Standard Laboratory Media Research Articles

Bacterial second messenger cyclic di-AMP in streptococci.

Cyclic dimeric adenosine monophosphate (c-di-AMP) has been well studied in bacteria, including those of the genus Streptococcus, since the first recognition of this dinucleotide in 2008. Streptococci possess a sole diadenylate cyclase, CdaA, and distinct c-di-AMP phosphodiesterases. Interestingly, cdaA is required for viability of some streptococcal species but not all when streptococci are grown in standard laboratory media. Bacteria of this genus also have distinct c-di-AMP effector proteins, diverse c-di-AMP-signaling pathways, and subsequent biological outcomes. In streptococci, c-di-AMP may influence bacterial growth, morphology, biofilm formation, competence program, drug resistance, and bacterial pathogenesis. c-di-AMP secreted by streptococci has also been shown to interact with the mammalian host and induces immune responses including type I interferon production. In this review, we summarize the reported c-di-AMP networks in seven species of the genus Streptococcus, which cause diverse clinical manifestations, and propose future perspectives to investigate the signaling molecule in these streptococcal pathogens.

Adding a new dimension: Multi-level structure and organization of mixed-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a 4-D wound microenvironment

Biofilms in wounds typically consist of aggregates of bacteria, most often Pseudomonas aeruginosa and Staphylococcus aureus, in close association with each other and the host microenvironment. Given this, the interplay across host and microbial elements, including the biochemical and nutrient profile of the microenvironment, likely influences the structure and organization of wound biofilms. While clinical studies, in vivo and ex vivo model systems have provided insights into the distribution of P. aeruginosa and S. aureus in wounds, they are limited in their ability to provide a detailed characterization of biofilm structure and organization across the host-microbial interface. On the other hand, biomimetic in vitro systems, such as host cell surfaces and simulant media conditions, albeit reductionist, have been shown to support the co-existence of P. aeruginosa and S. aureus biofilms, with species-dependent localization patterns and interspecies interactions. Therefore, composite in vitro models that bring together key features of the wound microenvironment could provide unprecedented insights into the structure and organization of mixed-species biofilms. We have built a four-dimensional (4-D) wound microenvironment consisting of a 3-D host cell scaffold of co-cultured human epidermal keratinocytes and dermal fibroblasts, and an in vitro wound milieu (IVWM); the IVWM provides the fourth dimension that represents the biochemical and nutrient profile of the wound infection state. We leveraged this 4-D wound microenvironment, in comparison with biofilms in IVWM alone and standard laboratory media, to probe the structure of mixed-species P. aeruginosa and S. aureus biofilms across multiple levels of organization such as aggregate dimensions and biomass thickness, species co-localization and spatial organization within the biomass, overall biomass composition and interspecies interactions. In doing so, the 4-D wound microenvironment platform provides multi-level insights into the structure of mixed-species biofilms, which we incorporate into the current understanding of P. aeruginosa and S. aureus organization in the wound bed.

Toxicity of di-2-ethylhexyl phthalate and tris (2-butoxyethyl) phosphate to a tropical micro-crustacean (Ceriodaphnia cornuta) is higher in Mekong River water than in standard laboratory medium.

Plasticizers such as di(2-ethylhexyl) phthalate (DEHP) and tris (2-butoxyethyl) phosphate (TBOEP) are manufactured chemicals produced in high volumes. These chemicals are frequently detected in the aquatic environment and cause toxic effects on organisms. In this study, we assessed the chronic impacts of DEHP and TBOEP, respectively, at the concentration of 100µg L-1 dissolved in the artificial medium (M4/4) and Mekong River water on life history traits of a tropical micro-crustacean, Ceriodaphnia cornuta, for 14days. DEHP and TBOEP substantially reduced the survival of C. cornuta. In M4/4 medium, both plasticizers strongly enhanced reproduction but did not influence the growth of C. cornuta. Mekong River water, plasticizers-exposed C. cornuta produced less neonates than those in the control. The detrimental impacts of DEHP and TBOEP on the fitness of C. cornuta were much stronger in natural river water than in M4/4. Our results suggest that plasticizers can cause adverse effects on tropical freshwater cladocerans, particularly in natural water. These results are of a deep concern, as national and international regulatory guidelines which are based on ecotoxicological tests using standard media may not fully capture these effects.

