A metabolomics and molecular networking approach to elucidate the structures of secondary metabolite produced by Serratia marcescens strains

Abstract: An integrated approach that combines reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry, untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MSE) and molecular networking (using the Global Natural Products Social molecular network platform) was used to elucidate the metabolic profiles and chemical structures of the secondary metabolites produced by pigmented (P1) and non-pigmented (NP1) Serratia marcescens (S. marcescens) strains. Tandem mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain (including 6 serratamolides, 10 glucosamine derivatives, prodigiosin and serratiochelin A) and 15 compounds for the NP1 strain (including 8 serratamolides, 6 glucosamine derivatives and serratiochelin A) using the MSE fragmentation profiles. The serratamolide homologues were comprised of a peptide moiety of two L-serine residues (cyclic or open-ring) linked to two fatty acid chains (lengths of C10, C12, or C12:1). Moreover, the putative structure of a novel open-ring serratamolide homologue was described. The glucosamine derivative homologues (i.e., N-butylglucosamine ester derivatives) consisted of four residues, including glucose/hexose, valine, a fatty acid chain (lengths of C13 – C17 and varying from saturated to unsaturated) and butyric acid. The putative structures of seven novel glucosamine derivative homologues and one glucosamine derivative congener (containing an oxo-hexanoic acid residue instead of a butyric acid residue) were described. Moreover, seven fractions collected during RPHPLC, with major molecular ions corresponding to prodigiosin, serratamolides (A, B, and C), and glucosamine derivatives (A, C, and E), displayed antimicrobial activity against a clinical Enterococcus faecalis S1 strain using the disc diffusion assay. The minimum inhibitory and bactericidal concentration assays however, revealed that prodigiosin exhibited the greatest antimicrobial potency, followed by glucosamine derivative A and then the serratamolides (A, B, and C)..

Human pathogenic bacteria detected in rainwater : risk assessment and correlation to microbial source tracking mark and traditional indicators

Abstract: Please refer to full text to view abstract

Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant

CITATION: Ndlovu, T., et al. 2017. Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant. AMB Express, 7:108, doi:10.1186/s13568-017-0363-8.The original publication is available at https://amb-express.springeropen.comPublication of this article was funded by the Stellenbosch University Open Access Fund.Biosurfactants are unique secondary metabolites, synthesised non-ribosomally by certain bacteria, fungi and yeast, with their most promising applications as antimicrobial agents and surfactants in the medical and food industries. Naturally produced glycolipids and lipopeptides are found as a mixture of congeners, which increases their antimicrobial potency. Sensitive analysis techniques, such as liquid chromatography coupled to mass spectrometry, enable the fingerprinting of different biosurfactant congeners within a naturally produced crude extract. Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, isolated from wastewater, were screened for biosurfactant production. Biosurfactant compounds were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). Results indicated that B. amyloliquefaciens ST34 produced C13–16 surfactin analogues and their identity were confirmed by high resolution ESI–MS and UPLC–MS. In the crude extract obtained from P. aeruginosa ST5, high resolution ESI–MS linked to UPLC–MS confirmed the presence of di- and monorhamnolipid congeners, specifically Rha–Rha–C10–C10 and Rha–C10–C10, Rha–Rha–C8–C10/Rha–Rha–C10–C8 and Rha–C8–C10/Rha–C10–C8, as well as Rha–Rha–C12–C10/Rha–Rha–C10–C12 and Rha–C12–C10/Rha–C10–C12. The crude surfactin and rhamnolipid extracts also retained pronounced antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains and the pathogenic yeast Candida albicans. In addition, the rapid solvent extraction combined with UPLC–MS of the crude samples is a simple and powerful technique to provide fast, sensitive and highly specific data on the characterisation of biosurfactant compounds.https://amb-express.springeropen.com/articles/10.1186/s13568-017-0363-8Publisher’s versio

Validation of large-volume batch solar reactors for the treatment of rainwater in field trials in sub-Saharan Africa

