Ultrastructural changes in Shigella flexneri cells during interaction with bacteriocinogenic Lactobacillus acidophilus

Results of electron-microscopic examination of Shigella flexneri cells, subjected to influence the bacteriocin-producing Lactobacillus acidophilus bacteria are presented. The response of shigellae to bacteriocinogenic lactobacilli was shown both on cellular and population levels. On population level the correlation of various morphological types of shigella cells with increase of involution, lysing and resting forms is revealed. At a cellular level the specific ultrastructural changes of shigella evidencing the significant destructive processes of the cells were revealed. In one case destabilization of shigella cellular wall was observed, that was manifested in expansion of periplasmic spaces and appearence of specific involution forms of the cells. In other cases, changes in the ultrastructural organization of shigella nucleoid were found out, manifested in disappearance of thin-fibrillar DNA and formation of electronic-dense globular structures of the cells.     

Study of the role of conditionally pathogenic enterobacteria in acute intestinal diseases]

Conditionally pathogenic enterobacteria were isolated in 34.2% of the patients with acute intestinal diseases (1559 cases examined in all). Such representatives of enterobacteria as Klebsiella, Hafnia, Citrobacter of 4, 8, and 22 serological groups. Proteus mirabilis of 6, 10, 13, 26, and 28 serological groups, and Proteus morganii mostly played the etiological role. In some of the patients conditionally pathogenic microbes only aggravated the main disease, taking part in the development of dysbacteriosis. In healthy individuals these microbes were much more rare and were encountered inconstantly.     

Partially chemically defined liquid medium development for intensive propagation of industrial fermentation lactobacilli strains

An economic liquid growth medium was synthesised for high-rate production of cellular mass, lactic acid and bacteriocin in lactobacilli. Three lactobacilli that are applied extensively in industry—Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586—were chosen to test the medium’s efficiency. These bacteria are chemoorganotrophs requiring rich, complex media for optimum growth. Contrary to the current practice of formulating a strain-specific medium, we attempted to prepare a universal broth that would allow easy formulation and optimisation. Man de Rogosa Sharp (MRS) medium, which can support the growth of lactobacilli, was found unsuitable for use in large quantities due to its high cost of preparation and its use of beef extract and peptone from poultry as nitrogen sources, which are not environmentally friendly and have potential health risks. The developed medium supported the growth of all the three bacteria equally, offering good maximum yields and incorporating only the chemical compounds needed, resulting in an improvement in the growth rate of the bacilli of between 50 % and 241 % compared to the same strains grown on MRS. Lactic acid production was between 28.6 and 35.74 g L^?1 and bacteriocin production ranged from 110 to 130 IU mL^?1.     

Bacteriocinogenic highly antagonistic strains of lactobacilli

Ability to secrete bacteriocines and microcines was studied in 25 cultures of lactobacilli isolated from intestine of healthy children. Sixteen (64%) of them produced microcines with wide spectrum of antagonistic activity. Susceptibility of microcines-secreting cultures to antibacterial preparations, different dilutions of hydrochloric acid and bile was studied along with their acid-producing ability. Five strains without DNA-se, RNA-se, gelatinase, lecitinase and caseinolytic activity were selected from Sixteen microcines-producing cultures. Three of the selected strains carried plasmid DNA and two didn't have plasmids. Bacteria were characterized by tolerance to hydrochloric acid and bile - minimal inhibitory concentrations for them were 1,25 and 10% respectively. Strains without plasmids were susceptible to majority of wide-spectrum antibiotics and resistant to fluorochinolones. Microcines-producing lactobacilli with wide spectrum of antagonistic activity against pathogenic and conditionally pathogenic microorganisms and tolerance to physiological concentrations of hydrochloric acid and bile have a potential to be used in manufacturing of probiotics.     

Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum

A protease-sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by α-amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non-bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.     

Taxonomic composition of bacteria associated with cultivated mollusks Crassostrea lugubris and Perna viridis and with the water of the Gulf of Nha Trang Lagoon, Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.     

Plasminogen activation in degradation and penetration of extracellular matrices and basement membranes by invasive bacteria

Methods to assess in vitro the role of plasminogen activation in enterobacterial degradation of extracellular matrices and their protein components as well as in penetration through basement membrane are described. Development of these methods was initiated after the findings that enterobacterial surface structures (fimbriae and the Pla surface protease) function in plasminogen activation as well as in laminin- and/or fibronectin-specific adhesion. Enterobacteria with these properties degrade radiolabeled laminin as well as metabolically labeled extracellular matrix from cultured endothelial or epithelial cells. Plasmin-coated bacteria also penetrate through the reconstituted basement membrane preparation Matrigel. The processes are dependent on plasminogen activation by the invasive bacteria. The results suggest a pathogenic similarity between enterobacteria and tumor cells in cellular metastasis through tissue barriers.     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.     

