biotype the cholera vibrios without prior isolation in pure culture. This is accomplished by using a dark-field microscope through which the chicken cell-positive cholera vibrios are observed to attach to the scattered erythrocytes and to propel them with a characteristic flipping motion.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Hyaluronic acid capsule: strategy for oxygen resistance in group A streptococci.

Unencapsulated variants of encapsulated, M-protein-positive group A streptococci are oxygen sensitive and secrete inhibitory concentrations of hydrogen peroxide when grown in aerated broth cultures. The organisms were equally sensitive to hydrogen peroxide, and neither exhibited catalase or peroxidase activity, suggesting that differences in oxygen sensitivity reflect dissimilarity in oxygen uptake. The encapsulated parental culture was found to grow in aggregates that take up oxygen more slowly than unencapsulated, oxygen-sensitive derivatives. Moreover, the latter grow in an unaggregated, homogenous suspension. The enzyme hyaluronidase was able to disrupt aggregates of the encapsulated strain increase the rate that these cells take up oxygen, and cause the accumulation of toxic concentrations of hydrogen peroxide earlier in their growth cycle. The evidence presented shows that the aggregation of streptococcal cells by their hyaluronic acid capsule provides this organism with a novel means to avoid self-destruction by oxygen metabolites--cells are shielded from oxygen. The reduced surface-to-volume ratio and limited diffusion of oxygen into the interior of aggregates are proposed as the protective mechanism.     

Use of a new method for restoring the type properties of atypical Vibrio cholerae

Subcultures with a number of signs characteristic of epidemically significant strains have been isolated from cholera vibrios, nonpathogenic and atypical in a number of properties, by a new in vitro method developed by the authors. This method makes it possible to increase the virulence of poorly agglutinating cultures of V. cholerae O1 and their agglutinability with cholera antisera.     

Development of antilipase immunoglobulin polymer diagnosticum for the detection of Vibrio cholerae eltor, possessing hemolytic and lipase activity

The possibility of using a heterogeneous, but structurally similar antigen--the commercial preparation of Pseudomonas sp. lipase (Sigma, USA)--for the development of polymer diagnosticum aimed at determination of lipase production in cholera vibrios was shown. The new diagnosticum (antilipase antibodies) on a polymer carrier was used in the serological volume agglomeration test for the detection of hemolytic atoxigenic V. eltor, obtained from environmental, objects, which produced lipase in 80% of cases. The differentiating capacity of the diagnosticum was confirmed on 120 V. eltor cultures isolated from environmental objects. The newly developed diagnosticum makes it possible to determine the lipase activity in cholera vibrios of different biovars and serovars.     

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H₂O₂ oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Use of analogs of primary metabolites in the selection of the producer of polymyxin B]

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.     

The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions]

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.