Standard Laboratory Media Research Articles

Aspergillus flavus Secondary Metabolites: More than Just Aflatoxins.

Aspergillus flavus is best known for producing the family of potent carcinogenic secondary metabolites known as aflatoxins. However, this opportunistic plant and animal pathogen also produces numerous other secondary metabolites, many of which have also been shown to be toxic. While about forty of these secondary metabolites have been identified from A. flavus cultures, analysis of the genome has predicted the existence of at least 56 secondary metabolite gene clusters. Many of these gene clusters are not expressed during growth of the fungus on standard laboratory media. This presents researchers with a major challenge of devising novel strategies to manipulate the fungus and its genome so as to activate secondary metabolite gene expression and allow identification of associated cluster metabolites. In this review, we discuss the genetic, biochemical and bioinformatic methods that are being used to identify previously uncharacterized secondary metabolite gene clusters and their associated metabolites. It is important to identify as many of these compounds as possible to determine their bioactivity with respect to fungal development, survival, virulence and especially with respect to any potential synergistic toxic effects with aflatoxin.

The Effect of Adding Blood on the Virulence Genes Expression of Staphylococcus aureus in Exponential and Stationary Growth Phase

Background: Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors, and the most important regulator system of virulence factors expression in these bacteria is the agr system. Expression of virulence factors is not the same under in vitro conditions in standard laboratory medium and in vivo in the host. Objectives: This study aimed at evaluating the effects of adding blood on the virulence genes expression of S. aureus. Methods: In this study, the expression levels of agrA, RNAIII, hla (encoding alpha-toxin), spa (encoding protein A), and mecA (encoding resistance of methicillin) genes were determined during growth of S. aureus isolates in BHI broth and BHI broth, containing 5 sheep blood during different growth phases. The gyrB gene was used as an internal control. Results: The agr system in the BHI broth, containing 5 sheep blood, was active. The expression levels of agrA, RNAIII, hla, and mecA in the stationary phase relative to exponential phase of growth was increased by 2.95, 5.7, 2.7 and 2.08 folds, respectively. However, the expression of spa gene was decreased by 0.78 folds. Conclusions: Aside from the growth phase, the expression levels of all of the genes in cultures containing blood relative to BHI broth alone were increased. During encounter with blood cells, the expression profile was similar to that seen in vivo conditions. © 2017, Jundishapur Journal of Microbiology.

Toxinogenicity and cytotoxicity of Alternaria, Aspergillus and Penicillium moulds isolated from working environments

There is currently limited research available on the secondary metabolites of moulds in workplaces. The aim of this study was to determine the mould contamination in museums (N = 4), composting plants (N = 4) and tanneries (N = 4) and the secondary metabolite profiles of Alternaria, Aspergillus and Penicillium isolates from these workplaces. Alternaria, Aspergillus and Penicillium species were identified using the ITS1/2 sequence of the rDNA region. Mould metabolites were quantitatively analysed on standard laboratory medium and mineral medium containing materials specific to each workplace using liquid chromatography-mass spectrometry. We also examined the cytotoxicity of the moulds using MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assays. Air microbiological contamination analyses showed a number of microorganisms, ranging from 2.4 × 103 CFU m−3 (composting plants) to 6.8 × 104 CFU m−3 (tanneries). We identified high percentages of Alternaria, Aspergillus and Penicillium moulds (air 57–59%, surfaces 10–65%) in all workplaces. The following moulds were the most cytotoxic (>90%): Alternaria alternata, A. limoniasperae, Aspergillus flavus, Penicillium biourgeianum, P. commune and P. spinulosum. The same mould species isolated from different working environments exhibited varying toxigenic and cytotoxic properties. Modifying the culture medium to simulate environmental conditions most often resulted in the inhibition of secondary metabolite production. Moulds isolated from the working environments produced the following mycotoxins (ng g−1): chanoclavines (0.28–204), cyclopiazonic acid (27.1–1045), fumigaclavines (0.33–10,640,000), meleagrin (0.57–13,393), roquefortins (0.01–16,660), rugulovasines (112–220), viridicatin (0.12–957), viridicatol (4.23–2753) and quinocitrinines (0.07–1104), which may have a negative impact on human health.

Effect of Human Burn Wound Exudate on Pseudomonas aeruginosa Virulence.