Towards recycling remediated cyanidation tailings water to the mineral biooxidation process – Impact on microbial community and its performance

Biooxidation is a well-established pretreatment technology used in refractory gold mineral processing to enhance metal value recovery. The technology is microbially-mediated and is dependent on appropriate quality water for optimum function. The influence of remediated cyanidation tailings effluent and increasing thiocyanate (SCN−) concentration on a biooxidation microbial community was evaluated in small-scale batch tests. Changes in ferrous iron (Fe2+) oxidation activity, microbial growth and microbial community structure were evaluated. Two detoxified tailings wastewater samples, one sourced from an industrial operation and another from a laboratory system, were evaluated and used as the liquid matrix for medium preparation. These were compared with a standard laboratory medium described for the cultivation of acidophilic biooxidation organisms. Additionally, the effect of increasing SCN− concentration over the range of 0–5 mg/L across the different nutrient matrices. Similar microbial growth and Fe2+ oxidation activity with a nominal shift in community structure were observed in nutrient media containing remediated tailings wastewater relative to experiments conducted using defined medium prepared with deionised water (dH2O). However, a lag in the onset of Fe2+ oxidation activity was observed in systems cultured in media containing remediated effluent. This was attributed to the coupled presence of anions, NO3– and SCN−, in conjunction with additional constituents within the remediated effluent matrix. Exposure to SCN− concentrations up to 1 mg/L across all nutrient media demonstrated marginal differences in Fe2+ oxidation performance, although the observed lag in Fe2+ oxidation activity was sustained in remediated effluent tests. At a SCN– concentration of 1.4 mg/L Fe2+ oxidation activity within the defined medium system was inhibited, however systems cultivated in remediated tailings water exhibited sustained Fe2+ oxidation performance at 1.4 mg SCN− mg/L. Exposure to SCN− concentrations above 1.4 mg/L resulted in negligible Fe2+ oxidation activity, lower cell concentrations and a significant increase in growth lag time in detoxified tailings water media. Microbial community structure data illustrated that Leptospirillum ferriphilum was the dominant Fe2+ oxidising species within all systems; and its decline in abundance at elevated SCN− concentrations corresponded to poor Fe2+ utilisation. The abundance (and inferred growth rate) of sulfur-oxidising Acidithiobacillus caldus remained constant across the SCN− range explored. Abundance of Acidiplasma cupricumulans was found to increase at SCN− concentrations above 1 mg/L; however, it did not contribute toward Fe2+ oxidation. Results from this study indicated that inclusion of remediated tailings wastewater within the biooxidation circuit would be feasible causing negligible disruption to process performance; however, SCN− concentrations should not exceed 1 mg/L.

Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium.

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

An Oral Fluorouracil Prodrug, Capecitabine, Mitigates a Gram-Positive Systemic Infection in Mice.

ABSTRACTNew classes of antibiotics are needed to fight bacterial infections, and repurposing existing drugs as antibiotics may enable rapid deployment of new treatments. Screens for antibacterials have been traditionally performed in standard laboratory media, but bacterial pathogens experience very different environmental conditions during infection, including nutrient limitation. To introduce the next generation of researchers to modern drug discovery methods, we developed a course-based undergraduate research experience (CURE) in which undergraduate students screened a library of FDA-approved drugs for their ability, in a nutrient-poor medium, to prevent the growth of the human Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium. The nine drugs identified all disrupt DNA metabolism in bacteria and eukaryotes. One of the hit compounds, capecitabine, is a well-tolerated oncology drug that is administered orally, a preferred treatment route. We demonstrated that capecitabine is more effective at inhibiting S. Typhimurium growth in nutrient-limited than in standard rich microbiological broth, an explanation for why the antibiotic activity of this compound has not been previously recognized. Capecitabine is enzymatically converted to the active pyrimidine analogue, fluorouracil (5-FU), and Gram-positive bacteria, including Staphylococcus aureus, are significantly more sensitive to 5-FU than Gram-negative bacteria. We therefore tested capecitabine for efficacy in a murine model of S. aureus peritonitis. Oral capecitabine administration reduced the colonization of tissues and increased animal survival in a dose-responsive manner. Since capecitabine is inexpensive, orally available, and relatively safe, it may have utility for treatment of intractable Gram-positive bacterial infections.IMPORTANCE As bacterial infections become increasingly insensitive to antibiotics, whether established, off-patent drugs could treat infections becomes an important question. At the same time, basic research has revealed that during infection, mammals starve pathogens for nutrients and, in response, bacteria dramatically alter their biology. Therefore, it may be fruitful to search for drugs that could be repurposed as antibiotics using bacteria grown with limited nutrients. This approach, executed with undergraduate student researchers, identified nine drugs known to interfere with the production and/or function of DNA. We further explored one of these drugs, capecitabine, a well-tolerated human oncology drug. Oral administration of capecitabine reduced infection with the human pathogen Staphylococcus aureus and increased survival in mice. These data suggest that capecitabine has potential as a therapy for patients with otherwise untreatable bacterial infections.

Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation.

Introduction Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).Hypothesis/Gap StatementWe hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.Aim We address the importance of PIA and Embp in biofilm grown in ‘humanized’ media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.Methodology S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.Results S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell–cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivopig lung, EVPL). Several in vitroassays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays. The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of <em>Pseudomonas aeruginosa</em> and <em>Staphylococcus aureus</em> Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo pig lung, EVPL). Several in vitro assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays. The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

CAUTI’s next top model – Model dependent Klebsiella biofilm inhibition by bacteriophages and antimicrobials

Klebsiella infections, including catheter associated urinary tract infections, are a considerable burden on health care systems. This is due to their difficulty to treat, caused by antimicrobial resistance and their ability to form biofilms. In this study, we investigated the use of a Klebsiella phage cocktail to reduce biofilm viability. We used two methodologies to investigate this, a standard 96-well plate assay and a more complicated Foley catheter-based model. The phage cocktail was used alone and in combination with clinically relevant antibiotic treatments. Viability was measured by both a resazurin based stain and colony forming unit counts, of cells sloughed off from the biofilm. We showed that phage infection dynamics and host survival vary significantly in different standard laboratory media, presumably due to the expression of different surface receptors and capsule composition by the bacteria effecting phage binding. This underscores the importance of a realistic model for developing phage therapy.We demonstrate that bacteriophage-based treatments are a viable option for preventing Klebsiella colonisation and biofilm formation on urinary catheters. Phage cocktails were able to significantly reduce the amount of biofilm that formed when they were present during early biofilm formation. The phages used in this study were unable to significantly reduce a pre-formed mature biofilm, despite encoding depolymerases. Phages applied together with antimicrobial treatments, showed synergistic interactions, in some cases the combined treatment was much more effective than antimicrobial treatments alone.We show that phage cocktails have the potential to prevent Klebsiella biofilms in catheters, if used early or as a preventative treatment and will work well alongside standard antibiotics in the treatment of catheter-associated urinary tract infections (CAUTI).

Control of spoilage fungi in yogurt using MicroGARD 200™, Lyofast-FPR2™ and HOLDBAC-YMC™ as bioprotectants

AbstractThe aim of this study was to assess the inhibitory effect of three commercial bioprotectant agents on the growth of yogurt-spoiling fungi. Mucor circinelloides, Mucor racemosus, Penicillium spp., Saccharomyces exiguus, and Candida intermedia, commonly involved in the spoilage of dairy products, were isolated from spoiled yogurt and were fully characterized using molecular and phenotypic methods. HOLDBAC-YMC™, Lyofast-FPR2™ and MicroGARD 200™ were used as antifungal products. An optimized experimental mixture design was applied to determine the proportion of each bioprotectant in terms of growth-inhibition response against the fungal strains in standard laboratory media. The results of the challenge tests showed that the application of bioprotectants inhibited the growth of the moulds in the range of 85–100% and the growth of yeast between 1.23 and 5.40 log cycles. The optimal combination of the bioprotectants was determined, tested in standard laboratory media and found to inhibit fungal growth. The antifungal effect of the optimal combination of the bioprotectants was validated in yogurt against the most resistant fungal species of the study, M. circinelloides and C. intermedia. The bioprotectants elicited antifungal effect in yogurt by completely inhibiting all of the tested fungi compared to controls. To the best of our knowledge, this is the first time a mixture of commercial bioprotectants has been tested on yogurt as a potential alternative for the biopreservation of yogurt in order to reduce spoilage of fermented dairy products and economic losses.

Survival strategy of Cronobacter sakazakii against ampicillin pressure: Induction of the viable but nonculturable state

In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 108 CFU/mL to an undetected level 7–30 days post-treatment. Meanwhile, viable cells were still approximately 104–105 cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.

Engineering endogenous fermentative routes in ethanologenic Escherichia coli W for bioethanol production from concentrated whey permeate

Whey permeate (WP) is a lactose-rich waste effluent, generated during cheese manufacturing and further valorization steps, such as protein extraction. The production of ethanol by WP fermentation has been proposed to increase cost-competitiveness of dairy waste processing. In previous work, the Escherichia coli W strain was selected for its efficient growth in dairy waste and it was engineered to convert lactose into ethanol as the main fermentation product from WP and concentrated WP (CWP). To improve its performance, here the lactate dehydrogenase, fumarate reductase and pyruvate formate lyase fermentative routes were disrupted, obtaining new deletion strains. In test tubes, growth and fermentation profiles obtained in standard laboratory media and CWP showed large differences, and were affected by oxygen, medium and ethanologenic gene expression level. Among the tested strains, the one with triple deletion was superior in both high-oxygen and low-oxygen test tube fermentations, in terms of ethanol titer, rate and yield. The improved performance was due to a lower inhibition by medium acidification rather than an improved ethanol flux. The parent and triple deletion strains showed similar performance indexes in pH-controlled bioreactor experiments. However, the deletion strain showed lower base consumption and residual waste, in terms of both dry matter and chemical oxygen demand after distillation. It thus represents a step towards sustainable dairy wastewater valorization for bioenergy production by decreasing process operation costs.

