Bacteria Micrococcus Research Articles

Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8

The potential production of rhamnolipids was demonstrated using the thermophilic eubacterium Thermus thermophilus HB8 and sunflower seed oil or oleic acid as carbon sources. Sunflower seed oil was directly hydrolyzed by secretion of lipase and became a favorable carbon source for rhamnolipids production. Rhamnolipids levels were attainted high values, comparable to those produced by Pseudomonas strains from similar sources. Rhamnolipids synthesis in oleic acid exhibited a long period of induction, while in sunflower seed oil, the synthesis is more rapid. Glucose resulted in a more protracted period of rhamnolipids production after exhaustion of each or both carbon sources. Both mono- and di-rhamnolipids were identified by thin-layer chromatography (TLC) in the total rhamnolipids extract. The molecular composition of the produced biosurfactant was evaluated by Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and LC-MS analysis. Furthermore, secretion of rhamnolipids was confirmed on agar plates. The antimicrobial activity of rhamnolipids was detected against the bacterium Micrococcus lysodeikticus using a lysoplate assay. These results demonstrate that rhamnolipids produced in these substrates can be useful in both environmental and food industry applications by using cheap oil wastes. The alternative use of this thermophilic microorganism opens a new perspective concerning the valorization of wastes containing plant oils or frying oils to reduce the cost of rhamnolipids production.

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NFκB is a family of transcription factors. In the present study, a Rel/NFκB family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical p I was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G +) bacteria ( Micrococcus lysodeikticus) and Gram-negative (G −) bacteria ( Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NFκB member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway.

Molecular characterization and expression of a crustin-like gene from Chinese mitten crab, Eriocheir sinensis

Crustin is a cysteine-rich antibacterial peptide which widely distributes within decapod crustaceans. In the present study, the cDNA encoding crustin-like peptide (designated CrusEs) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag analysis. The full-length cDNA of CrusEs was of 796 bp, containing a 5′ untranslated region (UTR) of 214 bp, a 3′ UTR of 267 bp with a poly(A) tail, and an open reading frame (ORF) of 315 bp encoding a polypeptide of 104 amino acids including a putative signal peptide of 21 amino acids. A WAP domain and the consensus framework existing in class I crustins were both identified in CrusEs, suggesting that CrusEs is a new member of type I crustins. Quantitative real-time RT-PCR was employed to examine the expression of CrusEs, and its mRNA transcript was mainly detected in haemocytes and gill. The CrusEs mRNA transcript in haemocytes was down-regulated after the challenge with Gram-positive bacteria Micrococcus luteus, while Gram-negative bacteria Listonella anguillarum did not induce significant variation of CrusEs mRNA. The mature peptide of CrusEs was cloned into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant CrusEs inhibited the growth of Gram-positive bacteria with low MIC. The results indicate that CrusEs is a potent antibacterial protein against Gram-positive bacteria infection, and it may play an important role in innate immune response of E. sinensis.

Virulence potential of some fungal isolates and their control-promise against the Egyptian cotton leaf worm, Spodoptera littoralis

