Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

Two strains of Lactobacillus crispatus (15L08 and 21L07) and one strain of Lactobacillus jensenii (5L08) were selected from amongst 100 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization and the production of H2O2 and/or bacteriocin-like compound. All three strains self-aggregated and adhered to vaginal epithelial cells, displacing well-known vaginal pathogens, such as Gardnerella vaginalis and Candida albicans. Lactobacillus crispatus 15L08 was characterized as a potential H2O2 producer. A high level of bacteriocin-like compound was synthesized by L. jensenii 5L08, with a bactericidal mode of action for G. vaginalis, C. albicans and Escherichia coli. However, H2O2-dependent activity alone was not sufficient to inhibit the growth of C. albicans. Simultaneous actions of H2O2 and bacteriocin-like compound produced by lactobacilli may be important for antagonizing pathogenic bacteria. These strains of lactobacilli may be excellent candidates for eventual use as probiotics to restore the normal microbial communities in the vaginal ecosystem.     

Influence of probiotic vaginal lactobacilli on in vitro adhesion of urogenital pathogens to vaginal epithelial cells

AIMS: Lactobacilli, the predominant micro-organisms of the vaginal microbiota, play a major role in the maintenance of a healthy urogenital tract by preventing the colonization of pathogenic bacteria. The aim of the present study was to assess the ability of four vaginal Lactobacillus strains, previously selected for their probiotic features, to block in vitro the adherence of three human urogenital pathogens to vaginal epithelial cells (VEC). METHODS AND RESULTS: Three types of assays were performed in order to determine the inhibitory effect of lactobacilli on adhesion of urogenital pathogens to VEC: blockage by exclusion (lactobacilli and VEC followed by pathogens), competition (lactobacilli, VEC and pathogens together) and displacement (pathogens and VEC followed by the addition of lactobacilli). Bacterial adhesion to VEC was quantified by microscopy (x1000) after Gram's stain. All the strains were able to inhibit by exclusion and competition the adhesion of Staphylococcus aureus to VEC but none was able to decrease the attachment of Escherichia coli by neither of the mechanisms assayed. Only Lactobacillus acidophillus CRL 1259 and Lactobacillus paracasei CRL 1289 inhibited the attachment of Group B streptococci (GBS) to VEC by exclusion and competition respectively. CONCLUSIONS: Lactobacillus of vaginal origin were able to inhibit the attachment of genitouropathogenic Staph. aureus and GBS to the vaginal epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the probiotic potential of these Lactobacillus strains as anti-infective agents in the vagina and encourage further studies about their capacity to prevent and manage urogenital tract infections in females.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Protease,peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

AIMS: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. METHODS AND RESULTS: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected strains could be used as adjunct starters to make high quality Feta cheese.     

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

AIMS: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. METHODS AND RESULTS: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1x10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nalr) and E. coli O78:K80 (EC34195, nalr). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1x10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. CONCLUSIONS: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.     

Assessing the viability of three Lactobacillus bacterial species protected in the cryoprotectants containing whey and maltodextrin during freeze-drying process

Freeze-drying of bacteria associates with different stresses such as osmotic pressure, temperature and oxidation, and decreases bacterial viability, which seem to reduce by applying cryoprotectants. The present study evaluated the effect of four cryoprotectants on decreasing the stress caused by freeze-drying process among three Lactobacillus species. Additionally, it highlighted the use of whey and maltodextrin as a substitute for peptone and sucrose in cryoprotectants respectively. The viability of lactobacilli was measured after freeze-drying, 1 month of storage at 25 and 4°C. Based on the results, the viability rate of bacteria in protectants during freeze-drying stage was dependent on their strains. The best viability of Lacticaseibacillus rhamnosus GG and Ligilactobacillus salivarius 20687 was, respectively, observed in the protectants containing sucrose and whey, while Lactiplantibacillus plantarum NRRL B-14768 viability was equal in all protectants. The number of live bacteria reduced significantly by storing bacteria for 1 month at 25°C compared to the 4°C storage. During the storage period, the viability of L. salivarius improved by adding sucrose in protectant. Due to the positive effect of whey and sucrose in the drying and storage stage, on bacterial viability, the protectant consisting of whey and sucrose is suggested for all of the species under study.     