The efficiency of two large-volume batch solar reactors [Prototype I (140 L) and II (88 L)] in treating rainwater on-site in a local informal settlement and farming community was assessed. Untreated [Tank 1 and Tank 2-(First-flush)] and treated (Prototype I and II) tank water samples were routinely collected from each site and all the measured physico-chemical parameters (e.g. pH and turbidity, amongst others), anions (e.g. sulphate and chloride, amongst others) and cations (e.g. iron and lead, amongst others) were within national and international drinking water guidelines limits. Culture-based analysis indicated that Escherichia coli, total and faecal coliforms, enterococci and heterotrophic bacteria counts exceeded drinking water guideline limits in 61%, 100%, 45%, 24% and 100% of the untreated tank water samples collected from both sites. However, an 8 hour solar exposure treatment for both solar reactors was sufficient to reduce these indicator organisms to within national and international drinking water standards, with the exception of the heterotrophic bacteria which exceeded the drinking water standard limit in 43% of the samples treated with the Prototype I reactor (1 log reduction). Molecular viability analysis subsequently indicated that mean overall reductions of 75% and 74% were obtained for the analysed indicator organisms (E. coli and enterococci spp.) and opportunistic pathogens (Klebsiella spp., Legionella spp., Pseudomonas spp., Salmonella spp. and Cryptosporidium spp. oocysts) in the Prototype I and II solar reactors, respectively. The large-volume batch solar reactor prototypes could thus effectively provide four (88 L Prototype II) to seven (144 L Prototype I) people on a daily basis with the basic water requirement for human activities (20 L). Additionally, a generic Water Safety Plan was developed to aid practitioners in identifying risks and implement remedial actions in this type of installation in order to ensure the safety of the treated water

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.Highlights: Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking; Wastewater analysis protocols are not optimized for trace analysis of viruses; False-positive and -negative errors have consequences for public health responses; Inter-laboratory studies utilizing standardized reference materials and protocols are needed.info:eu-repo/semantics/publishedVersio

The Acinetobacter baumannii website (Ab-web): a multidisciplinary knowledge hub, communication platform, and workspace

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with ten articles organized into two main sections, ‘Overview’ and ‘Topics’, and three themes, ‘epidemiology’, ‘antibiotic resistance’, and ‘virulence’. The ‘workspace’ section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas

Geographical distribution, characterisation and evaluation of Saccharomyces cerevisiae strains isolated from South African vineyards in the warmer, inland regions of the Western Cape