A preliminary virulence test of four fungal isolates, Beauveria bassiana IMI 382302, Beauveria bassiana IMI 386701, Trichoderma harzianum T24 and Aspergillus flavus Link against larvae of Spodoptera littoralis was performed. The most effective isolates against larvae of S. littoralis were B. bassiana 302 and T. harzianum T24, which also showed the lower percentage of pupation compared with the other two isolates under the same conditions of treatments. Three concentrations (1 × 106, 1 × 107 and 1 × 108 ml−1) of the aqueous conidial suspension of the four tested isolates were carried out against both larval and pupal stages of S. littoralis within five days post-treatment. T. harzianum T24 showed 80% larval mortality only when applied at the highest conidial concentration, while A. flavus showed 100% pupal mortality only, at all of its conidial concentrations. However, B. bassiana IMI 382302 showed relatively high dose-dependant larval and pupal mortalities, while strain IMI 386701 of B. bassiana showed a very weak mortality against pupae at higher concentrations, and no virulence against larvae was recorded. Enzymatic and antibiosis bioassays of the four fungal isolates showed relatively high activities against Fusarium spp. for most of the tested isolates. Clear zone of enzyme activity on agar plates proportionally increased with increasing the concentration of enzyme substrate and prolongation of the incubation period. Mtabolites produced in the agar culture inhibited the growth of Fusarium spp. and the productivity differed greatly among isolates or strains of the same isolate. Volatile and non-volatile compounds produced by A. flavus Link showed a higher inhibition activity against Fusarium spp. compared with the other fungal isolates. The humoral antifungal response of insect host is relatively high compared to the anti-bacterial one. Injection of larvae with the immune sensitive bacteria Micrococcus luteus (5 × 103 bacteria/larva) showed a detectable humoral response by 2 h, peaked around 12 h and became hardly detectable by 24 h post-injection. Injection of larvae with conidial suspension (5 × 103 conidia/larva) from each of the fungal isolates showed humoral antifungal activity against B. bassiana IMI 386701 and A. flavus only. This activity was detectable by 12 h, peaked around 36 h and became hardly detectable by 48 h post-injection. Although the humoral antifungal response was started slowly compared to the antibacterial one, it lasted for longer and enabled larvae to withstand the infection with these immune-sensitive fungal strains. No humoral activity was detected against B. bassiana IMI 382302, although however, weak activity was detected against T. harzianum T24 only at the low conidial concentration but not at the higher one (1 × 108 ml−1). Thus, this study concludes that larvae of S. littoralis showed immune-dependant sensitivity to T. harzianum T24 and B. bassiana IMI 382302. Therefore, this study may recommend these two fungal isolates as mycoinsecticides in the battle against cotton leaf worm in Egypt. Hence, they have been selected for future comprehensive bioassays in the laboratory under conditions similar to that in the field. This, in fact, may help for developing effective mycoinsecticides against this pest. Penetration mechanims of insect cuticle by entomopathogenic fungi will be discussed.

Expression of Acc-Royalisin Gene from Royal Jelly of Chinese Honeybee in Escherichia coli and Its Antibacterial Activity

Royalisin is an antibacterial peptide found in Royal Jelly. Two gene fragments of Chinese honeybee (Apis cerana cerana) head, 280 bp cDNA encoding pre-pro-Acc-royalisin (PPAR) of 95 amino acid residues, and 165 bp cDNA encoding mature Acc-royalisin (MAR) of 51 amino acid residues were cloned into the pGEX-4T-2 vector. They were then transformed individually into Escherichia coli for expression. Two expressed fusion proteins, glutathione S-transferase (GST)-PPAR of 36 kDa and GST-MAR of 32 kDa were obtained, which were cross reacted with GST antibody accounting for up to 16.3% and 15.4% of bacterial protein, respectively. In addition, 41% of GST-PPAR and nearly 100% of GST-MAR were soluble proteins. Both lysates of the two purified fusion proteins displayed antibacterial activities, similar to that of nisin against Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus. MAR peptide released from the thrombin-cleaved GST-MAR fusion protein has a stronger antibacterial activity than that of GST-MAR fusion protein.

Size-resolved culturable airborne bacteria sampled in rice field, sanitary landfill, and waste incineration sites

Size-resolved bacterial concentrations in atmospheric aerosols sampled by using a six stage viable impactor at rice field, sanitary landfill, and waste incinerator sites were determined. Culture-based and Polymerase Chain Reaction (PCR) methods were used to identify the airborne bacteria. The culturable bacteria concentration in total suspended particles (TSP) was found to be the highest (848 Colony Forming Unit (CFU)/m(3)) at the sanitary landfill sampling site, while the rice field sampling site has the lowest (125 CFU/m(3)). The closed landfill would be the main source of the observed bacteria concentration at the sanitary landfill. The rice field sampling site was fully covered by rice grain with wetted conditions before harvest and had no significant contribution to the airborne bacteria concentration. This might occur because the dry conditions favor suspension of soil particles and this area had limited personnel and vehicle flow. The respirable fraction calculated by particles less than 3.3 mum was highest (26%) at the sanitary landfill sampling site followed by waste incinerator (19%) and rice field (10%), which showed a lower level of respiratory fraction compared to previous literature values. We identified 58 species in 23 genera of culturable bacteria, and the Microbacterium, Staphylococcus, and Micrococcus were the most abundant genera at the sanitary landfill, waste incinerator, and rice field sites, respectively. An antibiotic resistant test for the above bacteria (Micrococcus sp., Microbacterium sp., and Staphylococcus sp.) showed that the Staphylococcus sp. had the strongest resistance to both antibiotics (25.0% resistance for 32 microg ml(-1) of Chloramphenicol and 62.5% resistance for 4 microg ml(-1) of Gentamicin).