Specific and non-specific interactions in bacterial adhesion to solid substrata

Abstract Based on a literature review, a hypothesis is forwarded on the mechanism of initial bacterial adhesion to solid substrata, which accounts both for the role of specific microscopic surface components as well as for the role of non-specific macroscopic surface properties (surface free energy, zeta potential or hydrophobicity). Three distinct regions in the adhesion process are suggested in which at large and intermediate separation distances adhesion is mediated by the macroscopic surface properties as surface free energy and surface charge, respectively. At small separation distances specific short-range interactions can occur, leading to a strong and irreversible bonding, provided the water film present in between the interaction surfaces can be removed. A major role of hydrophobic groups, supposed to be associated with bacterial surface appendages is suggested to be its dehydrating capacity, enabling the removal of the vicinal water film yielding small areas of direct contact between protruberant parts of the cell surface and the substratum.     

Structural similarity and functional diversity in diiron-oxo proteins

 Diiron-oxo proteins currently represent one of the most rapidly developing areas of bioinorganic chemistry. All of these proteins contain a four-helix bundle protein fold surrounding a (μ-carboxylato)diiron core, and most, if not all, of the diiron(II) sites appear to react with O₂ as part of their functional processes. Despite these common characteristics, an emerging functional diversity is one of the most striking aspects of this class of proteins. X-ray crystal structures of diiron(II) sites are now available for four of these proteins: hemerythrin (Hr), the hydroxylase protein of methane monooxygenase (MMOH), the R2 protein of Escherichia coli ribonucleotide reductase (RNR-R2), and a plant acyl-carrier protein Δ⁹-desaturase. The structure of the diiron(II) site in Hr, the sole O₂ carrier in the group, is clearly distinct from the other three, whose function is oxygen activation. The Hr diiron site is more histidine rich, and the oxygen-activating diiron sites contain a pair of (D/E)X_30–37EX₂H ligand sequence motifs, which is clearly not found in Hr. The Hr diiron site apparently permits only terminal O₂ coordination to a single iron, whereas the oxygen-activating diiron(II) centers present open or labile coordination sites on both irons of the center, and show a much greater coordinative flexibility upon oxidation to the diiron(III) state. Intermediates at the formal Fe^IIIFe^III and Fe^IVFe^IV oxidation levels for MMOH and formal Fe^IIIFe^IV oxidation level for RNR-R2 have been identified during reactions of the diiron(II) sites with O₂. An [Fe₂(μ-O)₂]^4+, 3+ "diamond core" structure has been proposed for the latter two oxidation levels. The intermediate at the Fe^IIIFe^IV oxidation level in RNR-R2 is kinetically competent to generate a stable, functionally essential tyrosyl radical. The Fe^IVFe^IV oxidation level is presumed to effect hydroxylation of hydrocarbons in MMOH, but the mechanism of this hydroxylation, particularly the involvement of discrete radicals, is currently controversial. The biological function of diiron sites in three members of this class, rubrerythrin, ferritin and bacterioferritin, remains enigmatic. Received: 31 July 1996 / Accepted: 4 October 1996     

Relationship between somatic cell count and bacterial pathogens in goat milk

The study aimed at evaluation of total count and pattern of somatic cells, as well as lactose content in relation to the type of bacterial pathogens in goat milk. The study was conducted on 66 Polish White Improved and Polish Fawn Improved dairy goats. A total of 487 milk samples were taken from day 30th, 60th and 200th of lactation for three years. The milk samples were divided into four groups: group 1 – containing no pathogens, group 2 – with minor pathogens up to 1000 CFU/mL such as coagulase-negative staphylococci (CNS), alpha-haemolytic streptococci, Enterococcus spp., Corynebacterium spp., group 3 – with minor pathogens (CNS) above 1 × 10³ CFU/mL of milk and group 4 – with major pathogens such as Streptococcus agalactiae, Staphylococcus intermedius and Staphylococcus aureus.In the majority of milk samples (64.9%) no pathogens were observed. The CNS were isolated from 25.3%, while the major pathogens were from 9.8% of milk samples. Both the major pathogens and high numbers of minor pathogens influenced the total somatic cell count (SCC) and lactose content. The percentage of leukocytes in the total somatic cells amounted to about 50% in the milk samples, which contained a high number of CNS or major pathogens. In the remaining samples this value reached only about 35%. Close relationship occurred between the presence of bacterial pathogens and total SCC, percentage of all leukocytes and their subpopulations in milk. The percentage of eosinophils and neutrophils in the total SCC were dramatically higher (p ≤ 0.0016) in samples of group 4 as compared to groups 1, 2 and 3. The percentage of monocytes was the highest in milk samples containing large numbers of minor pathogens. No relationship was found between the type of isolated bacterial pathogen and the percentage of lymphocytes in milk. In most samples, the presence of bacterial pathogens in goat milk led to the increase of the total SCC. However, the microbiological analysis showed that the bacterial pathogens were presented in about 20% of milk samples containing low SCC (below 1 × 10⁶/mL).     

Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds.

Epithelialization of normal acute wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore barrier function. The keratinocytes in the epidermis of chronic ulcers fail to execute this series of events. To better understand the epithelial dynamics of chronic ulcers, we used immunohistochemistry to evaluate proliferation, differentiation, adhesion, and migration in keratinocytes along the margin of chronic ulcers from patients with diabetes mellitus. We compared these features with keratinocytes from the migrating epithelial tongues of acute incisional and excisional wounds from normal volunteers. Keratinocytes at the chronic ulcer edge are highly proliferative (Ki67 proliferation marker), have an activated phenotype (K16), do not stain for keratins involved in epidermal differentiation (K10 and K2), and show a reduced expression of LM-3A32 (uncleaved, precursor of the alpha3 chain of laminin 5), a key molecule present on migrating epithelium. In contrast, keratinocytes in normal acute wound migrating epithelium do not express the proliferation marker Ki67 but do express K10, K2, and LM-3A32. A better understanding of molecular mechanisms involved in keratinocyte migration may lead to molecular targets for therapies for impaired wound healing.     

Transition from wind pollination to insect pollination in sedges: experimental evidence and functional traits

Transitions from wind pollination to insect pollination were pivotal to the radiation of land plants, yet only a handful are known and the trait shifts required are poorly understood. We tested the hypothesis that a transition to insect pollination took place in the ancestrally wind-pollinated sedges (Cyperaceae) and that floral traits modified during this transition have functional significance. We paired putatively insect-pollinated Cyperus obtusiflorus and Cyperus sphaerocephalus with related, co-flowering, co-occurring wind-pollinated species, and compared pairs in terms of pollination mode and functional roles of floral traits. Experimentally excluding insects reduced seed set by 56-89% in putatively insect-pollinated species but not in intermingled wind-pollinated species. The pollen of putatively insect-pollinated species was less motile in a wind tunnel than that of wind-pollinated species. Bees, beetles and flies preferred inflorescences, and color-matched white or yellow models, of putatively insect-pollinated species over inflorescences, or color-matched brown models, of wind-pollinated species. Floral scents of putatively insect-pollinated species were chemically consistent with those of other insect-pollinated plants, and attracted pollinators; wind-pollinated species were unscented. These results show that a transition from wind pollination to insect pollination occurred in sedges and shed new light on the function of traits involved in this important transition.     

Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY

Sequence alignments revealed that the conserved motifs of SOR_Sa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SOR_Sa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SOR_Sa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SOR_Sa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53–100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.     

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.     

Inferring temporal shifts in landuse intensity from functional response traits and functional diversity patterns: a study of Scotland's machair grassland

Plant functional response traits, which consistently respond to the environment, are useful for identifying drivers of vegetation change, particularly in response to disturbance gradients. Similarly, functional diversity indices have proven useful for investigating processes governing community assembly, particularly patterns of functional convergence/divergence. This study investigated the functional ecology of biodiverse, seminatural coastal grasslands (Scottish machair) at the national scale. We examined temporal shifts in functional response traits and functional diversity metrics using a series of null model, multivariate and regression analyses. The aim was to link temporal shifts in traits and diversity metrics to environmental variables in which to gauge the contribution of landuse change to plant functional composition and processes governing plant assembly. We observed significant shifts in the composition of 8 out of 12 functional response traits at the national scale, whereas at the regional scale all traits displayed at least one significant shift. Ordination of response traits found PC axis 1 (accounting for 39% of the variation) to be positively correlated to vegetation height and negatively correlated to specific leaf area, similar to that expected along a disturbance gradient. Significant changes in functional diversity indices were also observed at both national and regional scales, with varying convergence/divergence patterns observed across individual regions. We found functional richness (t = 4.87, p < 0.001) and divergence (t = 9.3, p < 0.001) to increase along PC axis 1, suggesting greater convergence and lower divergence along a disturbance gradient. This study demonstrates the potential for using functional diversity indices in combination with response traits as a sensitive method for detecting landuse change and its impacts on biodiversity. We conclude that landuse change, particularly management declines and intensification is a major driver governing change among the functional composition and functional diversity for machair grasslands, influencing convergence/divergence patterns, and subsequently community assembly processes.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.