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.     

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at ?20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5–6.5 and 5.0–5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.     

Interference of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in the In Vitro Conjugative Transfer of R-Plasmids

Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac– as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.     

Isolation and properties of a bacteriocin-producing Carnobacterium piscicola isolated from fish.

A facultative psychrotrophic lactic acid bacterium isolated from fresh fish was identified as Carnobacterium piscicola on the basis of carbohydrate utilization, G + C content and 16S RNA analysis. Its bacteriocin, designated carnocin UI49, is produced during the mid-exponential phase of growth at temperatures between 15 degrees C and 34 degrees C. Carnocin UI49 is active against a large number of closely-related lactic acid bacteria including carnobacteria, lactobacilli, pediococci and lactococci. Furthermore, the bacteriocin has a bactericidic mode of action which results in lysis of sensitive cells. Maximum bactericidal activity is observed at 34 degrees C with a decrease in activity down to 15 degrees C where it is completely abolished.     

The cost and benefit of quorum sensing‐controlled bacteriocin production in Lactobacillus plantarum

Bacteria eliminate competitors via ‘chemical warfare’ with bacteriocins. Some species appear to adjust bacteriocin production conditionally in response to the social environment. We tested whether variation in the cost and benefit of producing bacteriocins could explain such conditional behaviour, in the bacteria Lactobacillus plantarum. We found that: (a) bacterial bacteriocin production could be upregulated by either the addition of a synthetic autoinducer peptide (PLNC8IF; signalling molecule), or by a plasmid which constitutively encodes for the production of this peptide; (b) bacteriocin production is costly, leading to reduced growth when grown in poor and, to a lesser extent, in rich media; (c) bacteriocin production provides a fitness advantage, when grown in competition with sensitive strains; and (d) the fitness benefits provided by bacteriocin production are greater at higher cell densities. These results show how the costs and benefits of upregulating bacteriocin production can depend upon abiotic and biotic conditions.     

Etiological structure of acute intestinal infections caused by opportunistic bacteria in young children

Altogether 587 children with diagnosed acute enteric infections were examined. In 26.4% of them pathogenic enterobacteria were detected, and in 59%, opportunistic enterobacteria. In 38.2% of children these bacteria were found to be the etiological factor of the disease. A reverse relationship between the contamination of feces with pathogenic enterobacteria and the age of children was established.     

Black grain mycetoma: The ultrastructure of Madurella mycetomi

Madurella mycetomi, the fungus pathogen of black grain mycetoma, shows an elaborate development of mesosomes as its most striking ultrastructural feature. These occur in addition to mitochondria and endoplasmic reticulum. Condensations of hyphal cytoplasm and a recurring intrahyphal growth of new hyphal cells are features of the pigment-producing and pathogenic phases of the organism. The abundant pigment which histochemically resembled melanin was not traceable to any ultrastructural granule, nor was it built from a tyrosine precursor. The ultrastructural changes occurring in collagen through a sclerotizing action by the fungus are briefly noted.     

β-actin and γ-actin are each dispensable for auditory hair cell development but required for Stereocilia maintenance

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic β-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since β-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of β-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither β-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with β-actin- or γ-actin-deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for β-actin and γ-actin in maintaining hair cell stereocilia.     

Isolation and properties of a bacteriocin-producing Carnobacterium piscicola isolated from fish

A facultative psychrotrophic lactic acid bacterium isolated from fresh fish was identified as Carnobacterium piscicola on the basis of carbohydrate utilization, G + C content and 16S RNA analysis. Its bacteriocin, designated carnocin UI49, is produced during the mid-exponential phase of growth at temperatures between 15°C and 34°C. Carnocin UI49 is active against a large number of closely-related lactic acid bacteria including carnobacteria, lactobacilli, pediococci and lactococci. Furthermore, the bacteriocin has a bactericidic mode of action which results in lysis of sensitive cells. Maximum bactericidal activity is observed at 34°C with a decrease in activity down to 15°C where it is completely abolished.     

Characteristics of immune response in acute intestinal enterobacterial infections

Update literature on the problem of microorganism--host interactions with pathogenic enterobacteria as a model is reviewed. The activation of lymphoid cells and their protective role is shown to be due to the stimulating action of bacterial antigens. The role of cytokines and stimulation of their expression are discussed. Some phenomena of immunosuppression, phases of enteric mucosal immune response are described.