Antioxidant, antimicrobial, and tyrosinase inhibition activities of acetone extract of Ascophyllum nodosum

AbstractThe search for new antioxidants of natural origin derived from plants and seaweeds is still very important at present. In our study, the acetone extract of A. nodosum was investigated for its potential use as a natural antioxidant, natural feed additive with antibacterial activity and as a tyrosinase inhibitor. This study could be useful in the context of improved utilization of the A. nodosum extract in the food and cosmetics industry, being not only economically advantageous but also environmentally friendly. Extracts showed antioxidant activity with application of different methodologies: 1,1-diphenyl-2-picrilhydracil DPPH· radicals scavenging (39 %, 4 mg of freeze-dried sample), β-carotene-linoleic acid antioxidant assay (11 %, 4 mg of freeze-dried sample), O2· radicals scavenging activity (IC50 0.43 mg mL−1), OH· radicals scavenging activity (IC50 1.55 mg mL−1), and iron chelation ability (IC50 0.56 mg mL−1). The extract showed considerable antibacterial activity being more effective against gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus) than against gram-negative bacteria (Escherichia coli, Enterococcus aerogenes). Results of tyrosinase assay for the acetone extract of Ascophyllum nodosum presented 65.6 % inhibition of tyrosinase activity at the IC50 value of 0.1 mg mL−1. The outcomes of our study support potential utilization of this brown seaweed as a source of natural antioxidants. Antioxidant activity of the studied seaweed can be apparently explained by the free radicals scavenging activity, particularly related to the mechanisms of O2−· radicals scavenging activity, OH· radicals inactivation, and iron chelation ability.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Synthesis and evaluation of a series of aminocyanopyridines as antimicrobial agents

With the aim of developing potential antimicrobials, a series of 2-amino-3-cyanopyridines incorporating both sulfur and oxygen as part of the heteroaromatic ring (methyl thiophene, methyl furan) and fused cycloalkane groups were synthesized and characterized by FTIR, 1H-NMR, 13C-NMR, and bases of elemental analysis. All synthesized compounds were evaluated for their in vitro antibacterial and antifungal activity. Antibacterial and antifungal activities of aminocyanopyridines against Pseudomonas aeruginosa ATCC 29212, Bacillus subtilis RSKK 244, Bacillus megaterium (clinical isolate), the gram-positive bacterium Micrococcus luteus NRRLB 4375, and the fungus Candida albicans ATCC 90028 were studied. The relationship between the functional-group variation and the biological activity of the evaluated compounds is discussed.

The use of chromogenic bacteria as coloured substitutes for pathogens: A simple strategy during design and development of a new method for sample pretreatment

In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set-up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red-coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.

Dormant forms of Micrococcus luteus and Arthrobacter globiformis not platable on standard media

The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in the course of 3–9 month incubation to the level of 106–107 CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although preincubation of CRC suspensions in a buffer solution of 0.1 M K2HPO4 (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation of macrocolonies by a small portion of nonplateable CRC of M. luteus (50−80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 105 mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number (MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’ micrococcal CRCs to microcolony formation was observed (up to 2.3 × 107 mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary methods is of great importance for advanced monitoring studies.