Burn wound sepsis is currently the main cause of morbidity and mortality after burn trauma. Infections by notorious pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii impair patient recovery and can even lead to fatality. In this study, we investigated the effect of burn wound exudates (BWEs) on the virulence of those pathogens. BWEs were collected within 7days after burn trauma from 5 burn patients. We first monitored their effect on pathogen growth. In contrast to A.baumannii and S.aureus, P.aeruginosa was the only pathogen able to grow within these human fluids. Expression of typical virulence factors such as pyocyanin and pyoverdine was even enhanced compared the levels seen with standard laboratory medium. A detailed chemical composition analysis of BWE was performed, which enabled us to determine the major components of BWE and underline the metabolic modifications induced by burn trauma. These data are essential for the development of an artificial medium mimicking the burn wound environment and the establishment of an in vitro system to analyze the initial steps of burn wound infections. IMPORTANCE Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P.aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P.aeruginosa. The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the burn wound environment and for in vitro analysis of the initial step in the development of burn wound infections.

Optimization of <em>Lactobacillus fermentum</em> DSM 20049 Growth on Whey and Lupin Based Medium Using Response Surface Methodology

The aim of this study was to evaluate the potential use of whey components and lupin proteins as cheap nutrients in the formulation of optimum medium for lactic acid bacteria growth, for this purpose a Plackett and Burman design was used to screen variables (medium components and physical parameters) that have significant effects on the growth of Lactobacillus fermentum DSM 20049 in whey and lupin based medium. Statistical analysis of the results allowed selecting two factors having a significant positive effect on the bacterial growth (tween 80 and inoculum size). Central composite design was used to determine the optimum level of the selected factor. Under these optimized conditions, the composition of WLM (Whey and Lupin Medium) was the basic medium of whey and lupin supplemented with 1.56 mL/L of tween and inoculated at a rate of 5.25% with a maximum OD 600 value of 6.88. Based on the comparison of Lactobacillus fermentum DSM 20049 growth on the formulated medium with that observed on MRS (standard laboratory medium), the growth of the strain in WLM is much higher than that observed in MRS medium with a maximum OD 600 values of 7.04 on LWM and 4.08 on MRS medium, respectively and Lactobacillus fermentum DSM 20049 growth in the new medium was 72.54% higher than that on MRS. Thus, whey and lupin hydrolysates are good alternative sources of nitrogen that lead to a better growth of the strain and can be used for large-scale production.

84 An optimized in vitro antibiotic susceptibility testing method for Staphylococcus aureus small colony variants

Objectives S. aureus small colony variants (SCVs) frequently infect people with CF and are associated with worse lung disease. Because SCVs grow poorly and frequently revert to wild-type (WT) growth on standard laboratory media, there are currently no standardized methods for antibiotic susceptibility testing (AST) of SCVs to guide treatment. In this study, we sought to identify conditions that would support in vitro growth of SCVs, allowing for reproducible AST. Methods We tested growth and stability of SCVs on 11 agar media, selecting two for AST that yielded best SCV growth and lowest reversion rates. We performed AST by disk diffusion on 112 S. aureus CF respiratory isolates from 24 patients, comprising 85 SCVs and 27 isogenic WT isolates. Etest AST for a subset of isolates, 26 SCVs and 24 WT pairs, was also completed. Results Growth and reversion were optimal on brain-heart infusion agar (BHI) and cation-adjusted Mueller-Hinton agar supplemented with the 3 compounds for which most CF SCVs are auxotrophic: hemin, menadione, and thymidine (sMHA). AST yielded similar results for the majority of SCVs and isogenic WT pairs on both media. 81 SCVs and 19 WTs were not susceptible to at least 1 of the following: b-lactams, fluoroquinolones, macrolides, lincosamides, or rifampicin. All isolates were susceptible to quinupristin/dalfopristin, minocycline, and linezolid. Etest MICs were comparable to disk diffusion results. AST results also predicted MRSA status, which was confirmed by mecA PCR. Conclusion We have developed an optimized AST method for SCVs. With clinical correlation, AST profiles defined in this study may be useful in directing treatment for SCV infections.

Proteomic Investigation of the Response of Enterococcus faecalis V583 when Cultivated in Urine.

Enterococcus faecalis is a robust bacterium, which is able to survive in and adapt to hostile environments such as the urinary tract and bladder. In this label-free quantitative proteomic study based on MaxQuant LFQ algorithms, we identified 127 proteins present in the secretome of the clinical vancomycin-resistant isolate E. faecalis V583 and we compared proteins secreted in the initial phase of cultivation in urine with the secretome during cultivation in standard laboratory medium, 2xYT. Of the 54 identified proteins predicted to be secreted, six were exclusively found after cultivation in urine including the virulence factor EfaA (“endocarditis specific antigen”) and its homologue EF0577 (“adhesion lipoprotein”). These two proteins are both involved in manganese transport, known to be an important determinant of colonization and infection, and may additionally function as adhesins. Other detected urine-specific proteins are involved in peptide transport (EF0063 and EF3106) and protease inhibition (EF3054). In addition, we found an uncharacterized protein (EF0764), which had not previously been linked to the adaptation of V583 to a urine environment, and which is unique to E. faecalis. Proteins found in both environments included a histone-like protein, EF1550, that was up-regulated during cultivation in urine and that has a homologue in streptococci (HlpA) known to be involved in bacterial adhesion to host cells. Up-regulated secreted proteins included autolysins. These results from secretome analyses are largely compatible with previously published data from transcriptomics studies. All in all, the present data indicate that transport, in particular metal transport, adhesion, cell wall remodelling and the unknown function carried out by the unique EF0764 are important for enterococcal adaptation to the urine environment. These results provide a basis for a more targeted exploration of novel proteins involved in the adaptability and pathogenicity of E. faecalis.

