Influence of polyamine production and proteolytic activities of co-cultivated bacteria on histamine production by Morganiella morganii.

Consumption of temperature-abused marine fish containing elevated levels of histamine results in histamine poisoning. Histamine is a biogenic amine produced in fish by the action of certain groups of bacteria which are capable of producing an exogenous enzyme called histidine decarboxylase (HDC). Morganella morganii is one of the major causative organisms of histamine poisoning. In this study, the histamine forming potential of M. morganii (BSS142) was evaluated when it was co-incubated with proteolytic as well as polyamine forming bacteria. This experiment was designed to examine whether biotic factors such as proteolysis and the presence of other amines influenced histamine forming ability of BSS142. The study showed that the proteolytic activity of Aeromonas hydrophila as well as Pseudomonas aeruginosa greatly enhanced the histamine forming ability of M. morganii. Psychrobacter sangunis, a non proteolytic polyamine producer, negatively influenced histamine production by M. morganii.

Features of Hemolysin Biosynthesis by Morganella morganii.

We studied the influence of medium composition and aeration on the hemolytic activity of uropathogenic Morganella morganii strain MM 190. The maximum level of hemolysis was observed in LB (59%), DMEM supplemented with fetal bovine serum (62%), and urine (53%) under aeration conditions during the exponential growth phase. The presence of 2% urea in the medium suppressed hemolysin synthesis. Moreover, addition of bacterial culture fluid containing hemolysin to a monolayer of T-24 bladder carcinoma and OKP-GS kidney carcinoma cells led to 25 and 42% cell death, respectively. We found that the maximum expression of the hemolysin gene hlyA was observed in 2-h culture in LB medium, which correlated with the hemolytic activity of the bacteria in this medium and indicated the predominance of the short hlyCA transcript in the cells.

Microbial metabolites damage DNA.

Unexpected members of the gut microbiota produce diverse host cell genotoxins.

Commensal microbiota from patients with inflammatory bowel disease produce genotoxic metabolites.

Microbiota-derived metabolites that elicit DNA damage can contribute to colorectal cancer (CRC). However, the full spectrum of genotoxic chemicals produced by indigenous gut microbes remains to be defined. We established a pipeline to systematically evaluate the genotoxicity of an extensive collection of gut commensals from inflammatory bowel disease patients. We identified isolates from divergent phylogenies whose metabolites caused DNA damage and discovered a distinctive family of genotoxins-termed the indolimines-produced by the CRC-associated species Morganella morganii. A non-indolimine-producing M. morganii mutant lacked genotoxicity and failed to exacerbate colon tumorigenesis in mice. These studies reveal the existence of a previously unexplored universe of genotoxic small molecules from the microbiome that may affect host biology in homeostasis and disease.

Physical characterization and kinetic studies of Zn (II) biosorption by Morganella morganii ACZ05.

Through this investigation, we establish the mechanism and physical characterization of zinc (II) sequestration by Morganella morganii ACZ05 strain, which was isolated and characterized from soil polluted by effluents from electroplating industries. As far as we know, there is very little literature concerning zinc biosorption using an environmental strain of M. morganii. The SEM analysis shows the dark porous gaps in the aggregated cell-matrix of test bacterial biomass which is inferred as water channels usually seen in biofilms, as compared to metal-unexposed control. M. morganii is not known to produce biofilms unless in the rare nosocomial conditions. Here, SEM analysis shows the production of biofilms after exposure to zinc (II) at 500 ppm, which has not been previously reported. EDX analysis of bacterial biomass also specified the sorption of zinc (II) by the bacterial cells and the presence of new peaks for zinc in contrast to control. Both XRD and FTIR analysis observations strongly implicate the potential of physical adsorption as a mechanism for heavy metal resistance. Analysis of the cell surface by Atomic force microscopy and examination of the topography revealed cell aggregation occurs during biofilm production after zinc biosorption. Unlike other reports, regular models such as Langmuir isotherm and Freundlich isotherm were found insufficient to explain the physisorption of zinc (II) metal ions on complex multicomponent adsorbents such as the exopolymeric surface of the bacterial cells. However, adsorption kinetics of zinc (II) to the bacterial biomass was most effectively elucidated by a pseudo-second-order kinetic model, suggesting a certain kind of chemisorption that requires further study.

