Research Articles (Microbiology)

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50 years of Emmonsia disease in humans : the dramatic emergence of a cluster of novel fungal pathogens
(Public Library of Science, 2015) Schwartz, Ilan S.; Kenyon, Chris; Feng, Peiying; Govender, Nelesh P.; Dukik, Karolina; Sigler, Lynne; Jiang, Yanping; Stielow, J. Benjamin; Munoz, Jose F.; Cuomo, Christina A.; Botha, Alfred; Stchigel, Alberto M.; De Hoog, G. Sybren
New species of Emmonsia-like fungi, with phylogenetic and clinical similarities to Blastomyces and Histoplasma, have emerged as causes of systemic human mycoses worldwide. They differ from classical Emmonsia species by producing a thermally-dependent, yeast-like phase rather than adiaspores, and by causing disseminated infections, predominantly in immunocompromised patients and often with high case-fatality rates. Such differences will be important for clinicians to consider in diagnosis and patient management, and for microbiologists who may encounter these fungi with increasing frequency.
Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus
(Public Library of Science, 2013-11-08) Acuna, Lillian G.; Cardenas, Juan Pablo; Covarrubias, Paulo C.; Haristoy, Juan Jose; Flores, Rodrigo; Nunez, Harold; Riadi, Gonzalo; Shmaryahu, Amir; Valdes, Jorge; Dopson, Mark; Rawlings, Douglas E.; Banfield, Jillian F.; Holmes, David S.; Quatrini, Raquel
Background: Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings: Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance: For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.
Bacteria of the genus xenorhabdus, a novel source of bioactive compounds
(Frontiers Media, 2018-12-19) Dreyer, Jonike; Malan, Antoinette P.; Dicks, Leon Milner Theodore, 1961-
The genus Xenorhabdus of the family Enterobacteriaceae, are mutualistically associated with entomopathogenic nematodes of the genus Steinernema. Although most of the associations are species-specific, a specific Xenorhabdus sp. may infect more than one Steinernema sp. During the Xenorhabdus–Steinernema life cycle, insect larvae are infected and killed, while both mutualists produce bioactive compounds. These compounds act synergistically to ensure reproduction and proliferation of the nematodes and bacteria. A single strain of Xenorhabdus may produce a variety of antibacterial and antifungal compounds, some of which are also active against insects, nematodes, protozoa, and cancer cells. Antimicrobial compounds produced by Xenorhabdus spp. have not been researched to the same extent as other soil bacteria and they may hold the answer to novel antibacterial and antifungal compounds. This review summarizes the bioactive secondary metabolites produced by Xenorhabdus spp. and their application in disease control. Gene regulation and increasing the production of a few of these antimicrobial compounds are discussed. Aspects limiting future development of these novel bioactive compounds are also pointed out.
Bacterial diversity and production of sulfide in microcosms containing uncompacted bentonites
(2018) Grigoryan, Alexander A.; Jalique, Daphne R.; Medihala, Prabhakara; Stroes-Gascoyne, Simcha; Wolfaardt, Gideon M.; McKelvie, Jennifer; Korber, Darren R.
