Development of Antibiofilm Nanocomposites: Ag/Cu Bimetallic Nanoparticles Synthesized on the Surface of Graphene Oxide Nanosheets.

There are numerous issues associated with bacteria, particularly biofilms, which exhibit a strong resistance to antibiotics. This is currently considered an urgent global issue owing to the lack of effective treatments. Graphene oxide (GO) nanosheets are two-dimensional carbon materials that are available as a substrate for metal nanoparticles and have a lower release rate of metal ions than free metal nanoparticles by regulating the oxidation of metal nanoparticles, which is known to reduce the cytotoxicity caused by the free metal nanoparticles. Over centuries, metal particles, including Ag and Cu, have been considered as antibacterial agents. In this study, Ag and Cu bimetallic nanoparticles on a GO surface (Ag/Cu/GO) were synthesized using a chemical reduction method, and their antimicrobial effects against several bacterial species were demonstrated. Ag/Cu/GO nanocomposites were characterized by transmission electron microscopy and energy-dispersive X-ray spectroscopy. The in vitro cytotoxicity of an Ag/Cu/GO nanocomposite was evaluated in human dermal fibroblasts, and its antibacterial activity against Methylobacterium spp., Sphingomonas spp., and Pseudomonas aeruginosa (P. aeruginosa) was also tested. The synthesized Ag/Cu/GO nanocomposite was able to eradicate all three bacterial species at a concentration that was harmless to human cells. In addition, Ag/Cu/GO successfully removed a biofilm originated from the culturing of P. aeruginosa in a microchannel with a dynamic flow. In a small-animal model, a biofilm-infected skin wound was healed quickly and efficiently by the topical application of Ag/Cu/GO. The Ag/Cu/GO nanocomposites reported in this study could be used to effectively remove antibiotic-resistant bacteria and treat diseases in the skin or wound due to bacterial infections and biofilm formation.

Engineering copper nanoparticles synthesized on the surface of carbon nanotubes for anti-microbial and anti-biofilm applications.

Biofilms adhere to surfaces to produce extracellular polymeric substances (EPSs). EPSs grow and protect themselves from external stresses. Their formation causes a foul odor and may lead to chronic infectious diseases in animals and people. Biofilms also inhibit the contact between bacteria and antibiotics, thereby reducing their antibacterial activity. Thus, we describe novel nanostructures, a fusion of copper and multi-walled carbon nanotubes (MWCNTs), which increase antimicrobial activity against biofilms without being toxic to human cells. Simulations based on the stochastic response were performed to predict the efficiency of synthesizing nanostructures. The synthesized Cu/MWCNTs inhibit the growth of Methylobacterium spp., which forms biofilms; antimicrobial testing and cytotoxicity assessments showed that the Cu/MWCNTs were not cytotoxic to human cells. The Cu/MWCNTs come in direct contact with the bacterial cell surface, damage the cell wall, and cause secondary oxidation of reactive oxygen species. Furthermore, the Cu/MWCNTs release copper ions, which inhibit the quorum sensing in Methylobacterium spp., thereby inhibiting the expression of the genes that form biofilms. Additionally, we confirmed excellent electrical and thermal conductivity of Cu/MWCNTs as well as biofilm removal efficiency in the microfluidic channel.

Toblerols: Cyclopropanol-Containing Polyketide Modulators of Antibiosis in Methylobacteria.

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic.

Evaluation of Sulfadiazine Degradation in Three Newly Isolated Pure Bacterial Cultures.

This study is aimed to assess the biodegradation of sulfadiazine (SDZ) and characterization of heavy metal resistance in three pure bacterial cultures and also their chemotactic response towards 2-aminopyrimidine. The bacterial cultures were isolated from pig manure, activated sludge and sediment samples, by enrichment technique on SDZ (6 mg L-1). Based on the 16S rRNA gene sequence analysis, the microorganisms were identified within the genera of Paracoccus, Methylobacterium and Kribbella, which were further designated as SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47. The three identified pure bacterial strains degraded up to 50.0, 55.2 and 60.0% of SDZ (5 mg L-1), respectively within 290 h. On the basis of quadrupole time-of-flight mass spectrometry and high performance liquid chromatography, 2-aminopyrimidine and 4-hydroxy-2-aminopyrimidine were identified as the main intermediates of SDZ biodegradation. These bacteria were also able to degrade the metabolite, 2-aminopyrimidine, of the SDZ. Furthermore, SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47 also showed resistance to various heavy metals like copper, cadmium, chromium, cobalt, lead, nickel and zinc. Additionally, all three bacteria exhibited positive chemotaxis towards 2-aminopyrimidine based on the drop plate method and capillary assay. The results of this study advanced our understanding about the microbial degradation of SDZ, which would be useful towards the future SDZ removal in the environment.

Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently.

The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations.

Killing effect of peppermint vapor against pink-slime forming microorganisms.

The killing effect of peppermint vapor (PMV) against pink-slime forming microorganisms, Methylobacterium mesophilicum as a bacterium and Rhodotorula mucilaginosa as a yeast, was investigated by the agar vapor assay. In this method, microbial cells were spread over the agar surface exposed to PMV in a petri dish, and then transferred into a recovery liquid. When 60µl of the peppermint liquid was added to a paper disc, a marked killing effect of PMV was observed after 48h against M. mesophilicum and after 168h against R. mucilaginosa. M. mesophilicum and R. mucilaginosa were found to be more resistant to PMV than Escherichia coli and Candida albicans, used as reference microorganisms, respectively. With the addition of 0.03% sodium pyruvate as a hydrogen peroxide scavenger in agar, the killing effect of PMV against E. coli and C. albicans was decreased, whereas it was little changed against M. mesophilicum and R. mucilaginosa. In fact, the properties of the killing effect of hydrogen peroxide solution at 0.2-1.0mM was in accord with those of PMV. M. mesophilicum and R. mucilaginosa were more resistant to the oxidant than E. coli and C. albicans, respectively. Results obtained suggested that reactive oxygen species (ROS) may be involved in the killing action of PMV and therefore pink-slime formers are more resistant to PMV than non-pink-slime formers because of the presence of carotenoids as an antioxidant in cells. We also suggest that the use of PMV appeared to be a potential tool for the control of pink-slime forming microorganisms occurring in wet areas of houses such as the bathroom and washing room.

Characteristics of Meningitis Due to Methylobacterium mesophilicum: A Rare Case.

Methylobacterium mesophilicum is a gram-negative bacillus most often isolated in medical establishments. In humans, M. mesophilicum is considered a conditionally pathogenic flora. Infections in humans generally occur in immunodeficient individuals. This article describes a rare case of M. mesophilicum infection that developed into acute meningitis in a 26-year-old patient. Decreased neutrophil bactericidal activity was also detected. To our knowledge, there have been no previous reports of meningitis due to M. mesophilicum.

[Functional activity of the modA, gene in Methylobacterium dichloromethanicum DM4].

The putative METDI2644 (modA2) gene of Methylobacterium dichloromethanicum DM4, present in the 126-kbp chromosomal fragment associated with dichloromethane (DCM) degradation was investigated. While this gene is presumed to encode the periplasmic substrate-binding subunit of the molybdate ABC transporter, its conceptual translation also exhibits similarity to the proteins containing the ostA conservative domain and responsible for resistance of gram-negative bacteria to organic solvents. Reverse transcription polymerase chain reaction (RT-PCR) revealed the RNA transcripts of this gene in the cells grown on either DCM or methanol. The mobilizable suicide vector pK18mob was used to obtain a knockout mutant with the METDI2644 gene inactivated by insertion of the gentamycin cassette. The mutant pregrown on methanol exhibited lower growth rate on DCM than the wild-type strain DM4. The difference was not alleviated by addition of sodium molybdate. Our results suggest that the METDI2644 gene product plays a role in cell adaptation to DCM degradation.

Dominant colonization and inheritance of Methylobacterium sp. strain OR01 on perilla plants.