Editorial on: Bacterial pathogens in the non-clinical environment.

Editorial on: Bacterial pathogens in the non-clinical environment.

Identification and characterization of outer membrane vesicle-associated proteins in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes.

Vibrio cholerae Classical Biotype Is Converted to the Viable Non-Culturable State when Cultured with the El Tor Biotype

A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.

Non-Traditional Antibacterial Screening Approaches for the Identification of Novel Inhibitors of the Glyoxylate Shunt in Gram-Negative Pathogens

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.

Hyperosmotic Stress Response of Campylobacter jejuni

The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter(-1). C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.

Influence of chemical and physical conditions on the production of bacteriocin by Aeromonas hydrophila

Aeromonas hydrophila have been isolated as a cause of a cute gastroenteritis in 23 (5.6%) of 410 patients. Other bacterial enteropathogens have been isolated from 387 patients with diarrhea, were 19 different strains. A. hydrophila occurred more commonly in children with acute diarrhea, the results showed that 18(78.26%) isolates of A. hydrophila found in children under 10 years old ,distributed to 10(43.47%) in male and 8(34.78%) in female ,and in adults with diarrhea 5 (21.73%). In the other hand, we noticed frequency of isolation was higher in male 14(60.86%) when compared with 9(39.14%) in female. Six strains of A. hydrophila have been observed to have bacteriocin activity against 12 of 23 different A. hydrophila ,as well as Staphylococcus aureas, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacea and Shigella dysenteria. The results showed Bacteriocin-like substances (BLS11) had isoinhibitory activity on 10 same A. hydrophila species and heteroinhibitory activity effects on all pathogenic bacterial strains used, while BLS5 showed isoinhibitory activity on 2 same A. hydrophila species and heteroinhibitory activity by effecting on gram negative only, and BLS3& BLS12 showed activity on E. coli isolates only, and none of BLS1& BLS10(isoinhibitory activity on 1 A.hydrophila respectively) had effect on all pathogenic bacteria. Among the standard laboratory media used Brain Heart Infusion broth (BHI) showed the maximum production and poor yields resulted from growth in Peptone Glyserol (PG) and Nutrient broth. We selected BLS11 to their wide range effect on same species and enteric pathogenic strains, to study the Influence of chemical and physical conditions on the production of BLS by A.hydrophila. The BLS11 preparations from A.hydrophila11 strains of A. hydrophila were tolerant to all three treatments of surfactant. In the other hand, effect of organic acid on BLS production BLS11 has been studied and showed no remarkable difference in zone of inhibition when used acetone as affecter element, while both of isopropanol and ethanol have narrow inhibition zone range when compared with control strain. These results indicated that most A. hydrophila might be harboring plasmid mediated bacteriocin like substance, and there are no relation between BLS production and number of plasmid bands present in bacteria.

Influence of chemical and physical conditions on the production of bacteriocin by Aeromonas hydrophila

