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Antibiotic resistance concerns have been compounded by a report that soil bacteria can catabolise antibiotics, i.e. break down and use them as a sole carbon source. To date this has not been verified or reproduced, therefore in this study soil bacteria were screened to verify and reproduce this hypothesis. Survival in high concentrations of antibiotics was initially observed; however, on further analysis these bacteria either did not degrade the antibiotics or they used an intrinsic resistance mechanism (β-lactamases) to degrade the β-lactams, as demonstrated by high-performance liquid chromatography. These results did not verify or reproduce the hypothesis that bacteria subsist on antibiotics or catabolise antibiotics as previously reported. This study identified that bacteria with a catabolising phenotype did not degrade streptomycin or trimethoprim and therefore could not utilise the antibiotics as a nutrient source. Therefore, we conclude that soil bacteria do not catabolise antibiotics., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Microbial colonisation on clinical and industrial surfaces is currently of global concern and silane based sol-gel coatings are being proposed as potential solutions. Sol-gels are chemically inert, stable and homogeneous and can be designed to act as a reservoir for releasing antimicrobial agents over extended time periods. In the present study, silver nitrate (AgN) and a series of silver coumarin complexes based on coumarin-3-carboxylatosilver (AgC) and it is 6, 7 and 8 hydroxylated analogues (Ag6, Ag7, Ag8) were incorporated into sol-gel coatings. The comparative antibacterial activity of the coatings was determined against meticillin resistant Staphylococcus aureus (MRSA) and multidrug resistance Enterobacter cloacae WT6. The percentage growth inhibitions were found in the range of 9.2 (±2.7)-66.0 (±1.2)% at low silver loadings of 0.3% (w/w) with E. cloacae being the more susceptible. Results showed that among the Ag coumarin complexes, the Ag8 doped coating had the highest antibiofilm property. XPS confirmed the presence of silver in the nanoparticulate state (Ag(0)) at the coating surface where it remained after 4 days of exposure to bacterial culture. Comparative cytotoxicity studies revealed that the Ag-complex coatings were less toxic than the AgN coating. Thus, it can be concluded that a sol-gel matrix with Ag-coumarin complexes may provide non-toxic surfaces with antibacterial properties., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pantoea agglomerans is a cosmopolitan plant epiphytic bacterium that includes some of the most effective biological antagonists against the fire blight pathogen Erwinia amylovora, a major threat to pome fruit production worldwide. Strain E325 is commercially available as Bloomtime Biological™ in the USA and Canada. New quantitative PCR (qPCR) assays were developed for species- and strain -specific detection in the environment, and for detection of indigenous strains carrying the biocontrol antibacterial peptide biosynthesis gene paaA. The qPCR assays were highly specific, efficient and sensitive, detecting fewer than three cells per reaction or 700 colony forming units per flower, respectively. The qPCR assays were tested on field samples, giving first indications to the incidence of P. agglomerans E325 related strains, total P. agglomerans and pantocin A producing bacteria in commercial orchards. These assays will facilitate monitoring the environmental behavior of biocontrol P. agglomerans after orchard application for disease protection, proprietary strain-tracking, and streamlined screening for discovery of new biocontrol strains., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pantoea vagans C9-1 is a biocontrol strain that produces at least two antibiotics inhibiting the growth of Erwinia amylovora, the causal agent of fire blight disease of pear and apple. One antibiotic, herbicolin I, was purified from culture filtrates of P. vagans C9-1 and determined to be 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine, also known as N(ß)-epoxysuccinamoyl-DAP-valine. A plasposon library was screened for mutants that had lost the ability to produce herbicolin I. It was shown that mutants had reduced biocontrol efficacy in immature pear assays. The biosynthetic gene cluster in P. vagans C9-1 was identified by sequencing the flanking regions of the plasposon insertion sites. The herbicolin I biosynthetic gene cluster consists of 10 coding sequences (CDS) and is located on the 166-kb plasmid pPag2. Sequence comparisons identified orthologous gene clusters in Pantoea agglomerans CU0119 and Serratia proteamaculans 568. A low incidence of detection of the biosynthetic cluster in a collection of 45 Pantoea spp. from biocontrol, environmental, and clinical origins showed that this is a rare trait among the tested strains.