Liquid Crystal Emulsions as the Basis of Biological Sensors for the Optical Detection of Bacteria and Viruses

AbstractA versatile sensing method based on monodisperse liquid crystal (LC) emulsion droplets detects and distinguishes between different types of bacteria (Gram +ve and −ve) and viruses (enveloped and non‐enveloped). LCs of 4‐cyano‐4'‐pentylbiphenyl transition from a bipolar to radial configuration when in contact with Gram −ve bacteria (E. coli) and lipid‐enveloped viruses (A/NWS/Tokyo/67). This transition is consistent with the transfer of lipid from the organisms to the interfaces of the micrometer‐sized LC droplets. In contrast, a transition to the radial configuration is not observed in the presence of Gram +ve bacteria (Bacillus subtilis and Micrococcus luteus) and non‐enveloped viruses (M13 helper phage). The LC droplets can detect small numbers of E. coli bacteria (1–5) and low concentrations (104 pfu mL−1) of A/NWS/Tokyo/67 virus. Monodisperse LC emulsions incubated with phosholipid liposomes (similar to the E. coli cell wall lipid) reveal that the orientational change is triggered at an area per lipid molecule of ∼46 Å2 on an LC droplet (∼1.6 × 108 lipid molecules per droplet). This approach represents a novel means to sense and differentiate between types of bacteria and viruses based on their cell‐wall/envelope structure, paving the way for the development of a new class of LC microdroplet‐based biological sensors.

Dielectrophoretic Separation of Airborne Microbes and Dust Particles Using a Microfluidic Channel for Real-Time Bioaerosol Monitoring

Airborne microbes such as fungi, bacteria, and viruses are a threat to public health. Robust and real-time detection systems are necessary to prevent and control such dangerous biological particles in public places and dwellings. For direct and real-time detection of airborne microbes, samples must be collected and typically resuspended in liquid prior to detection; however, environmental particles such as dust are also trapped in such samples. Therefore, the isolation of target bacteria or a selective collection of microbes from unwanted nonbiological particles prior to detection is of great importance. Dielectrophoresis (DEP), the translational motion of charge neutral matter in nonuniform electric fields, is an emerging technique that can rapidly separate biological particles in microfluidics because low voltages produce significant and contactless forces on particles without any modification or labeling. In this paper, we propose a new method for the separation of airborne microbes using DEP with a simple and novel curved electrode design for separating bacteria in a solution containing beads or dust that are taken from an airborne environmental sample. Using this method, we successfully isolated 90% of the airborne bacterium Micrococcus luteus from a mixture of bacteria and dust using a microfluidic device, consisting of novel curved electrodes that attract bacteria and repel or leave dust particles. As there has been little research on analyzing environmental samples using microfluidics and DEP, this work describes a novel strategy for a rapid and direct bioaerosol monitoring system.

Mapping of Apidaecin Regions Relevant for Antimicrobial Activity and Bacterial Internalization

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.

Functional and molecular immune response of Mediterranean mussel ( Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria

The effect of live bacteria ( Micrococcus lysodeikticus and Vibrio anguillarum), and PAMPs (poly I:C, zymosan, LPS, LTA and CpG) on the production of intermediate toxic radicals (respiratory burst activity and production of nitric oxide) and mytilin B, myticin C and lysozyme gene expression was studied in vivo and in vitro. In vitro, bacteria were able to modulate the haemocytes' respiratory burst activity, being significantly increased after 6 h of incubation. The effect of pathogen-associated molecular patterns (PAMPs) was also studied. Zymosan produced an increase of the PMA-mediated response but an inhibition of the zymosan-mediated response. A significant increase of nitric oxide production was found at all the sampled time points (1, 3 and 6 h) in comparison with controls on both, the Gram-positive and Gram-negative bacteria. The in vivo responses measured on haemocytes after M. lysodeikticus injection were faster than those induced by V. anguillarum. However, V. anguillarum induced stronger in vitro effects. Mytilin B, myticin C and lysozyme in vitro gene expression, occurred at short times after infection. The maximum in vitro expression was detected 3 h post-infection. The differences between M. lysodeikticus and V. anguillarum in different measured parameters may suggest that different signalling pathways might be involved. Moreover, among all assayed PAMPs, LPS elicited the highest response.