Aeromonas hydrophila have been isolated as a cause of a cute gastroenteritis in 23 (5.6%) of 410 patients. Other bacterial enteropathogens have been isolated from 387 patients with diarrhea, were 19 different strains. A. hydrophila occurred more commonly in children with acute diarrhea, the results showed that 18(78.26%) isolates of A. hydrophila found in children under 10 years old ,distributed to 10(43.47%) in male and 8(34.78%) in female ,and in adults with diarrhea 5 (21.73%). In the other hand, we noticed frequency of isolation was higher in male 14(60.86%) when compared with 9(39.14%) in female. Six strains of A. hydrophila have been observed to have bacteriocin activity against 12 of 23 different A. hydrophila ,as well as Staphylococcus aureas, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacea and Shigella dysenteria. The results showed Bacteriocin-like substances (BLS11) had isoinhibitory activity on 10 same A. hydrophila species and heteroinhibitory activity effects on all pathogenic bacterial strains used, while BLS5 showed isoinhibitory activity on 2 same A. hydrophila species and heteroinhibitory activity by effecting on gram negative only, and BLS3& BLS12 showed activity on E. coli isolates only, and none of BLS1& BLS10(isoinhibitory activity on 1 A.hydrophila respectively) had effect on all pathogenic bacteria. Among the standard laboratory media used Brain Heart Infusion broth (BHI) showed the maximum production and poor yields resulted from growth in Peptone Glyserol (PG) and Nutrient broth. We selected BLS11 to their wide range effect on same species and enteric pathogenic strains, to study the Influence of chemical and physical conditions on the production of BLS by A.hydrophila. The BLS11 preparations from A.hydrophila11 strains of A. hydrophila were tolerant to all three treatments of surfactant. In the other hand, effect of organic acid on BLS production BLS11 has been studied and showed no remarkable difference in zone of inhibition when used acetone as affecter element, while both of isopropanol and ethanol have narrow inhibition zone range when compared with control strain. These results indicated that most A. hydrophila might be harboring plasmid mediated bacteriocin like substance, and there are no relation between BLS production and number of plasmid bands present in bacteria.

Proteomics of Pseudomonas aeruginosa Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.

Anaerococcus lactolyticus causing a mycotic aortic aneurysm: Category: Clinical lesson

Anaerobic bacteria are a rare cause of mycotic aortic aneurysm (MAA). One reason for this may be that the organisms are often fastidious and do not grow well on standard laboratory media. The advent of molecular techniques such as the identification of RNA from the 16S ribosomal subunit of bacteria by polymerase chain reaction (16S PCR) has offered the opportunity to identify organisms from clinical specimens that are culture negative. This allows better management of patients with difficult or unusual infections, including those caused by anaerobic organisms.

Mycotoxin production by different ochratoxigenic Aspergillus and Penicillium species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8μg/g) and by A. niger BFE632 (10.6μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.

Global Changes in Staphylococcus aureus Gene Expression in Human Blood

Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.

Novel wine-mediated FLO11 flocculation phenotype of commercial Saccharomyces cerevisiae wine yeast strains with modified FLO gene expression

Depending on the genetic background of Saccharomyces strains, a wide range of phenotypic adhesion identities can be directly attributed to the FLO11-encoded glycoprotein, which includes asexual flocculation, invasive growth and pseudohyphal formation, flor formation and adhesion to biotic and abiotic surfaces. In a previous study, we reported that HSP30-mediated stationary-phase expression of the native chromosomal FLO11 ORF in two nonflocculent commercial Saccharomyces cerevisiae wine yeast strains, BM45 or VIN13 did not generate a flocculent phenotype under either standard laboratory media or synthetic MS300 must fermentation conditions. In the present study, the BM45- and VIN13-derived HSP30p-FLO11 wine yeast transformants were observed to be exclusively and strongly flocculent under authentic red wine-making conditions, thus suggesting that this specific fermentation environment specifically contributes to the development of a flocculent phenotype, which is insensitive to either glucose or mannose. Furthermore, irrespective of the strain involved this phenotype displayed both Ca(2+)-dependent and Ca(2+)-independent flocculation characteristics. A distinct advantage of this unique FLO11-based phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by BM45-F11H and VIN13-F11H transformants were significantly less turbid than those produced by their wild-type parental strains.

Evidence for evolutionary constraints in Drosophila metal biology

Mutations in single Drosophila melanogaster genes can alter total body metal accumulation. We therefore asked whether evolutionary constraints maintain biologically abundant metal ions (iron, copper, manganese and zinc) to similar concentrations in different species of Drosophilidae, or whether metal homeostasis is a highly adaptable trait as shown previously for triglyceride and glycogen storage. To avoid dietary influences, only species able to grow and reproduce on a standard laboratory medium were selected for analysis. Flame atomic absorption spectrometry was used to determine metal content in 5-days-old adult flies. Overall, the data suggest that the metallome of the nine species tested is well conserved. Meaningful average values for the Drosophilidae family are presented. Few statistically significant differences were noted for copper, manganese and zinc between species. In contrast, Drosophila erecta and Drosophila virilis showed a 50% increase above average and a 30% decrease below average in iron concentrations, respectively. The changes in total body iron content correlated with altered iron storage in intestinal ferritin stores of these species. Hence, the variability in iron content could be accounted for by a corresponding adaptation in iron storage regulation. We suggest that the relative expression of the multitude of metalloenzymes and other metal-binding proteins remains overall similar between species and likely determines relative metal abundances in the organism. The availability of a complete and annotated genome sequence of different Drosophila species presents opportunities to study the evolution of metal homeostasis in closely related organisms that have evolved separately for millions or dozens of million years.