Aims: This study examined the diversity and sulfide-producing activity of microorganisms in microcosms containing commercial clay products (e.g., MX-80, Canaprill and National Standard) similar to materials which are currently considered for use in the design specifications for deep geologic repositories (DGR) for spent nuclear fuel. Methods and results: In anoxic microcosms incubated for minimum of 60 days with 10 g l-¹ NaCl, sulfide production varied with temperature, electron donor and bentonite type. Maximum specific sulfide production rates of 0.189 d-¹, 0.549 d-¹ and 0.157 d-¹ occurred in lactate-fed MX-80, Canaprill and National Standard microcosms, respectively. In microcosms with 50 g l-¹ NaCl, sulfide production was inhibited. Denaturing gradient gel electrophoresis (DGGE) profiling of microcosms revealed the presence of bacterial classes Clostridia, Bacilli, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Sphingobacteriia and Erysipelotrichia. Spore-forming and non-spore-forming bacteria were confirmed in microcosms using high-throughput 16S rRNA gene sequencing. Sulfate-reducing bacteria of the genus Desulfosporosinus predominated in MX-80 microcosms; whereas, Desulfotomaculum and Desulfovibrio genera contributed to sulfate-reduction in National Standard and Canaprill microcosms. Conclusions: Commercial clays microcosms harbour a sparse bacterial population dominated by spore-forming microorganisms. Detected sulfate- and sulfur-reducing bacteria presumably contributed to sulfide accumulation in the different microcosm systems. Significance and impact of study The use of carbon-supplemented, clay-in-water microcosms offered insights into the bacterial diversity present in as-received clays, along with the types of metabolic and sulfidogenic reactions that might occur in regions of a DGR (e.g., interfaces between the bulk clay and host rock, cracks, fissures, etc.) that fail to attain target parameters necessary to inhibit microbial growth and activity.
The biochemistry of malic acid metabolism by wine yeasts – a review
(South African Society for Enology and Viticulture, 2006) Saayman, M.; Viljoen-Bloom, M.
L-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the “fixed acidity” that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A variety of factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast’s intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species.
Bioenergy and African transformation
(BioMed Central, 2015-02) Lynd, Lee R.; Sow, Mariam; Chimphango, Annie F. A.; Cortez, Luis A. B.; Brito Cruz, Carlos H.; Elmissiry, Mosad; Laser, Mark; Mayaki, Ibrahim A.; Moraes, Marcia A. F. D.; Nogueira, Luiz A. H.; Wolfaardt, Gideon M.; Woods, Jeremy; Van Zyl, Willem H.
Among the world’s continents, Africa has the highest incidence of food insecurity and poverty and the highest rates of population growth. Yet Africa also has the most arable land, the lowest crop yields, and by far the most plentiful land resources relative to energy demand. It is thus of interest to examine the potential of expanded modern bioenergy production in Africa. Here we consider bioenergy as an enabler for development, and provide an overview of modern bioenergy technologies with a comment on application in an Africa context. Experience with bioenergy in Africa offers evidence of social benefits and also some important lessons. In Brazil, social development, agricultural development and food security, and bioenergy development have been synergistic rather than antagonistic. Realizing similar success in African countries will require clear vision, good governance, and adaptation of technologies, knowledge, and business models to myriad local circumstances. Strategies for integrated production of food crops, livestock, and bioenergy are potentially attractive and offer an alternative to an agricultural model featuring specialized land use. If done thoughtfully, there is considerable evidence that food security and economic development in Africa can be addressed more effectively with modern bioenergy than without it. Modern bioenergy can be an agent of African transformation, with potential social benefits accruing to multiple sectors and extending well beyond energy supply per se. Potential negative impacts also cut across sectors. Thus, institutionally inclusive multi-sector legislative structures will be more effective at maximizing the social benefits of bioenergy compared to institutionally exclusive, single-sector structures.
Biofilm dynamics : linking in situ biofilm biomass and metabolic activity measurements in real-time under continuous flow conditions
(Nature Research, 2020) Klopper, Kyle Brent; De Witt, Riaan N.; Bester, Elanna; Dicks, Leon Milner Theodore, 1961-; Wolfaardt, Gideon M.
The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass–function relationships and aid in the development of biofilm control strategies.
Biologically based methods for control of fumonisin-producing fusarium species and reduction of the fumonisins
(Frontiers Media, 2016) Alberts, Johanna F.; Van Zyl, Willem H.; Gelderblom, Wentzel C. A.
Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with selected biologically based treatments, mild chemical and physical treatments could reduce fumonisin contamination effectively. In rural subsistence farming communities, simple, practical, and culturally acceptable hand-sorting, maize kernel washing, and dehulling intervention methods proved to be effective as a last line of defense for reducing fumonisin exposure. Biologically based methods for control of fumonisin-producing Fusarium spp. and decontamination of the fumonisins could have potential commercial application, while simple and practical intervention strategies could also impact positively on food safety and security, especially in rural populations reliant on maize as a dietary staple.