A study into measuring the antibacterial activity of lysozyme‐containing foods

PurposeThe purpose of this paper is to provide food science or biology students with a simple and reliable method of determining the antibacterial activity of a range of foods and biological materials that contain lysozyme.Design/methodology/approachThe antibacterial effects of the materials reported to contain lysozyme were assayed by gel‐diffusion using the lysozyme‐sensitive bacterium Micrococcus lysodeikticus. The antibacterial effects of the selected test materials, namely fresh hen egg‐white, human saliva, Brussels sprouts, papaya and figs were compared against standard solutions of proprietary analytical crystalline hen egg‐white lysozyme.FindingsThe antibacterial activity of the test substances was similar to the effects of their lysozyme concentrations as quoted by other workers.Research limitations/implicationsAntibacterial activity was higher in avian egg‐white and human saliva than in the assayed plant material. Measurement of the activity in the plant material was at the limits of the sensitivity of the method.Practical implicationsThe two main practical methods for measuring lysozyme are either a gel‐diffusion assay or a spectrophotometric procedure. Gel‐diffusion assay provides a convenient procedure for student investigative work as it has a limited requirement for method development. Additionally, it is simple, cheap, reproducible and does not require specialist equipment. Further possible investigations for students are suggested.Originality/valueThe paper builds on established techniques to provide a procedure that is appropriate for student use for determining lysozyme activity in a variety of biological materials.

ASSOCIATED BACTERIA INCREASE THE PHYTOEXTRACTION OF CADMIUM AND ZINC FROM A METAL-CONTAMINATED SOIL BY MYCORRHIZAL WILLOWS

In order to enhance phytoremediation efficiency, we investigated the effects of dual inoculation with ectomycorrhizal fungi and the ectomycorrhiza associated bacteria Micrococcus luteus and Sphingomonas sp. on the growth and metal accumulation of willows (Salix viminalis x caprea) on contaminated soil. The bacterial strains were previously collected from sporocarps of ectomycorrhizal fungi. The bacteria increased plant growth and the mycorrhizal dependency of willows colonized with the ectomycorrhizal fungus Hebeloma crustuliniforme. The total cadmium (Cd) and zinc (Zn) accumulation in the shoot biomass was increased after inoculation with the fungal strain Hebeloma crustuliniforme in combination with Micrococcus luteus up to 53% and in combination with Sphingomonas sp. up to 62%, respectively. The dual inoculation in combination with Laccaria laccata did not increase the accumulation of Cd and Zn in the willows. We conclude that associated bacteria can enhance the ectomyorrhiza formation and growth of willows and, thereby, the Cd and Zn accumulation in the plant biomass. The results suggest that bacterial support of root growth promoting ectomycorrhizal fungi may be a promising approach to improve the remediation of metal-contaminated soils by using willows.

Grafting of Lysozyme and/or Poly(ethylene glycol) to Prevent Biofilm Growth on Stainless Steel Surfaces

In the aim of protecting stainless steel surfaces against protein and/or bacterial adhesion, thin films including the glycosidase hen egg white lysozyme (HEWL) and/or the synthetic polymer poly(ethylene glycol) (PEG) were covalently coated onto flat substrates by wet chemical processes. Chemical grafting of both species was carried out by covalent binding to surfaces pretreated by the polyamine poly(ethylene imine) (PEI). Surfaces were characterized at each step of functionalization by means of reflection-absorption infrared spectroscopy by modulation of polarization (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to determine the atomic and molecular composition of the interfaces, respectively. Then, the ability of the so-modified surfaces to prevent protein adsorption and bacterial adhesion together with their biocide properties were demonstrated by three local tests employing bovine serum albumin (BSA), and the bacteria Listeria ivanovii and Micrococcus luteus. A new test was implemented to assess the local enzymatic properties of HEWL. Cografting of PEG and HEWL resulted in a surface with both antiadhesion and antibacterial properties.