The colonisation of clinical and industrial surfaces with microorganisms, including antibiotic-resistant strains, has promoted increased research into the development of effective antibacterial and antifouling coatings. This study describes the preparation of metal nitrate (Ag, Cu, Zn) doped methyltriethoxysilane (MTEOS) coatings and the rapid assessment of their antibacterial activity using polyproylene microtitre plates. Microtitre plate wells were coated with different volumes of liquid sol-gel and cured under various conditions. Curing parameters were analysed by thermogravimetric analysis (TGA) and visual examination. The optimum curing conditions were determined to be 50-70°C using a volume of 200 μl. The coated wells were challenged with Gram-positive and Gram-negative bacterial cultures, including biofilm-forming and antibiotic-resistant strains. The antibacterial activities of the metal doped sol-gel, at equivalent concentrations, were found to have the following order: silver>zinc>copper. The order is due to several factors, including the increased presence of silver nanoparticles at the sol-gel coating surface, as determined by X-ray photoelectron spectroscopy, leading to higher elution rates as measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The use of microtitre plates enabled a variety of sol-gel coatings to be screened for their antibacterial activity against a wide range of bacteria in a relatively short time. The broad-spectrum antibacterial activity of the silver doped sol-gel showed its potential for use as a coating for biomaterials., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antibiotics are essential for control of bacterial diseases of plants, especially fire blight of pear and apple and bacterial spot of peach. Streptomycin is used in several countries; the use of oxytetracycline, oxolinic acid and gentamicin is limited to only a few countries. Springtime antibiotic sprays suppress pathogen growth on flowers and leaf surfaces before infection; after infection, antibiotics are ineffective. Antibiotics are applied when disease risk is high, and consequently the majority of orchards are not treated annually. In 2009 in the United States, 16,465 kg (active ingredient) was applied to orchards, which is 0.12% of the total antibiotics used in animal agriculture. Antibiotics are active on plants for less than a week, and significant residues have not been found on harvested fruit. Antibiotics have been indispensable for crop protection in the United States for more than 50 years without reports of adverse effects on human health or persistent impacts on the environment.

This study examined differences in antibiotic-resistant soil bacteria and the presence and quantity of resistance genes in soils with a range of management histories. We analyzed four soils from agricultural systems that were amended with manure from animals treated with erythromycin and exposed to streptomycin and/or oxytetracycline, as well as non-manure-amended compost and forest soil. Low concentrations of certain antibiotic resistance genes were detected using multiplex quantitative real-time PCR (qPCR), with tet(B), aad(A), and str(A) each present in only one soil and tet(M) and tet(W) detected in all soils. The most frequently detected resistance genes were tet(B), tet(D), tet(O), tet(T), and tet(W) for tetracycline resistance, str(A), str(B), and aac for streptomycin resistance, and erm(C), erm(V), erm(X), msr(A), ole(B), and vga for erythromycin resistance. Transposon genes specific for Tn916, Tn1549, TnB1230, Tn4451, and Tn5397 were detected in soil bacterial isolates. The MIC ranges of isolated bacteria for tetracycline, streptomycin, and erythromycin were 8 to >256 μg/ml, 6 to >1,024 μg/ml, and 0.094 to >256 μg/ml, respectively. Based on 16S rRNA gene similarity, isolated bacteria showed high sequence identity to genera typical of soil communities. Bacteria with the highest MICs were detected in manure-amended soils or soils from agricultural systems with a history of antibiotic use. Non-manure-amended soils yielded larger proportions of antibiotic-resistant bacteria, but these had lower MICs, carried fewer antibiotic resistance genes, and did not display multidrug resistance (MDR).

Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre- and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Pantoea vagans is a commercialized biological control agent used against the pome fruit bacterial disease fire blight, caused by Erwinia amylovora. Compared to other biocontrol agents, relatively little is currently known regarding Pantoea genetics. Better understanding of antagonist mechanisms of action and ecological fitness is critical to improving efficacy., Principal Findings: Genome analysis indicated two major factors Contribute to biocontrol activity: competition for limiting substrates and antibacterial metabolite production. Pathways for utilization of a broad diversity of sugars and acquisition of iron were identified. Metabolism of sorbitol by P. vagans C9-1 may be a major metabolic feature in biocontrol of fire blight. Biosynthetic genes for the antibacterial peptide pantocin A were found on a chromosomal 28-kb genomic island, and for dapdiamide E on the plasmid pPag2. There was no evidence of potential virulence factors that could enable an animal or phytopathogenic lifestyle and no indication of any genetic-based biosafety risk in the antagonist., Conclusions: Identifying key determinants contributing to disease suppression allows the development of procedures to follow their expression in planta and the genome sequence contributes to rationale risk assessment regarding the use of the biocontrol strain in agricultural systems.