High-Pressure Inactivation of Histamine-Forming Bacteria Morganella morganii and Photobacterium phosphoreum.

ABSTRACT: The effects of high hydrostatic pressure (HHP) treatments on histamine-forming bacteria (HFB) Morganella morganii and Photobacterium phosphoreum in phosphate buffer and tuna meat slurry were investigated using viability counting and scanning electron microscopy. The first-order model fits the destruction kinetics of high pressure on M. morganii and P. phosphoreum during the pressure hold period. The D-values of M. morganii (200 to 600 MPa) and P. phosphoreum (100 to 400 MPa) in phosphate buffer ranged from 16.4 to 0.08 min and 26.4 to 0.19 min, respectively, whereas those in tuna meat slurry ranged from 51.0 to 0.09 min and 71.6 to 0.19 min, respectively. M. morganii had higher D-values than P. phosphoreum at the same pressure, indicating it was more resistant to HHP treatment. HFB had a higher D-value in tuna meat slurry compared with that in phosphate buffer, indicating that the HFB were more resistant to pressure in tuna meat slurry. The Zp values (pressure range that results in a 10-fold change in D-value) of M. morganii and P. phosphoreum were 162 and 140 MPa in phosphate buffer and 153 and 105 MPa in tuna meat slurry, respectively. Damage to the cell wall and cell membrane by HHP treatments can be observed by scanning electron microscopy. To our knowledge, this is the first report to demonstrate that HHP can be applied to inactivate the HFB M. morganii and P. phosphoreum by inducing morphological changes in the cells.

Histamine Production Behaviors of a Psychrotolerant Histamine-Producer, Morganella psychrotolerans, in Various Environmental Conditions.

Histamine food poisoning is a major safety concern related to seafood consumption worldwide. Morganella psychrotolerans is a novel psychrotolerant histamine-producer. In this study, the histamine production behaviors of M. psychrotolerans and two other major histamine-producers, mesophilic Morganella morganii and psychrotrophic Photobacterium phosphoreum, were compared in seafood products, and histamine accumulation by M. psychrotolerans was characterized at various pH and temperature levels in culture broth. The growth of M. psychrotolerans and P. phosphoreum increased similarly at 4 °C in canned tuna, but M. psychrotolerans produced much higher levels of histamine than P. phosphoreum. Histamine accumulation by M. psychrotolerans was induced at lower environmental pH condition at 4 and 20 °C. The optimal temperature and pH for producing histamine by crude histidine decarboxylase of M. psychrotolerans were 30 °C and pH 7, respectively. The activity of the crude HDC extracted from M. psychrotolerans cells at 10 °C retained 45% of the activity at 30 °C. Histidine decarboxylase gene expression of M. psychrotolerans was induced by low pH conditions. These results suggest that M. psychrotolerans are also a very important histamine-producer leading to histamine poisoning associated with seafood below the refrigeration temperature.

Morganella morganii bacteria produces phenol as the sex pheromone of the New Zealand grass grub from tyrosine in the colleterial gland.

Costelytra zealandica (Coleoptera: Scarabeidae) is a univoltine endemic species that has colonised and become a major pest of introduced clover and ryegrass pastures that form about half of the land area of New Zealand. Female beetles were previously shown to use phenol as their sex pheromone produced by symbiotic bacteria in the accessory or colleterial gland. In this study, production of phenol was confirmed from the female beetles, while bacteria were isolated from the gland and tested for attractiveness towards grass grub males in traps in the field. The phenol-producing bacterial taxon was identified by partial sequencing of the 16SrRNA gene, as Morganella morganii. We then tested the hypothesis that the phenol sex pheromone is biosynthesized from the amino acid tyrosine by the bacteria. This was shown to be correct, by addition of isotopically labelled tyrosine ((13)C) to the bacterial broth, followed by detection of the labelled phenol by SPME-GCMS. Elucidation of this pathway provides specific evidence how the phenol is produced as an insect sex pheromone by a mutualistic bacteria.

