Potential for <Emphasis Type="Italic">luxS</Emphasis> related signalling in marine bacteria and production of autoinducer-2 in the genus <Emphasis Type="Italic">Shewanella</Emphasis>

Background  

The autoinducer-2 (AI-2) group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat.     

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

Background  

In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.     

Evolutionary relationships of <Emphasis Type="Italic">Fusobacterium nucleatum</Emphasis> based on phylogenetic analysis and comparative genomics

Background  

The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Induction of temperate cyanophage AS-1 by heavy metal - copper

Background  

It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle.     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

The role of iron uptake in pathogenicity and symbiosis in <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> TT01

Background  

Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺ and Fe²⁺ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.     

Diet shapes the ability of human intestinal microbiota to degrade phytate – in vitro studies

Aims

Investigation of intestinal bacterial groups involved in phytate degradation and the impact of diets with different phytate contents on phytase activity.

Methods and Results

Faecal samples of adults on conventional (n = 8) or vegetarian (n = 8) diets and breastfed infants (n = 6) were used as an inoculum for modified media supplemented with phytate. Populations of Gram‐positive anaerobes (GPA), lactic acid bacteria (LAB), Proteobacteria–Bacteroides (P‐B), coliforms and anaerobes were studied. The PCR‐DGGE analysis revealed a random distribution of DGGE profiles in the dendrograms of GPA, P‐B and coliforms, and a partially diet‐specific distribution in the DGGE dendrograms of LAB and anaerobes. The degradation of phytic acid (PA) was determined with HPLC method in supernatants of the cultures. Regardless of the diet, the Gram‐positive anaerobes and LAB displayed the lowest ability to degrade phytate, whereas the coliforms and P‐B cultures produced higher amounts of intermediate myo‐inositol phosphates. Bacterial populations grown in a nonselective medium were the most effective ones in phytate degradation. It was the vegetarians' microbiota that particularly degraded up to 100% phytate to myo‐inositol phosphate products lower than InsP₃.

Conclusions

A diet rich in phytate increases the potential of intestinal microbiota to degrade phytate. The co‐operation of aerobic and anaerobic bacteria is essential for the complete phytate degradation.

Significance and Impact of the Study

This study provides insights on the effect of diet on specific metabolic activity of human intestinal microbiota.     

Biochanin A improves fibre fermentation by cellulolytic bacteria

Aims

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity.

Methods and Results

When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short‐chain fatty acids were produced and pH declined. BCA (30 μg ml^?1) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent.

Conclusions

These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition.

Significance and Impact of the Study

BCA could serve as a feed additive to improve cellulosis when cattle are consuming high‐fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.     

Identification of genes involved in the mutualistic colonization of the nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> by the bacterium <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>

Background  

Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.     

Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora

Background  

Despite their antimicrobial potential, vaginal lactobacilli often fail to retain dominance, resulting in overgrowth of the vagina by other bacteria, as observed with bacterial vaginosis. It remains elusive however to what extent interindividual differences in vaginal Lactobacillus community composition determine the stability of this microflora. In a prospective cohort of pregnant women we studied the stability of the normal vaginal microflora (assessed on Gram stain) as a function of the presence of the vaginal Lactobacillus index species (determined through culture and molecular analysis with tRFLP).     

Properties and identification of antibiotic drug targets

Background  

We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from E. coli only and 4243 non-drug targets from E. coli to identify differences in their properties and to predict new potential drug targets.     

Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae

Aims

American foulbrood, caused by the Gram‐positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested.

Methods and Results

Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed.

Conclusions

The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae.

Significance and Impact of the Study

The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.     

Influence of Physico-Chemical Factors on the Oligomerization and Biological Activity of Bacteriocin AS-48

Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml⁻¹, but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120°C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria. Received: 3 July 2000\t/\tAccepted: 11 August 2000     

Brucin,an antibacterial peptide derived from fruit protein of Fructus Bruceae,Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l^?1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

Significance and Impact of the Study

An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L.) Merr. by pepsin‐catalysed hydrolysis. Its inhibitory activity towards the Gram‐positive bacteria was higher than penicillin G and chloramphenicol. The result suggested that Brucin may be applied for the treatment of the disease caused by Strep. pyogenes^*.     

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus > V. anguillarum > M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+ 35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+ 16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus > V. anguillarum > M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.     

Comparison of the in vitro and in vivo susceptibilities of Burkholderia mallei to Ceftazidime and Levofloxacin

Background  

Burkholderia mallei is a zoonotic Gram negative bacterium which primarily infects solipeds but can cause lethal disease in humans if left untreated. The effect of two antibiotics with different modes of action on Burkholderia mallei strain ATCC23344 was investigated by using in vitro and in vivo studies.     

Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora

Background  

The microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species.