A broad host range reporter plasmid for the analysis of divergent promoter regions
(Academy of Science of South Africa, 2008) Jiwaji, Meesbah; Matcher, Gwynneth Felicity; Dorrington, Rosemary Ann
Although many vectors exist for Escherichia coli and closely related species, there are few broad host range vectors that can be conjugated into a large variety of Gram-negative bacteria. We have constructed a broad host range vector, pMJ445, that facilitates the analysis of divergent promoters in Gram-negative bacteria. The vector was validated using two intergenic regions derived from gene clusters involved in hydantoin hydrolysis, from the environmental isolates Pseudomonas putida and Agrobacterium tumefaciens. The DNA sequences analysed were capable of activating expression of the reporter enzymes, β-glucuronidase and β-galactosidase, present on pMJ445, indicating the presence of divergent promoters in the sequences selected. In addition, we demonstrated that pMJ445 can be applied to gene regulation studies.
Canary in the coliform mine : exploring the industrial application limits of a microbial respiration alarm system
(Public Library of Science, 2021-03-04) Stone, Wendy; Louw, Tobi M.; Booysen, Marthinus J.; Wolfaardt, Gideon M.; Zhang, Dawei
Fundamental ecological principles of ecosystem-level respiration are extensively applied in greenhouse gas and elemental cycle studies. A laboratory system termed CEMS (Carbon Dioxide Evolution Measurement System), developed to explore microbial biofilm growth and metabolic responses, was evaluated as an early-warning system for microbial disturbances in industrial settings: in (a) potable water system contamination, and (b) bioreactor inhibition. Respiration was detected as CO₂ production, rather than O₂ consumption, including aerobic and anaerobic metabolism. Design, thresholds, and benefits of the remote CO₂ monitoring technology were described. Headspace CO₂ correlated with contamination levels, as well as chemical (R² > 0.83–0.96) and microbiological water quality indicators (R² > 0.78–0.88). Detection thresholds were limiting factors in monitoring drinking water to national and inter- national standards (0 CFU/100 mL fecal coliforms) in both open- (>1500 CFU/mL) and closed-loop CO₂ measuring regimes (>100 CFU/100 mL). However, closed-loop detection thresholds allow for the detection of significant contamination events, and monitoring less stringent systems such as irrigation water (<100 CFU/mL). Whole-system respiration was effectively harnessed as an early-warning system in bioreactor performance monitoring. Models were used to deconvolute biological CO₂ fluctuations from chemical CO₂ dynamics, to optimize this real-time, sustainable, low-waste technology, facilitating timeous responses to biological disturbances in bioreactors.
Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant
(SpringerOpen, 2017) Ndlovu, Thando; Rautenbach, Marina; Vosloo, Johann Arnold; Khan, Sehaam; Khan, Wesaal
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.
Characterisation of Metarhizium majus (Hypocreales : Clavicipitaceae) isolated from the Western Cape Province, South Africa
(Public Library of Science, 2021-03-19) Mathulwe, Letodi L.; Jacobs, Karin; Malan, Antoinette P.; Birkhofer, Klaus; Addison, Matthew F.; Addison, Pia
Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.
Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates
(BioMed Central, 2005-05) Rawlings, Douglas E.