A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections (CAUTIs).

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system.

Screening of phenylpyruvic acid producers and optimization of culture conditions in bench scale bioreactors.

Alpha keto acids are deaminated forms of amino acids that have received significant attention as feed and food additives in the agriculture and medical industries. To date, their production has been commonly performed at shake-flask scale with low product concentrations. In this study, production of phenylpyruvic acid (PPA), which is the alpha keto acid of phenylalanine was investigated. First, various microorganisms were screened to select the most efficient producer. Thereafter, growth parameters (temperature, pH, and aeration) were optimized in bench scale bioreactors to maximize both PPA and biomass concentration in bench scale bioreactors, using response surface methodology. Among the four different microorganisms evaluated, Proteus vulgaris was the most productive strain for PPA production. Optimum temperature, pH, and aeration conditions were determined as 34.5 °C, 5.12, and 0.5 vvm for PPA production, whereas 36.9 °C, pH 6.87, and 0.96 vvm for the biomass production. Under these optimum conditions, PPA concentration was enhanced to 1,054 mg/L, which was almost three times higher than shake-flask fermentation concentrations. Moreover, P. vulgaris biomass was produced at 3.25 g/L under optimum conditions. Overall, this study demonstrated that optimization of growth parameters improved PPA production in 1-L working volume bench-scale bioreactors compared to previous studies in the literature and was a first step to scale up the production to industrial production.

Heat resistance of histamine-producing bacteria in irradiated tuna loins.

Consumption of foods high in biogenic amines leads to an illness known as histamine, or scombrotoxin, poisoning. The illness is commonly associated with consumption of fish with high levels of histamine ( $ 500 ppm). The objective of this study was to determine and compare the heat resistance of five histamine-producing bacteria in irradiated albacore tuna loins. Heat-resistance parameters (D- and z-values) were determined for Morganella morganii, Raoultella planticola, Hafnia alvei, and Enterobacter aerogenes. D- or z-values were not determined for Photobacterium damselae, which was the most heat-sensitive organism in this study. P. damselae declined > 5.9 log CFU/g after a heat treatment of 50°C for 10 min, 54°C for 3 min, and 56°C for 0.5 min. M. morganii was the most heat-resistant histamine-producing bacteria in albacore tuna loins, followed by E. aerogenes, H. alvei, and R. planticola. M. morganii and E. aerogenes had the highest D(50°C), 49.7 ± 17.57 and 51.8 ± 17.38 min, respectively. In addition, M. morganii had the highest D-values for all other temperatures (54, 56, and 58°C) tested. D- and zvalues were also determined for M. morganii in skipjack tuna. While no significant (P > 0.05) difference was observed between D(54°C) and D(56°C) of M. morganii in either albacore or skipjack tuna, the D(58°C) (0.4 ± 0.17 min) was significantly lower (P < 0.05) in skipjack than in albacore (0.9 ± 0.24 min). The z-values for all organisms tested were in the range of 3.2 to 3.8°C. This study suggests that heat treatment designed to control M. morganii in tuna loins is sufficient for controlling histamine-producing bacteria in canned-tuna processing environments.

Bioautography-guided isolation of antibacterial compounds of essential oils from Thai spices against histamine-producing bacteria.

The outbreak of histamine fish poisoning has been being an issue in food safety and international trade. The growth of contaminated bacterial species including Morganella morganii which produce histidine decarboxylase causes histamine formation in fish during storage. Histamine, the main toxin, causes mild to severe allergic reaction. At present, there is no well-established solution for histamine fish poisoning. This study was performed to determine the antibacterial activity of essential oils from Thai spices against histamine-producing bacteria. Among the essential oils tested, clove, lemongrass and sweet basil oils were found to possess the antibacterial activity. Clove oil showed the strongest inhibitory activity against Morganella morganii, followed by lemongrass and sweet basil oils. The results indicated that clove, lemongrass and sweet basil oils could be useful for the control of histamine-producing bacteria. The attempt to identify the active components using preparative TLC and GC/MS found eugenol, citral and methyl chavicol as the active components of clove, lemongrass and sweet basil oils, respectively. The information from this study would be useful in the research and development for the control of histamine-producing bacteria in fish or seafood products to reduce the incidence of histamine fish poisoning.