Thesis (M.Sc.) -- University of Stellenbosch, 1999.ENGLISH SUMMARY: The natural or spontaneous fermentation of grape must sugars, by yeast that originate from the grapes and winery equipment, to ethanol, carbon dioxide and other minor, but important metabolites, produces wine. The early stages of fermentation are usually carried out by the yeast genera with low fermentative activity, such as Hanseniaspora, Kloeckera, Candida and Pichia. An increase in the alcohol content of the wine causes the suppression of these genera, leaving Saccharomyces cerevisiae, which plays a significant and dominating role, to complete the fermentation process. For this reason S. cerevisiae is then generally accepted and known as the "wine yeast" in the wine industry. Wines made in different areas from the same grape variety with apparently similar fruit composition and processing, often taste noticeably different. It may be that differences in the microflora, naturally present in the two areas, play an important role in producing flavour differences in the wines. The genetic diversity of the yeast flora in nature was proven, by a number of microbiological and oenological studies, to be greatly influenced by a series of parameters, which among others includes the age of the vineyard, grape variety, viticultural and oenological practices, geographic location and climatic conditions. This project, as part of a larger research programme described by Pretorius, Van der Westhuizen & Augustyn (1999), was directly aimed at isolating and characterising indigenous S. cerevisiae yeast strains from vineyards based in the warmer, inland wine regions of the Western Cape in South Africa. Research by Van der Westhuizen, Augustyn & Pretorius (1999a) and Van der Westhuizen et al. (1999b) on the wine yeast strains present on grapes in the cooler, coastal areas of the same province enabled us to compare these strains isolated from these two contrasting climatic zones on a molecular, physiological and biochemical level. The ideal or ultimate aim of any such study would be to determine the entire sequence of the bases of the yeast nucleic acid, for each of these isolates. Molecular biological, physiological and biochemical techniques can, however, be used as indicators of the homology of the chromosomes of strains or isolates obtained. The specific aims and approaches to this study were as follows: Firstly approximately 1 kg of grapes were sampled from each of the 19 sites in the warmer, inland wine regions. Areas sampled included: Villiersdorp, Robertson (2 sites), Nuy, Du Toits Kloof, Bonnievale, Ashton, Montagu (2 sites), Rawsonville, Riebeeck-Kasteel (2 sites), Porterville (2 sites), Wolseley, Malmesbury (2 sites), Sianghoek and Tulbagh. These grapes were aseptically crushed and the must allowed to spontaneously ferment at 15°C. Thirty single colonies per fermentation were isolated when the residual sugar was less than 4 g/l. Characterisation of these S. cerevisiae isolates was done using molecular biological fingerprinting techniques such as electrophoretic karyotyping by pulsed field gel electrophoresis [Contour-Clamped Homogenous Electric Field, (CHEF)] and Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using five specific Operon Kit C (OPC) primers. Comparison of chromosomal banding patterns per sampling site revealed the presence of 30 S. cerevisiae strains hereafter designated W1-W30. Initially it was thought a total of 21 yeast strains (based on CHEF banding pattern results only) were isolated from the warmer, inland regions. However, by comparing the CHEF-DNA analysis and RAOP-PCR data it was possible to distinguish between all of the strains isolated, except W1 and W5. Comparing these chromosomal banding patterns to the banding patterns collected in the Nietvoorbij database revealed that no single strain recovered was indigenous to both the warmer areas of this study and the cooler areas sampled by Van der Westhuizen at al. (1999a, 1999b). Further comparison and characterisation of these strains by means of various physiological and biochemical techniques such as the ability to flocculate; ferment various carbon sources; survive stress resistance at 55°C and -20°C by monitoring cell growth after 24 and 48 hours, respectively; and produce extracellular enzymes, was determined. Even though no distinct differences could be observed in the physiological and biochemical behaviour of strains isolated from the warmer, inland and cooler, coastal wine regions, it was shown that strains W1 and W5, which had the same DNA banding patterns, were in fact different as W1 was galactose negative and W5 was galactose positive. Strain W1 could also grow invasively into the agar medium whereas W5 could not. This comparison revealed that 30 unique strains were in fact isolated from the warmer, inland wine regions of the Western Cape. Investigations into possible survival mechanisms of S. cerevisiae was done by evaluating their ability to form pseudohyphae/grow invasively. Results indicated that these factors could contribute to the organisms over-wintering ability, but clearly other mechanisms must also be involved. Molecular biological, physiological and biochemical characterisation techniques should then be used in collaboration with each other to obtain an overall, positive and extensive profile of any yeast species or strain. These results also then prove invaluable in not only providing information, but also elucidating any possible differences between strains isolated from different climatic zones. The fermentation potential of these 30 unique strains was evaluated and of these strains three are included in the breeding programme at the ARC-Institute for Fruit, Vine and Wine, Nietvoorbij, aimed at improving wine yeast.AFRIKAANSE OPSOMMING: Wyn word geproduseer wanneer natuurlike of spontane gisting van druiwesuikers deur giste wat natuurlik op druiwe en keldertoerusting voorkom, na etanol, koolstofdioksied en ander minder beduidende, maar belangrike metaboliete omgeskakel word. Die vroee stadia van gisting word gewoonlik deur gisgenera met 'n lae fermentasie-aktiwiteit, 5005 Hanseniaspora, Kloeckera, Candida en Pichia, uitgevoer. 