Microorganisms are used in large-scale heap or tank aeration processes for the commercial extraction of a variety of metals from their ores or concentrates. These include copper, cobalt, gold and, in the past, uranium. The metal solubilization processes are considered to be largely chemical with the microorganisms providing the chemicals and the space (exopolysaccharide layer) where the mineral dissolution reactions occur. Temperatures at which these processes are carried out can vary from ambient to 80°C and the types of organisms present depends to a large extent on the process temperature used. Irrespective of the operation temperature, biomining microbes have several characteristics in common. One shared characteristic is their ability to produce the ferric iron and sulfuric acid required to degrade the mineral and facilitate metal recovery. Other characteristics are their ability to grow autotrophically, their acid-tolerance and their inherent metal resistance or ability to acquire metal resistance. Although the microorganisms that drive the process have the above properties in common, biomining microbes usually occur in consortia in which cross-feeding may occur such that a combination of microbes including some with heterotrophic tendencies may contribute to the efficiency of the process. The remarkable adaptability of these organisms is assisted by several of the processes being continuous-flow systems that enable the continual selection of microorganisms that are more efficient at mineral degradation. Adaptability is also assisted by the processes being open and non-sterile thereby permitting new organisms to enter. This openness allows for the possibility of new genes that improve cell fitness to be selected from the horizontal gene pool. Characteristics that biomining microorganisms have in common and examples of their remarkable adaptability are described.
Characterization of killer yeast isolates from chenin blanc grapes and grape skins
(South African Society for Enology and Viticulture, 1991) Jacobs, C. J.; Fourie, I.; Van Vuuren, H. J. J.
Wild-type killer yeast strains isolated from six South African wineries were identified using classical taxonomic methods. They were further characterized according to their cross-reactions with reference killer yeasts (K1-Kn) and by electrophoresis of their double-stranded RNA molecules. All isolates belonged to the K2 phenotype and were identified as strains of Saccharomyces cerevisiae and Saccharomyces bayanus. The killer strains differed substantially in their ability to kill a sensitive wine yeast (Geisenheim GS-1). This phenomenon may be attributed to strain differences among the killer yeasts as was shown by electrophoresis of total soluble cell proteins and gas chromatographic analysis of cellular fatty acids.
Characterization of Leucocin B-KM432Bz from Leuconostoc pseudomesenteroides isolated from Boza, and comparison of its efficiency to Pediocin PA-1
(PLoS, 2013-08) Makhloufi, Kahina Maya; Carre-Mlouka, Alyssa; Peduzzi, Jean; Lombard, Carine; Van Reenen, Carol Ann; Dicks, Leon Milner Theodore, 1961-; Rebuffat, Sylvie
A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EIIt Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.
A Chromogenic substrate for a β-xylosidase-coupled assay of α-glucuronidase
(Elsevier, 2000-08) Biely, Peter; Hirsch, Jan; La Grange, Daniel C.; Van Zyl, Willem H.; Prior, Bernard A.
-Nitrophenyl 2-(4-O-methyl-α- -glucopyranuronosyl)-β- -xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-α- -glucopyranosyluronate)-β- -xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145–149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic α-glucuronidase activity. A new precise α-glucuronidase assay was developed by coupling the α-glucuronidase-catalyzed formation of 4-nitrophenyl β- -xylopyranoside with its efficient hydrolysis by β-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the β-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of β-xylosidase. The activity values of β-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the α-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.
The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli
(American Society for Microbiology, 2000-05) Butcher, Bronwyn G.; Deane, Shelly M.; Rawlings, Douglas E.
The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli- derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.
Ciprofloxacin-eluting nanofibers inhibits biofilm formation by pseudomonas aeruginosa and a Methicillin-resistant Staphylococcus aureus
(Public Library of Science, 2015-04) Ahire, Jayesh J.; Neveling, Deon P.; Hattingh, Melanie; Dicks, Leon Milner Theodore, 1961-; Microbiology
Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital- acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC (minimal inhibitory concentration) levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly(D,Llactide) (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillinresistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers.
Cloning of the Bacillus pumilus beta-xylosidase gene (xynB ) and its expression in Saccharomyces cerevisiae
(Springer-Verlag, 1997-07) La Grange, Daniel C.; Pretorius, Isak S.; Van Zyl, Willem H.
A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the beta-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2P-MFa1SxynB-ADH2T was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional beta-xylosidase. The total beta-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45±50 °C and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min.
Co-detection of virulent escherichia coli genes in surface water sources
(Public Library of Science, 2015) Ndlovu, Thando; Le Roux, Marcellous; Khan, Wesaal; Khan, Sehaam
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.

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