Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

Antimicrobial Activity of Silver Nanoparticles in a Carboxymethyl Chitin Matrix Obtained by the Microwave Hydrothermal Method

Silver nanoparticles have been obtained in a matrix of 6-O-carboxymethyl chitin in the presence of D-glucose as a reducing agent by microwave hydrothermal synthesis. The TEM results show that the silver nanoparticles have a spherical shape; the particle size range is 3–20 nm. The resulting colloidal solution of silver nanoparticles had a strong bacterial effect on gram-positive bacteria Staphylococcus aureus ATCC 21027 (=209 P), Bacillus subtilis ATCC 6633, and, to a lesser extent, on gram-negative bacteria Escherichia coli АТСС 25922. The synthesized silver nanoparticles showed pronounced fungistatic activity against A. niger INA 00760.     

Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.     

Efficient inhibition of some multi-drug resistant pathogenic bacteria by bioactive metabolites from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> S<Subscript>5</Subscript>I<Subscript>4</Subscript> isolated from archaeological soil in Egypt

Sixty-eight bacterial cultures were isolated from 5 archaeological soils in Egypt. It is necessary to characterize bacteria from ancient temples to develop protection programs for such archaeological places. Purified bacterial cultures were then tested for their capability to inhibit some multi-drug resistant (MDR) pathogenic bacteria including Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli and Klebsiella pneumoniae. Among the most active 10 antibacterial isolates, only one isolate designated as S₅I₄ was selected, characterized and identified as belonging to Bacillus amyloliquefaciens. The strain identification was confirmed by amplification of its 16S rRNA gene. The partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted in GenBank with accession number AB813716. The physical and nutritional parameters were optimized to improve the production of antimicrobial agents by the B. amyloliquefaciens S₅I₄. The maximum antagonistic effect of this strain against the tested MDR pathogenic bacteria was achieved in presence of 1% galactose and 0.5% yeast extract at 37°C and pH 7.0 after 48 h incubation. The antibacterial compounds of B. amyloliquefaciens S₅I₄ were extracted, purified and characterized using spectroscopic analysis (IR, UV, proton NMR and MS). The compound having inhibitory activity was identified as butanedioic acid, octadecyl,1(1carboxy1methylethyl) 4octyl ester.     

Characterization of bacteriophages specificity for antibiotic-resistant <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

This study aimed to propose a new approach to understand the binding interaction between bacteriophages and antibiotic-resistant Salmonella typhimurium. The antibiotic susceptibilities of S. typhimurium strains were determined using a broth dilution method. The phage adsorption rates were determined to evaluate the lytic ability of bacteriophages against S. typhimurium strains. Bacterial outer membrane proteins and lipopolysaccharide (LPS) were analyzed to evaluate the antibiotic-induced alteration in bacterial cell surface receptors. The relative expression levels of outer membrane-, flagella-, porin-, and O-antigen-related genes were estimated using a qPCR assay. Compared to ST^WT, the ST^CIP exhibited a reduced susceptibility to cefotaxime (32-fold), ciprofloxacin (32-fold), meropenem (16-fold), and norfloxacin (64-fold). PBST35 produced adsorption rates of 82–95% at ST^WT, ST^CIP, and ST^CCARM within the first 10 min of infection. Compared to ST^WT, ST^CIP exhibited less protein bands between 24 and 36 kDa, corresponding to the low adsorption rates of P22 and PBST10. The relative expression levels of outer membrane-related genes (btuB, ompC, and tolC), flagellar-related genes (fliC, fljB, and fliK), porin-related gene (fhuA), and O-antigen-related genes (rfaL) were decreased in ST^CIP. The alteration in bacteriophage-binding receptors resulted in the low adsorption rate. Our findings provide new insights for effective treatment against antibiotic-resistant bacteria. The results would help to develop new therapeutic strategy as a prospective alternative control of antibiotic-resistant bacteria.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

In vitro assessment of antimicrobial activity of aqueous and alcoholic extracts of moss <Emphasis Type="Italic">Atrichum undulatum</Emphasis> (Hedw.) P. Beauv.

Bryophytes, the shade loving plants, have tremendous medicinal properties. The aqueous and alcoholic extracts of Atrichum undulatum (Hedw.) P. Beauv. were analysed for antimicrobial properties against the fungi Aspergillus fumigatus and Fusarium oxysporum and the bacteria Escherichia coli, Bacillus mycoides, Proteus mirabilis, Staphylococcus aureus and Salmonella typhi. The study is an attempt to investigate the medicinal properties of Atrichum undulatum (Hedw.) P. Beauv. using disc-diffusion method. No inhibition was observed against A. fumigatus and P. mirabilis. For bacteria S. typhi and E. coli (20 and 15 mm), aqueous and alcoholic extracts of Atrichum showed significant inhibition. However, alcoholic extract was found remarkably effective against bacteria rather than aqueous extract.     

