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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0363-8) contains supplementary material, which is available to authorized users.

A broad body of literature has been published regarding roof-harvested rainwater quality around the world. In particular, the presence of fecal indicator bacteria and pathogenic microorganisms has raised concerns regarding the acceptability of rainwater for potable and non-potable uses. As the use of molecular assays has improved understanding of the diverse microbial communities present in rainwater tanks and their role in providing benefits or harm to human health, a comprehensive review is needed to summarize the state of the science in this area. To provide a summary of microbial contaminants in rainwater tanks and contextual factors, a comprehensive review was conducted here to elucidate the uses of rainwater, factors affecting water quality, concentrations of fecal indicators and pathogens, the attribution of pathogens to host sources using microbial source tracking, microbial ecology, human health risks determined using epidemiological approaches and quantitative microbial risk assessment, and treatment approaches for mitigating risks. Research gaps were identified for pathogen concentration data, microbial source tracking approaches for identifying the sources of microbial contamination, limitations to current approaches for assessing viability, treatment, and maintenance practices. Frameworks should be developed to assess and prioritize these factors in order to optimize public health promotion for roof-harvested rainwater.

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Ethidium monoazide (EMA) quantitative polymerase chain reaction (qPCR), propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR were compared for the determination of microbial cell viability. Additionally, varying EMA and PMA concentrations were analysed to determine which dye and concentration allowed for the optimal identification of viable cells. Viable, heat treated (70 °C for 15 min) and autoclaved cultures of Legionella pneumophila, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Enterococcus faecalis were utilised in the respective viability assays. Analysis of the viable and heat-treated samples indicated that variable log reductions were recorded for both EMA [log reductions ranging from 0.01 to 2.71 (viable) and 0.27 to 2.85 (heat treated)], PMA [log reductions ranging from 0.06 to 1.02 (viable) and 0.62 to 2.46 (heat treated)] and DNase treatment [log reductions ranging from 0.06 to 0.82 (viable) and 0.70 to 2.91 (heat treated)], in comparison to the no viability treatment controls. Based on the results obtained, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat-treated samples) in comparison to the no viability treatment control. In addition, the results recorded for the 6 μM EMA concentration were comparable to the results obtained for both the 50 μM PMA and the DNase treatment. The use of EMA-qPCR (6 μM) may therefore allow for the rapid identification and quantification of multiple intact opportunistic pathogens in water sources, which would benefit routine water quality monitoring following disinfection treatment.

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BackgroundThe antimicrobial resistance of clinical, environmental and control strains of the WHO “Priority 1: Critical group” organisms, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa to various classes of antibiotics, colistin and surfactin (biosurfactant) was determined.MethodsAcinetobacter baumannii was isolated from environmental samples and antibiotic resistance profiling was performed to classify the test organisms [A. baumannii (n = 6), P. aeruginosa (n = 5), E. coli (n = 7) and K. pneumoniae (n = 7)] as multidrug resistant (MDR) or extreme drug resistant (XDR). All the bacterial isolates (n = 25) were screened for colistin resistance and the mobilised colistin resistance (mcr) genes. Biosurfactants produced by Bacillus amyloliquefaciens ST34 were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). The susceptibility of strains, exhibiting antibiotic and colistin resistance, to the crude surfactin extract (cell-free supernatant) was then determined.ResultsAntibiotic resistance profiling classified four A. baumannii (67%), one K. pneumoniae (15%) and one P. aeruginosa (20%) isolate as XDR, with one E. coli (15%) and three K. pneumoniae (43%) strains classified as MDR. Many of the isolates [A. baumannii (25%), E. coli (80%), K. pneumoniae (100%) and P. aeruginosa (100%)] exhibited colistin resistance [minimum inhibitory concentrations (MICs) ≥ 4 mg/L]; however, only one E. coli strain isolated from a clinical environment harboured the mcr-1 gene. UPLC-MS analysis then indicated that the B. amyloliquefaciens ST34 produced C13–16 surfactin analogues, which were identified as Srf1 to Srf5. The crude surfactin extract (10.00 mg/mL) retained antimicrobial activity (100%) against the MDR, XDR and colistin resistant A. baumannii, P. aeruginosa, E. coli and K. pneumoniae strains.ConclusionClinical, environmental and control strains of A. baumannii, P. aeruginosa, E. coli and K. pneumoniae exhibiting MDR and XDR profiles and colistin resistance, were susceptible to surfactin analogues, confirming that this lipopeptide shows promise for application in clinical settings.

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 RP-HPLC, 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). These results provide crucial insight into the secondary metabolic profiles of pigmented and non-pigmented S. marcescens strains and confirms that S. marcescens strains are a promising natural source of novel antimicrobial metabolites.

McNemar’s test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar’s test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.