Aqueous phase synthesis of copper nanoparticles: a link between heavy metal resistance and nanoparticle synthesis ability in bacterial systems.

We demonstrate aqueous phase biosynthesis of phase-pure metallic copper nanoparticles (CuNPs) using a silver resistant bacterium Morganella morganii. This is particularly important considering that there has been no report that demonstrates biosynthesis and stabilization of pure copper nanoparticles in the aqueous phase. Electrochemical analysis of bacterial cells exposed to Cu(2+) ions provides new insights into the mechanistic aspect of Cu(2+) ion reduction within the bacterial cell and indicates a strong link between the silver and copper resistance machinery of bacteria in the context of metal ion reduction. The outcomes of this study take us a step closer towards designing rational strategies for biosynthesis of different metal nanoparticles using microorganisms.

Detection of KPC-2 and qnrS1 in clinical isolates of Morganella morganii from China.

Seven carbapenem-nonsusceptible Morganella morganii isolates, which have similar antibiotic susceptibility profiles, were isolated over a 5-month period. MICs of imipenem, meropenem, and ertapenem were 8, 1, and 0.25 to 0.5 µg/mL, respectively. Pulsed-field gel electrophoresis indicated that 6 isolates were indistinguishable or closely related. Carbapenem resistance can be transferred from M. morganii to Escherichia coli by conjugation. All M. morganii isolates and E. coli transconjugants produced KPC-2 and carried the qnrS1 gene. Production of KPC-2 mainly contributed to the carbapenem resistance in M. morganii. KPC-2-producing M. morganii clonally spread in a hospital in China.

[Inhibitory effect of essential oils, food additives, peracetic acid and detergents on bacterial histidine decarboxylase].

The aim of this study is to examine whether various essential oils, food additives, peracetic acid and detergents inhibit bacterial histidine decarboxylase. Crude extract of Morganella morganii NBRC3848 was prepared and incubated with various agents. Histidine decarboxylase activity was significantly inhibited (p<0.05) by 26 of 45 compounds tested. Among the 26 agents, sodium hypochlorite completely decomposed both histidine and histamine, while peracetic acid caused slight decomposition. Histidine and histamine were stable in the presence of the other 24 agents. These results indicated that 25 of the agents examined were inhibitors of histidine decarboxylase.

[Antibacterial activity of essential oil vapor for histamine-producing bacteria].

In this study, we evaluated the antibacterial activity of essential oil vapors against histamine-producing bacteria Morganella morganii NBRC3848 and Raultella planticola NBRC3317. We measured the minimum inhibitory dose (MID) of 14 essential oils towards these two strains. Allyl isothiocyanate (AIT) and salicylaldehyde (SA) vapors showed higher antibacterial activity than the other 12 essential oil vapors. Both AIT and SA vapors suppressed growth of total aerobic bacteria and histamine-producing bacteria in bigeye tuna and mackerel meat during storage at 12°C. These vapors also inhibited histamine accumulation in bigeye tuna meat and mackerel meat. Thus, application of AIT and SA vapors is effective for preventing increase of histamine-producing bacteria and histamine formation in fish meat.

[Antibacterial effect of food additives and detergents against histamine-producing bacteria on food contact material surfaces].

We investigated the antibacterial activity of food additives and detergents against histamine-producing bacteria on food contact material surfaces. Based on minimum inhibitory concentration (MIC) testing with Morganella morganii NBRC3848, Raoultella planticola NBRC3317 and Enterobacter aerogenes NCTC10006, we screened nine food additives and four detergents with relatively high inhibitory potency. We prepared food contact material surfaces contaminated with histamine-producing bacteria, and dipped them into fourteen agents (100 µg/mL). Sodium hypochlorite, benzalkonium chloride, benzethonium chloride, n-hexadecyltrimethylammonium chloride and 1-n-hexadecylpyridinium chloride showed antibacterial activity against histamine-producing bacteria. We prepared low concentrations of the five agents (10 and 50 µg/mL) and tested them in the same way. Sodium hypochlorite showed high antibacterial activity at 10 µg/mL, and the other four showed activity at 50 µg/mL. So, washing the material surface with these reagents might be effective to prevent histamine food poisoning owing to bacterial contamination of food contact surfaces.