Influence of FtsZ proteins from some mycoplasma species on the division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

FtsZ is a highly conserved protein that performs one of the key roles in cell division of most prokaryotes. At the present time it is believed that the main role of FtsZ in the cells of the microorganisms studied (Escherichia coli, Bacillus subtilis, etc.) is to control the process of cell wall synthesis at the site of future septum. In this regard, the presence of FtsZ in the cells of mycoplasmas – bacteria that lack the cell wall, looks intriguing. In the present work we investigated the influence of FtsZ proteins of three mycoplasmas – Mycoplasma hominis, Mycoplasma gallisepticum and Acholeplasma laidlawii – on the cytokinesis of E. coli. FtsZ proteins were able to interact with the E. coli division machinery, including inhibiting the cytokinesis process, while demonstrating various patterns of distribution in the cell. The data obtained support the hypothesis that FtsZ plays a significant role in the division of mycoplasma cells.     

The antibacterial activity of <Emphasis Type="Italic">E. coli</Emphasis> bacteriophage lysin lysep3 is enhanced by fusing the <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> bacteriophage endolysin binding domain D8 to the C-terminal region

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.     

Madurastatin B3, a rare aziridine derivative from actinomycete <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. LS150010 with potent anti-tuberculosis activity

Since the discovery of the first antibiotic, natural products have played an important role in chemistry, biology and medicine. To explore the potential of bioactive compounds from microbes isolated from the southeast of Tibet, China, a crude extract library was constructed and screened against Staphylococcus aureus. The strain Nocardiopsis sp. LS150010 was scaled up and subjected to further chemical studies, resulting in the identification of N-salicyloyl-2-aminopropan-1,3-diol (2) and its rare aziridine derivative, madurastatin B3 (1). Their structures were determined by detailed analysis of 1D, 2D NMR and HRMS data. Compounds 1 and 2 displayed significant inhibitory activity against S. aureus and methicillin resistant S. aureus, with MIC values of 6.25 µg/mL. Compound 1 also showed potent inhibitory activity against Bacillus subtilis and Escherichia coli, as well as activity in a Mycobacterium tuberculosis Bacillus Calmette-Guérin infected THP-1 cell model.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

TatC-dependent translocation of pyoverdine is responsible for the microbial growth suppression

Infections are often not caused by a colonization of Pseudomonas aeruginosa alone but by a consortium of other bacteria. Little is known about the impact of P. aeruginosa on the growth of other bacteria upon coinfection. Here, cellree culture supernatants obtained from P. aeruginosa suppressed the growth of a number of bacterial strains such as Corynebacterium glutamicum, Bacillus subtilis, Staphylococcus aureus, and Agrobacterium tumefaciens, but had little effect on the growth of Escherichia coli and Salmonella Typhimurium. The growth suppression effect was obvious when P. aeruginosa was cultivated in M9 minimal media, and the suppression was not due to pyocyanin, a well-known antimicrobial toxin secreted by P. aeruginosa. By performing transposon mutagenesis, PA5070 encoding TatC was identified, and the culture supernatant of its mutant did not suppress the growth. HPLC analysis of supernatants showed that pyoverdine was a secondary metabolite present in culture supernatants of the wild-type strain, but not in those of the PA5070 mutant. Supplementation of FeCl₂ as a source of iron compromised the growth suppression effect of supernatants and also recovered biofilm formation of S. aureus, indicating that pyoverdine-mediated iron acquisition is responsible for the growth suppression. Thus, this study provides the action of TatC-dependent pyoverdine translocation for the growth suppression of other bacteria, and it might aid understanding of the impact of P. aeruginosa in the complex community of bacterial species upon coinfection.     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Antimicrobial effect of OKCEL® H-D prepared from oxidized cellulose

The antimicrobial effect of OKCEL® H-D, a topical, absorbable hemostatic textile prepared from oxidized cellulose, was tested. Testing by dilution and diffusion methods was conducted on a spectrum of 27 select microorganisms, including also antibiotic-resistant strains. OKCEL® H-D showed inhibitory effects on nearly all tested bacteria. In testing using the dilution suspension method, the majority of bacteria showed decrease in cell density by 7–8 orders of magnitude after just 6 h of exposure. For clinical isolates of antibiotic-resistant strains, a reduction occurred after 24 h of exposure. In testing the antimicrobial effects of OKCEL® H-D by the dilution method was least effective on spore-forming Bacillus subtilis, for which no antimicrobial effect was detected after 48 h, and on Mycobacterium smegmatis, for which the number of cells decreased by four orders of magnitude only after 24 h. By the diffusion method, inhibition zones were recorded for nearly all test microorganisms except for Staphylococcus aureus, M. smegmatis, and Listeria monocytogenes. No growth beneath the tested OKCEL® H-D material was recorded, however, even for the latter-named bacteria strains, which attests to its good inhibitory effect.     

Inhibitory effects of YCW and MOS from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella pullorum</Emphasis> adhesion to Caco-2 cells

Background

For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.

Methods

YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.

Results

The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.

Conclusions

These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.