Silver nanoparticles (AgNPs) were synthesised by reducing silver salts using NaBH(4) followed by capping with varying concentrations of beta-cyclodextrin (beta-CD) and were physically characterised. Antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was determined by a microtitre well method. The AgNPs were spherical under transmission electron microscopy, whilst dynamic light scattering showed average diameters of capped particles to be smaller (4-7 nm) than their uncapped equivalents (17 nm). Capped particles demonstrated superior photostability when exposed to intense ultraviolet radiation for 4h as well as significantly (P<0.05) higher (up to 3.5-fold) antibacterial activity. The influence of beta-CD concentration was seen to delay bacterial growth, indicating that a Trojan horse mechanism may be occurring owing to bacterial carbohydrate affinity, thereby enhancing silver ion absorption., (Copyright (c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Coatings that demonstrate reduced attachment of crystalline precipitates and the medical device colonising Staphylococcus epidermidis were prepared by the immobilisation of silver doped perfluoropolyether-urethane siloxane thin films on glass substrates. The presence of stratified hydrophobic perfluoropolyether groups protects the coating surface from the attachment of crystalline hydrophilic species such as chlorides and phosphates, whilst silver ion release inhibited attachment of S. epidermidis and subsequent biofilm formation in vitro. The release of silver ions protects the perfluoro groups from the hydrophobic interactions of S. epidermidis cells, which can reduce the hydrophobicity of the protective coating. These coatings also exhibited significant antibacterial activity against planktonic Acinetobacter baumannii and S. epidermidis bacterial strains. Detailed elemental and chemical surface analysis obtained using X-ray photoelectron spectroscopy (XPS) provided useful information on the effect of bacterial incubation on key indicator hydrophobic and hydrophilic functional groups. XPS analysis indicated preferential adsorption of S. epidermidis cells at the hydrophobic sites along the polymeric chain. These dual-action hygienic coatings can be employed to protect against contamination environmental surfaces and bacterial colonisation on implanted medical devices., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The colonisation of clinical and industrial surfaces with pathogenic microorganisms has prompted increased research into the development of effective antibacterial and antifouling coatings. There is evidence that implanted biomedical surfaces coated with metallic silver can be inactivated by physiological fluids, thus reducing the bioactivity of the coating. In this work, we report the biofilm inhibition of Staphylococcus epidermidis using a room temperature processed silver doped perfluoropolyether-urethane coating. The release of silver ions from these fluoropolymers over a six-day period inhibited bacterial encrustation - as observed by scanning electron microscopy (SEM). X-ray photoelectron spectroscopy (XPS) analysis indicated differences in carbon, fluorine and sodium surface composition between silver doped and undoped fluoropolymers after exposure to nutrient rich media. These silver doped perfluoropolyether coatings also exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii; suggesting potential use in preventing transmission of pathogenic and opportunistic microbes on environmental surfaces in healthcare facilities. The broad-spectrum antibacterial activity of these silver release coatings may be exploited on biomaterials surfaces to combat the development of resistant Gram-negative Enterobacteriaceae that can occur during prophylactic treatment for urinary tract infections.

Streptomycin is used in plant agriculture for bacterial disease control, particularly against fire blight in pome fruit orchards. Concerns that this may increase environmental antibiotic resistance have led to bans or restrictions on use. Experience with antibiotic use in animal feeds raises the possible influence of formulation-delivered resistance genes. We demonstrate that agricultural streptomycin formulations do not carry producer organism resistance genes. By using an optimized extraction procedure, Streptomyces 16S rRNA genes and the streptomycin resistance gene strA were not detected in agricultural streptomycin formulations. This diminishes the likelihood for one potential factor in resistance development due to streptomycin use.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+. Pyochelin production was increased by Co2+, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose. The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.