Characterisation of meropenem-resistant Bacillus sp. FW 1 isolated from biogas digestate.

Recently a Bacillus sp. strain FW 1 was isolated from biogas digestate and shown to have novel resistance to meropenem (MEM), of critical importance in human medicine. MEM-resistance has so far only been described for one species within the genus Bacillus, that is, Bacillus cereus. Bacillus is an abundant representative of the microbial community in biogas digesters and consequently, the finding indicates a risk of spreading such resistance when using the digestate as fertiliser. In this study, the Bacillus strain was characterised and classified as Heyndrickxia oleronia (previous Bacillus oleronius), previously not described to harbour MEM-resistance. The mechanism of resistance was explored by metallo-ß-lactamase (MBL) production, mapping of carbapenemase genes and genome analysis. The transferability of MEM-resistance in strain FW 1 was investigated by plasmid transformation/conjugation, combined with genome analysis. The results confirmed MBL production for both strain FW 1 and the type strain H. oleronia DSM 9356T . However, elevated MEM resistance was found for strain FW 1, which was suggested to be caused by the production of unclassified carbapenemase, or overexpression of MBL. Moreover, the results suggest that the MEM-resistance of strain FW 1 is not transferable, thus representing a limited risk of MEM-resistance spread to the environment when using digestate on arable land.

Tetramic acid based alkaloids from Aspergillus amoenus Roberg strain UP197 - antibiotic properties and new pyranterreones.

The fungus Aspergillus amoenus Roberg strain UP197 was shown to produce antibacterial tetramic acid based alkaloids. Two new compounds, pyranterreone I and J (1 and 2), were isolated and characterized, in addition to the known compounds cordylactam, 7-hydroxycordylactam, pyranterreone C, D, F and G. Neither the pyranterreones nor the cordylacctams had previously been tested for antimicrobial activity. Thus, all isolated compounds were tested against a panel of clinically important bacteria and fungi. Pyranterreone C was active against Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) between 1 and 8 µg/mL, whereas the MICs for all other compounds were >32 µg/mL. Pyranoterreone C was cytotoxic towards HepG2 cells, and since pyranterreone C reacted rapidly with the nucleophile cysteine, it is likely that the observed antibacterial activity is due to the chemical reactivity rather than enzymatic affinity, making it unsuitable for development as an antibacterial drug.

Selective Isolation of Multidrug-Resistant Pedobacter spp., Producers of Novel Antibacterial Peptides.

Twenty-eight multidrug-resistant bacterial strains closely related or identical to Pedobacter cryoconitis, Pedobacter lusitanus and Pedobacter steynii were isolated from soil samples by selection for multidrug-resistance. Approximately 3-30% of the selected isolates were identified as Pedobacter, whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed Pedobacter to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The Pedobacter isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of Pedobacter, and most of these compounds were found to be peptides. The Pedobacter strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by Pedobacter sp. Other Pedobacter strains produced analogous peptides with a sequence variation. Further strains of Pedobacter produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700-960 Da in size). A 16S rRNA phylogenetic tree for the Pedobacter isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

In-peptide amino acid racemization via inter-residue oxazoline intermediates during acidic hydrolysis.

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr-L-2,3-diaminopropanoic acid-D-Phe-L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D2O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu-2,3-diaminopropanoic acid-Leu-L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala-L-Phe-L-Val/3-hydroxyvaline-L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid.

Isopedopeptins A-H: Cationic Cyclic Lipodepsipeptides from Pedobacter cryoconitis UP508 Targeting WHO Top-Priority Carbapenem-Resistant Bacteria.

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 µg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 µg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

Isolation of antibiotic-resistant bacteria in biogas digestate and their susceptibility to antibiotics.

Antibiotics are widely used to prevent and treat diseases and promote animal growth in the livestock industry, and therefore antibiotic residues can end up in biogas digestate from processes treating animal manure (AM) and food waste (FW). These digestates represent a potential source of spread of antimicrobial resistance (AMR) when used as fertilisers. This study evaluated AMR risks associated with biogas digestates from two processes, using AM and FW as substrate, by isolation and identification of antibiotic-resistant bacteria (ARB) and testing their susceptibility to different antibiotics. ARB from the digestates were isolated by selective plating. The antibiotic susceptibility profile of isolates was determined using ampicillin, ceftazidime, meropenem, vancomycin, ciprofloxacin, rifampicin, chloramphenicol, clindamycin, erythromycin, tetracycline, gentamicin or sulfamethoxazole/trimethoprim, representing different antibiotic classes with differing mechanisms of action. In total, 30 different bacterial species belonging to seven genera were isolated and classified. Bacillus and closely related genera, including Paenibacillus, Lysinibacillus and Brevibacillus, were the dominant ARB in both digestates. Most of the ARB strains isolated were non-pathogenic and some were even known to be beneficial to plant growth. However, some were potentially pathogenic, such as an isolate identified as Bacillus cereus. Many of the isolated species showed multi resistance and the AM digestate and FW digestate both contain bacterial species resistant to all antibiotics tested here, except gentamicin. A higher level of resistance was displayed by the FW isolates, which may indicate higher antibiotic pressure in FW compared with AM digestate. Overall, the results indicate a risk of AMR spread when these digestates are used as fertiliser. However, most of the ARB identified are species commonly found in soil, where AMR in many cases is abundant already, so the contribution of digestate-based fertiliser to the spread of AMR may still be very limited.

Antibacterial pyrrolidinyl and piperidinyl substituted 2,4-diacetylphloroglucinols from Pseudomonas protegens UP46.

