The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Synthesis of lipopolysaccharides in the bacterium Yersinia pseudotuberculosis: effect of the pVM82 plasmid and growth temperature

The composition and structure of lipopolysaccharides (LPS) of three isogenic strains of Yersinia pseudotuberculosis serovar O:1b (without plasmids (82-) and with plasmids pVM82 (82+) or p57 (57+)) grown at 8 or 37 degrees C were studied by chemical and immunochemical methods, SDS-polyacrylamide gel electrophoresis, and 13C-NMR spectroscopy. At the lower temperature, the (82-) and (82+) strains synthesized S-form of LPS with similar structure characterized by high acylation and immunochemical activity. On the other hand, LPS of the (82+) strain had shorter carbohydrate chains than LPS of the (82-) strain. The contents of LPS were decreased in cells of the plasmid-free strain grown at the higher temperature. LPS isolated from these cells were of the R-form and had low acylation and immunochemical activity. Total LPS content in cells of the (82+) strain did not significantly depend on the growth temperature. LPS of the warm variant of these bacteria contained a polysaccharide fragment and had moderate immunochemical activity. The cells of the (57+) strain at both growth temperatures had low LPS contents and produced LPS of low acylation without O-specific chains (cold variant) or containing O-polysaccharide with low polymerization degree (bacteria grown at 37 degrees C). The data indicate that in the absence of the plasmids, LPS synthesis is encoded by the chromosomal genes in pseudotuberculosis bacteria. Expression of the genes involved in LPS synthesis is regulated by the temperature of bacterial growth. Genes responsible for temperature-dependent regulation of LPS biosynthesis are located on chromosomal DNA. The pVM82 plasmid includes two gene groups; one group is localized in a 57-mD fragment of DNA and inhibits LPS synthesis, suppressing temperature-dependent regulation of the synthesis. The genes located in a 25-mD fragment of the pVM82 plasmid are de-repressors of the 57-mD fragment, and they restore the ability of pseudotuberculosis bacteria to synthesize relatively long LPS at both growth temperatures.     

Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.     

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.     

Effects of Culture Method and Growth Phase on Free Lipid Composition of Yersinia pseudotuberculosis

The influence of culture method (free-floating cells in liquid nutrient broth or bacteria attached to agar surface on solid agarized medium of the same formulation) and bacterial age on the composition of free lipids in Yersinia pseudotuber-culosis (O:Ib serovar, strain KS 3058) grown in the cold (5°C) has been investigated. The specific growth rate of the bacteria on solid medium was about threefold less than that in liquid medium. The qualitative composition of phospholipids and fatty acids only slightly depended on the bacterial culture method. At the same time, the colonially growing cultures contained somewhat more total lipids, they synthesized more phospholipids, in the linear growth phase they contained more lysophosphatides, and they had higher fatty acid unsaturation index and higher pathogenic potential than their planktonic counterparts grown in otherwise identical conditions. The bacterial growth phase influenced the amount of 3-hydroxytetrade-canoic acid and, indirectly, that of lipopolysaccharide. The dynamics of changes in the amount of this acid with bacterial age was opposite in the surface and broth cultures.     

Glucose as a growth medium factor regulating lipid composition of Yersinia pseudotuberculosis

Effects of glucose and growth temperature on Yersinia pseudotuberculosis O:1b serovar lipid composition have been studied. These growth parameters were shown to have drastic effects on biosynthetic processes in the pseudotuberculosis bacteria. The temperature effect is the most universal, extending to cell growth and to free lipid and lipopolysaccharide content and composition; it is most conspicuous in the bacteria cultivated on glucose-containing nutrient broth. The effect of glucose is selective, affecting only free lipids and depending on temperature (glucose favors phospholipid (PL) synthesis in the cold and inhibits it at 37°C); the effect of glucose is more evident in the cold. Determination of the contents of individual PL in percent dry bacterial weight indicates that the most obvious effect of glucose and/or growth temperature is on phosphatidylethanolamine (PE) content: on both media and at both temperatures an overall decrease in PL content stems from the inhibition of PE synthesis and is attended by decreasing ratio of neutral to acidic lipids.     

Influence of Structural Properties and Kinetic Constraints on Bacillus cereus Growth

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter⁻¹. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

Improved Solid Medium for the Detection and Enumeration of Pectolytic Bacteria

An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.     

Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica

An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4°C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in tryptone—soya broth yielded better results than in—frequently used—phosphate buffer, pH 7.6.While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29°C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results.It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.     

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

Exposure of Salmonella senftenberg 4969 to sublethal heating in phosphate buffer, pH 7·0, at 52· produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37· in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favourable environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.     

New medium for isolating iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage.

A new solid medium is described for growing iron and heterotrophic bacteria from acid mine drainage (AMD). Examination of AMD from five states revealed several kinds of colonies of iron-oxidizing bacteria: (i) smooth, (ii) smooth with secondary growth sectors or branching, (iii) star-shaped, (iv) radiating lobe, and (v) flat-rough. All AMD samples yielded whitish colonies that could not use ferrous iron, sulfur, or hydrogen, nor could they grow on nutrient agar, brain heart infusion agar, or Trypticase soy agar. Glucose and sucrose supported growth if the sugar-salts medium was at pH 3.0. The new iron medium has several advantages over others: (i) easy preparation, (ii) rapid growth, (iii) larger colonies, (iv) differentiation of colony morphology, and (v) detection of a new group of heterotrophic acidophilic bacteria.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Heterogeneity of lipopolysaccharides from Yersinia pseudotuberculosis: chemical characterization of various molecular types

Prior studies have shown some unusual changes in the lipopolysaccharides (LPSs) from Yersinia pseudotuberculosis that occur when the microbe is grown at low temperature; the specific features of these LPSs in comparison with the LPSs from other enteropathogens may be due to unusual thermal adaptation mechanisms. To gain insight into this question, the chemical composition of Y. pseudotuberculosis LPS has been determined. The data indicate that two different S-form LPS species are produced in "cold"-grown bacteria. These have an identical set of bands after SDS-PAGE, similar elution profiles during gel-filtration on a Sephadex G-200 column in the presence of sodium deoxycholate, identical monosaccharide and fatty acid compositions, and similar polymerization degrees, but they have different acylation degree. On the whole, the macromolecularly different LPS populations, varying not only in their smooth or rough nature and hydrophobicity, but also in their localization in the outer membrane and, probably, their interactions with other cell components, are synthesized in "cold"-grown Y. pseudotuberculosis. The biological sense of the heterogeneity and its connection with psychrophilic and pathogenic properties of pseudotuberculosis organisms are discussed.     

Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.     

A comparison of solid and liquid media for measuring the sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media, and the time needed to recover resistance

Sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media was measured by viable counts in liquid and on agar-solidified versions of these media and on nutrient media. All solid media, including the supposedly non-inhibitory nutrient agar, were more inhibitory to injured cells than the corresponding liquid media. Catalase or pyruvate increased counts on nutrient agar to the level obtained in nutrient broth. Therefore nutrient agar plus pyruvate was the most suitable reference medium against which to compare recoveries on other media. Although recoveries of injured cells varied widely depending on the composition and physical state of the medium, this had a minor effect on estimates of repair time because resistance to all selective media was regained by the end of the lag phase.     

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR

In the last three decades, several monkeys reared in outdoor/indoor-outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.     

Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.