[Extracellular slime production and adhesion of Morganella morganii strains to polystyrene]. / Wytwarzanie sluzu pozakomórkowego, a adhezja paleczek Morganella morganii do polistyrenu.

The aim of this study was the evaluation of the ability of extracellular slime production and adhesive properties of M. morganii strains. This study included 50 of M. morganii strains isolated from clinical samples. All of these strains were isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital in 2008-2009. Five (10.0%) out of 50. M. morganii strains demonstrated extracellular slime production. Adherence to polystyrene revealed 36 (72.0%) of M. morganii strains in it 6 strains (12.0%) adhered strongly, medium - 12 (24.0%) and weakly - 18 (36.0%).

Characterisation of histamine-producing bacteria from farmed blackspot seabream (Pagellus bogaraveo) and turbot (Psetta maxima).

Turbot (Psetta maxima) and blackspot seabream (Pagellus bogaraveo) represent two of the most important emerging farmed fish species in European countries. However, no information of the presence and development of histamine-producing bacteria on them has been reported so far. Accordingly, the aim of this study was to isolate and identify the main histamine-producing bacteria in farmed turbot and blackspot seabream. For this study, 24 isolates (12 from turbot and 12 from blackspot seabream) were preliminarily selected on Niven medium. Two of these isolates were confirmed as prolific histamine producers by HPLC. Thus, Pseudomonas fragi (isolated from turbot) and Pseudomonas syringae (isolated from blackspot seabream) were able to produce 272±69ppm and 173±45ppm of histamine in vitro, respectively, after incubation at 30°C/24h. While turbot fillets proved to be quite resistant to histamine formation at temperatures below 10°C, blackspot seabream fillets inoculated with P. syringae and the prolific histamine former Morganella morganii accumulated 696±84 and 760±59ppm histamine, respectively, under such conditions. Genetic identification based on 16S rRNA sequencing was performed in parallel with the investigation of characteristic mass spectral profiles of the isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS analyses provided species-specific fingerprints, which allow rapid identification and classification of the isolates. Six genus-specific mass peaks in the range of 2218-4434 m/z were shared by both strains. Bacterial identification was achieved by the identification of six species-specific mass peaks in the ranges of 2534-7183 m/z and 2536-9113 m/z for P. fragi and P. syringae, respectively.

Structural characterization and MHCII-dependent immunological properties of the zwitterionic O-chain antigen of Morganella morganii.

Morganella morganii is a commensal Gram-negative bacterium that has long been known to produce an antigen bearing phosphocholine groups. We determined the structure of this O-chain antigen and found that its repeating unit also contains a free amino group and a second phosphate: This alternating charge character places the M. morganii O-chain polysaccharide into a small family of zwitterionic polysaccharides (ZPSs) known to induce T-cell-dependent immune responses via presentation by class II major histocompatibility complex (MHCII) molecules. In vitro binding assays demonstrate that this O-chain interacts with MHCII in a manner that competes with binding of the prototypical ZPS antigen PSA from Bacteroides fragilis, despite its lack of a helical structure. Cellular studies also showed that the M. morganii polysaccharide induces activation of CD4(+) T-cells. Antibody binding experiments using acid hydrolyzed fragments representing the monomer and higher oligomers of the repeating unit showed that the phosphocholine group was the dominant element of the epitope with an overall affinity (K(D)) of about 5 × 10(-5) M, a typical value for an IgM anti-carbohydrate antibody but much lower than the affinity for phosphocholine itself. These data show that the structure of the M. morganii polysaccharide contains a unique zwitterionic repeating unit which allows for immune recognition by T-cells, making it the first identified T-cell-dependent O-chain antigen.