Pink-pigmented facultative methylotrophs (PPFMs) are major inhabitants of the phyllosphere. In a preceding study, we found that perilla plants harbor a dominant population of PPFMs on their leaves and seeds, and that the closest relative of PPFMs (Methylobacterium sp. strain OR01 as representative strain) isolated from red perilla seeds was M. fujisawaense DSM5686(T). In the present study, the specific interaction between red perilla and Methylobacterium species was investigated. All the PPFMs isolated from red perilla seeds harvested in the Ohara area of Kyoto, Japan in 2009, 2010, and 2011 and the PPFMs isolated from red perilla leaves planted at four geographically different places in Japan had 16S rRNA sequences identical to that of strain OR01. Direct transmission of PPFMs from seeds to leaves and the competitiveness of strain OR01 were confirmed. This report is the first step toward understanding the species-level specificity of the interaction between perilla plants and Methylobacterium species.

Stress tolerance of Methylobacterium biofilms in bathrooms.

A comprehensive survey of microbial flora within pink biofilms in bathrooms was performed. Pink biofilms develop relatively rapidly in bathrooms, can be difficult to remove, and are quick to recur. Bacterium-sized cells were found to be predominant in 42 pink biofilms in Japan using a scanning electron microscope. Methylobacterium strains were detected from all samples in bathrooms by an isolation method. To explain this predominance, 14 biofilm samples were analyzed by fluorescence in situ hybridization. Methylobacterium was indicated to be the major genus in all biofilms. The isolated Methylobacterium survived after contact with 1.0% cleaning agents, including benzalkonium chloride for 24 h. Their tolerance did not differ under biofilm-like conditions on fiber reinforced plastics (FRP), a general material of bath tubs, floors, and walls. Also, the strains exhibited higher tolerance to desiccation than other isolated species on FRP. Some Methylobacterium survived and exhibited potential to grow after four weeks of desiccation without any nutrients. These specific characteristics could be a cause of their predominance in bathrooms, an environment with rapid flowing water, drying, low nutrients, and occasional exposure to cleaning agents.

[Inactivation of the chlorine-resistant bacteria isolated from the drinking water distribution system].

Inactivation experiments of seven strains of chlorine-resistant bacteria, isolated from a drinking water distribution system, were conducted with four kinds of disinfectants. All the bacteria showed high resistance to chlorine, especially for Mycobacterium mucogenicum. The CT value of 99.9% inactivation for M. mucogenicum, Sphingomonas sanguinis and Methylobacterium were 120 mg x (L x min)(-1), 7 mg x (L x min)(-1) and 4 mg x (L x min)(-1), respectively. The results of inactivation experiments showed that chlorine dioxide and potassium monopersulfate could inactive 5 lg of M. mucogenicum within 30 min, which showed significantly higher efficiency than free chlorine and monochloramine. Free chlorine was less effective because the disinfectant decayed very quickly. Chloramination needed higher concentration to meet the disinfection requirements. The verified dosage of disinfectants, which could effectively inactivate 99.9% of the highly chlorine-resistant M. mucogenicum within 1 h, were 3.0 mg/L monochloramine, 1.0 mg/L chlorine dioxide (as Cl2), and 1.0 mg/L potassium monopersulfate (as Cl2). It was suggested that the water treatment plants increase the concentration of monochloramine or apply chlorine dioxide intermittently to control the disinfectant-resistant bacteria.

Evidence of Methylobacterium spp. and Hyphomicrobium sp. in azaspiracid toxin contaminated mussel tissues and assessment of the effect of azaspiracid on their growth.

A flagellar protein belonging to the genus Methylobacterium or Agrobacterium was previously observed by proteomics in azaspiracids (AZA) toxic mussels. Here, we report the isolation of two different Methylobacterium spp. (NTx1 and Tx1) from non-toxic and AZA toxic mussels, respectively, which when co-cultured with AZA exhibited significantly different growth responses - isolate Tx1 growth rate was enhanced, whereas growth of isolate NTx1 was adversely affected, compared to non-AZA supplemented control cultures. A Hyphomicrobium sp. (Tx2) also isolated from the toxic mussels achieved greater cell density in AZAs supplemented cultures.

Genotyping and chlorine-resistance of Methylobacterium aquaticum isolated from water samples in Japan.