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG (1) and 6-(piperidin-2-yl)DAPG (2). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentration (MIC) 2 and 4 µg ml-1, respectively, for 1, and 2 µg ml-1 for both pathogens for 2. The MICs for 1 and 2, against all tested Gram-negative bacteria, were >32 µg ml-1. The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 µg ml-1, respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2, and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2, but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.

Antibacterial Isoquinoline Alkaloids from the Fungus Penicillium Spathulatum Em19.

In the search for new microbial antibacterial secondary metabolites, two new compounds (1 and 2) were isolated from culture broths of Penicillium spathulatum Em19. Structure determination by nuclear magnetic resonance and mass spectrometry identified the compounds as 6,7-dihydroxy-5,10-dihydropyrrolo[1,2-b]isoquinoline-3-carboxylic acid (1, spathullin A) and 5,10-dihydropyrrolo[1,2-b]isoquinoline-6,7-diol (2, spathullin B). The two compounds displayed activity against both Gram-negative and -positive bacteria, including Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus. Compound 2 was more potent than 1 against all tested pathogens, with minimal inhibitory concentrations down to 1 µg/mL (5 µM) against S. aureus, but 2 was also more cytotoxic than 1 (50% inhibitory concentrations 112 and 11 µM for compounds 1 and 2, respectively, towards Huh7 cells). Based on stable isotope labelling experiments and a literature comparison, the biosynthesis of 1 was suggested to proceed from cysteine, tyrosine and methionine via a non-ribosomal peptides synthase like enzyme complex, whereas compound 2 was formed spontaneously from 1 by decarboxylation. Compound 1 was also easily oxidized to the 1,2-benzoquinone 3. Due to the instability of compound 1 and the toxicity of 2, the compounds are of low interest as possible future antibacterial drugs.

Antibacterial 3,6-Disubstituted 4-Hydroxy-5,6-dihydro-2H-pyran-2-ones from Serratia plymuthica MF371-2.

Bioassay-guided fractionation of culture extracts of Serratia plymuthica strain MF371-2 resulted in the isolation of two new antibacterial compounds with potent activity against Gram-positive bacteria, including Staphylococcus aureus LMG 15975 (MRSA). A spectroscopic investigation, in combination with synthesis, enabled the characterization of the compounds as 3-butyryl-4-hydroxy-6-heptyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone A, 1) and 3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone B, 2). The MIC values for 1 and 2 against S. aureus LMG 15975 were determined to be 1-2 µg mL-1 and 0.8 µg mL-1, respectively. Compound 2 was found to have potent activity against many strains of S. aureus, including several mupirocin-resistant strains, other species of Staphylococcus, and vancomycin-resistant enterococci. Compound 2 was slightly cytotoxic for human cells, with CC50 values between 4.7 and 40 µg mL-1, but the CC50/MIC ratio was ≥10 for many tested combinations of human cells and bacteria, suggesting its possible use as an antibacterial agent. Several analogues were synthesized with different alkyl groups in the 3- and 6-positions (6-13), and their biological properties were evaluated. It was concluded that the activity of the compounds increased with the lengths of the alkyl and acyl substituents.

Labradorins with Antibacterial Activity Produced by Pseudomonas sp.

The urgent need for new antibacterial drugs has led to renewed interest in microorganisms, which historically have been the main source of previously discovered antibiotics. The present study describes the discovery of two new antibacterial oxazolylindole type alkaloids, labradorins 5 (1) and 6 (2), which were isolated and characterized from two isolates of Pseudomonas sp., along with four previously known tryptophane derived alkaloids. The structures of 1 and 2 were determined by NMR spectroscopy and MS, and confirmed by synthesis. During bioassay-guided isolation using several human bacterial pathogens, 1 and 2 displayed activity towards Staphylococcus aureus and Acinetobacter baumannii. The minimal inhibitory concentrations (MIC) of compounds 1 and 2 against S. aureus were 12 µg·mL-1 and 50 µg·mL-1, respectively, whereas the MICs against A. baumannii were >50 µg·mL-1. The CC50 values of compound 1 towards a liver cell line (HEP-G2) and a T-cell line (MT4) were 30 µg·mL-1 and 20 µg·mL-1, respectively, and for compound 2 were >100 µg·mL-1 and 20 µg·mL-1, respectively. Due to the limited potency of compounds 1 and 2, along with their toxicity, the compounds do not warrant further development towards new antibiotics.

Anti-fungal and anti-bacterial activity of some herbal remedies from Tanzania.

Plants are not only important to the millions of people to whom traditional medicine serves as the only opportunity for health care and to those who use plants for various purposes in their daily lives, but also as a source of new pharmaceuticals. During interviews with the Pare people from Northeastern Tanzania, 29 plants that are used for medicinal purposes as well as 41 plants used for non-medicinal purposes were reported. Six medicinally used plants were selected for bioactivity analysis. Extracts of Coccinia adoensis, Cineraria grandiflora, Pavonia urens, Marattia fraxinea, Clutia abyssinica var. usambarica, and Vangueria infausta were made using ethyl acetate, methanol, cold water and boiling water. The antimicrobial activity was tested on Candida albicans, Aspergillus fumigatus, Fusarium culmorum, Staphylococcus aureus, Pseudomonas syringae, and Erwinia amylovora. All plants showed activity against several test organisms.

Physiological and genetic characterization of fluorescent Pseudomonas associated with Cantharellus cibarius.

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog and 16S-rDNA are insufficient until databases for different ecosystems are created.