'n Toename in die alkoholinhoud veroorsaak egter 'n onderdrukking van hierdie giste en daarom speel Saccharomyces cerevisiae 'n betekenisvolle en dominante rol deurdat dit die gistingsproses voltooi. Om hierdie rede word S. cerevisiae algemeen aanvaar en erken as die "wyngis" in die wynbedryf. Wyne afkomstig vanaf dieselfde kultivar en met skynbaar eenderse geursamestelling, maar van verskillende areas, proe dikwels opmerklik verskillend. Dit is moontlik dat die verskille in mikroflora wat in die onderskeie streke voorkom, 'n belangrike rol speel om geurverskille in die wyn na vore te bring. 'n Verskeidenheid mikrobiologiese en wynkundige studies het egter bewys dat die genetiese diversiteit van die gisflora in 'n groot mate deur verskeie fa ktore, 5005 onder andere die ouderdom van die wingerd, die druifvarieteit, geografiese ligging en klimaatstoestande, beinvloed word. Hierdie projek vorm deel van 'n groter navorsingsprogram, 5005 beskryf deur Pretorius, Van der Westhuizen & Augustyn (1999). Die doel van hierdie projek het behels die isolasie en karakterisering van natuurlike S. cerevisiae-giste van wingerde in die warmer, binnelandse streke van die Wes-Kaap in Suid-Afrika. Navorsing deur Van der Westhuizen, Augustyn & Pretorius (1999a) and Van der Westhuizen et al. (1999b) op wyngiste wat op druiwe van die koeler, kusstreek van dieselfde provinsie voorkom, het ens in staat gestel om rasse van die twee verskillende klimaatstreke op 'n molekulere, fisiologiese en biochemiese vlak te vergelyk. Die uiteindelike doel van sodanige studie sou wees om die totale nukleiensuurbasissekwensie van aile isolate te bepaal. Molekulere, fisiologiese en biochemiese tegnieke kan egter gebruik word om 'n aanduiding van die homologie van chomosome van die onderskeie isolate te gee. Die spesifieke doel en benadering van die studie was soos volg: Ongeveer 1 kg druiwe is van elk van die 19 onderskeie lokaliteite in die warmer, binnelandse areas geneem. Plekke van monstememing het die volgende ingesluit: Villiersdorp, Robertson (2 plekke), Nuy, Du Toitskloof, Bonnievale, Ashton, Montagu (2 plekke), Rawsonville, Riebeeck-Kasteel (2 plekke), Porterville (2 plekke), Wolseley, Malmesbury (2 plekke), Sianghoek en Tulbagh. Hierdie druiwe is asepties gepers en toegelaat om spontaan by 15°C te gis. Nadat elke individuele gisting voltooi is en die residuele suiker minder as 4 g/l was, is 30 enkelkolonies van elke gisting geisoleer. Die karakterisering van S. cerevisiae-isolate is deur middel van molekulere-biologiese vingerafdruktegnieke uitgevoer, naamlik elektroforetiese kariotipering deur middel van pulsveld-jelelektroforese [Kontoer-GeklampdeHomogene- Elektrieseveld-Elektroforese (CHEF]; Lukraak Geamplifiseerde Polimorfiese DNA-Polimerasekettingreaksie (RAPD-PKR) met vyf spesifieke Operon Kit C (OPC) -voorvoerders. Die vergelyking van chromosoombandpatrone per monstememingsarea het 30 S. cerevisae-rasse (W1-W30) opgelewer. Aanvanklik is vermoed dat net 21 gisrasse (op grond van slegs CHEF-bandpatrone) in die warmer, binnelandse streke geisoleer is. Met 'n vergelyking tussen die CHEF-DNA- en die RAPD-PKR-analises was dit moontlik om tussen aile rasse, behalwe W1 en W5, te onderskei. 'n Vergelyking van die chromosoombandpatrone van aile isolate in die Nietvoorbij-databasis, het aangedui dat geen ras natuurlik in beide die warmer en koeler streke van die Wes-Kaap voorgekom het nie. Verdere vergelyking en karakterisering van hierdie rasse deur middel van verskeie fisiologiese en biochemiese tegnieke is gedoen en het betrekking op die vermoe om te flokkuleer, benutting van verskeie koolstofbronne en selgroei na 24 en 48 uur na onderwerping aan strestoestande by 55°C en -20°C. Die hidrolise en produksie van ekstrasellulere ensieme is ook bepaal. Ten spyte daarvan dat geen duidelike verskille in die fisiologiese en biochemiese kenmerke van rasse van die warmer, binnelandse gebiede en koeler kusstreek waargeneem kon word nie, het dit aangetoon dat rasse W1 en W5, wat dieselfde DNAband patrone het, wei ten opsigte van suikerbenutting verskil. Ras W1 is galaktose-negatief, in teenstelling met W5 wat galaktose-positief was. Ras W1 kon ook penetrerend in die agarmedium in groei en W5 kon nie. Hierdie resultate bevestig uiteraard dat 30 unieke rasse wei in die warmer, binnelandse streek van die Wes-Kaap geisoleer is. Moontlike oorlewingsmeganismes van S. cerevisiae, soos die vorming van pseudohifes wat in die agarmedium kan ingroei, is ook ondersoek. Resultate wat verkry is, bewys dat hierdie faktore tot die giste se oorwinteringsvermoe kan bydra, maar dit is duidelik dat daar ook ander meganismes betrokke is. Molekulere, fisiologiese en biochemiese karakteriseringstegnieke moet dus aanvullend tot mekaar gebruik word ten einde 'n geheelbeeld te verkry van die spesie of ras wat bestudeer word. Hierdie resultate wys duidelik dat dit nie net van onskatbare waarde is ten einde inligting te voorsien nie, maar dat dit ook enige moontlike verskille tussen rasse afkomstig uit verskillende klimaatsgebiede kan aandui. Die gistingspotensiaal van die 30 unieke rasse is ook gevalueer. Drie van hierdie rasse is ingesluit in die telingsprogram van die LNR-Instituut vir Vrugte, Wingerd en Wyn te Nietvoorbij, wat ten doel het om wyngiste te verbeter.Maste