For microbial ecological analysis, 14 strains of Methylobacterium aquaticum isolated from water samples were subjected to clustering analysis on the basis of ribotyping and RAPD-PCR tests. The ribopatterns after digestion with EcoRI obtained from 14 strains of M. aquaticum were used to divide the strains into two groups (Groups I and II) with a similarity of 55%. From the analysis of RAPD patterns using primer 208, the 14 strains were divided into 3 groups (A-C) based on a homology of 45% or greater, and from that using primer 272, there were 4 groups (A-D) based on a homology of 50% or greater. The chlorine resistance (99.9% CT values) of these isolates was also experimentally confirmed, and we attempted to define the connection between chlorine resistance and the geno-cluster. The average CT value of group I was 0.89 mg•min/l and the average of group II was 0.69 mg•min/l. No remarkable differences in the CT values for the groups were found.

Metal tolerating methylotrophic bacteria reduces nickel and cadmium toxicity and promotes plant growth of tomato (Lycopersicon esculentum L).

Inoculation of plants with microorganisms may reduce the toxicity of heavy metals to plants in contaminated soils. In this study, we have shown that the plant growth promoting bacteria Methylobacterium oryzae strain CBMB20 and Burkholderia sp. strain CBMB40 from rice reduce the toxicity of Ni and Cd in tomato and promote plant growth under gnotobiotic and pot culture experiments. The bacterial strains bound considerable amounts of Ni(II) and Cd(II) in their growing and resting cells and showed growth in the presence of NiCl2 and CdCl2. In gnotobiotic assay, inoculation with the bacterial strains reduced the ethylene emission and increased the tolerance index of the seedlings against different concentrations of NiCl2/CdCl2. In pot experiments carried out with non-polluted, Ni and Cd supplemented Wonjo-Mix bed soil, the results clearly demonstrated reduction in the accumulations of Ni(II) and Cd(II) in roots and shoots, with significant increase in the plant growth attributes with bacterial inoculations compared to untreated control. Strain CBMB20 performed better than CBMB40 in reducing the heavy metal accumulations in plants. Our results suggest conclusively, that protection against the heavy metals toxicity is rendered by these bacterial strains by reducing their uptake and further translocation to shoots in plants and promote the plant growth by other PGP characteristics.

Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov.

Various pink-pigmented facultative methylotrophic (PPFM) bacteria (strains iEII3, iEIV1, iEI6, iEII1, iEIII3 iEIII4, iEIII5, iRII1, iRII2, iRIII1, iRIV1 and iRIV2) were obtained from the rhizosphere and endosphere of hyperaccumulating plant Thlaspi goesingense grown in Redschlag, Austria [R. Idris, R. Trifonova, M. Puschenreiter, W.W. Wenzel, A. Sessitsch, Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense, Appl. Environ. Microbiol. 70 (2004) 2667-2677]. Due to their unexpected diversity, abundance and nickel tolerance they were further characterized by detailed 16S rRNA gene analysis, DNA-DNA hybridization, fatty acid analysis, heavy metal tolerance, screening for known Ni resistance genes and phenotypic analysis. These strains were found to exhibit different multiple heavy metal resistance characteristics to Ni, Cd, Co, Zn and Cr. On the basis of their physiological and genotypic properties, strains could be grouped with Methylobacterium extorquens and M. mesophilicum. One endophyte, strain iEII3, was found to belong to a novel species for which the name M. goesingense is proposed.

Isolation and identification of methylobacterium species from the tap water in hospitals in Japan and their antibiotic susceptibility.

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90 = 1 microg/ml) and tetracycline (MIC90 = 8 microg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

[Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria.

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.

[Biotransformation of organic substances by an immobilized associative bacterial culture]. / Biotransformatsiia organicheskikh veshchestv immobilizovannoi assotsiativnoi bakterial'noi kul'turoi.

Bacterial strains were screened to transform to end-products (carbon monoxide and water) elevated concentrations of acetone, acidic acid, and ethanol in a biocatalyst with an immobilized bacterial association cultivated on solid foam polyvinyl acetate (FPVA). The innocuous association amalgamated Paracoccus denitrificans VKM V-1324, Pseudomonas esterophilus VKM V-1736D and Achromobacter parvulus VKM V-1541D. The biocatalyst was tested with the help of classic methods and equipment for microbes cultivation. Microbial growth was assessed in the optical density units. Comparative evaluation of the biocatalytic activity was performed on gas-chromatographer Pue Unicam-104. Based on test results, specific rates of oxidation of acidic acid, ethanol and acetone per one g of FPVA were 0.75, 0.72, and 0.67 mg/hr, respectively.