Evaluation of pooled human urine and synthetic alternatives in a dynamic bladder infection in vitro model simulating oral fosfomycin therapy

The impact of the bladder environment on fosfomycin activity and treatment response is uncertain. Standard laboratory media does not reflect the biomatrix of urine, where limited nutritional factors are important for growth and antimicrobial kill rates. We compared fosfomycin activity against Enterobacteriaceae in laboratory media, human urine and synthetic alternatives. Sixteen clinical isolates (8-Escherichia coli, 4-Enterobacter cloacae, 4-Klebsiella pneumoniae) were studied with broth microdilution (BMD) susceptibility, static time-kill assays and dynamic testing in a bladder infection model simulating a 3 g oral fosfomycin dose. Mueller-Hinton broth (MHB) with and without 25 mg/L glucose-6-phosphate (G6P), pooled midstream urine (MSU), pooled 24 h urine collection (24 U), artificial urine medium (AUM) and synthetic human urine (SHU) were compared. BMD susceptibility, bacterial growth and response to static fosfomycin concentrations in urine were best matched with SHU and were distinctly different when tested in MHB with G6P. Fosfomycin exposure in the bladder infection model was accurately reproduced (bias 4.7 ± 6.2%). Under all media conditions, 8 isolates (2-E. coli, 2-E. cloacae, 4-K. pneumoniae) re-grew and 4 isolates (4-E. coli) were killed. The remaining isolates (2-E. coli, 2-E. cloacae) re-grew variably in urine and synthetic media. Agar dilution MIC failed to predict re-growth, whereas BMD MIC in media without G6P performed better. Emergence of resistance was restricted in synthetic media. Overall, SHU provided the best substitute for urine for in vitro modelling of antimicrobial treatment of uropathogens, and these data have broader utility for improved preclinical testing of antimicrobials for urinary tract infections.

Group A Streptococcus Transcriptome Analysis.

Global gene expression analyses in bacteria have undergone a dramatic transformation. Prior to the development of high-throughput sequencing technologies, real-time PCR or microarray studies were the mainstay of assessing differences in gene expression in bacteria. Real-time PCR remains a critical tool for targeted gene expression analyses. However, microarray studies have given way to the plethora of advantages in RNA sequencing (RNA-seq) for the determination of global gene expression (i.e., transcriptome). Increased accessibility to high-throughput sequencing and user-friendly bioinformatics data analysis software have made RNA-seq technology use more widespread. Here, we provide comprehensive methods to perform RNA sequencing of Streptococcus pyogenes strains grown in vitro in standard laboratory media, including cell growth, RNA extraction, ribosomal RNA depletion, and library construction. Considerations for library sequencing and data analysis are also provided.

A population shift between two heritable cell types of the pathogen Candida albicans is based both on switching and selective proliferation

Differentiated cell types often retain their characteristics through many rounds of cell division. A simple example is found in Candida albicans, a member of the human microbiota and also the most prevalent fungal pathogen of humans; here, two distinct cell types (white and opaque) exist, and each one retains its specialized properties across many cell divisions. Switching between the two cell types is rare in standard laboratory medium (2% glucose) but can be increased by signals in the environment, for example, certain sugars. When these signals are removed, switching ceases and cells remain in their present state, which is faithfully passed on through many generations of daughter cells. Here, using an automated flow cytometry assay to monitor white-opaque switching over 96 different sugar concentrations, we observed a wide range of opaque-to-white switching that varied continuously across different sugar compositions of the medium. By also measuring white cell proliferation rates under each condition, we found that both opaque-to-white switching and selective white cell proliferation are required for entire populations to shift from opaque to white. Moreover, the switching frequency correlates with the preference of the resulting cell type for the growth medium; that is, the switching is adjusted to increase in environments that favor white cell proliferation. The widely adjustable, all-or-none nature of the switch, combined with the long-term heritability of each state, is distinct from conventional forms of gene regulation, and we propose that it represents a strategy used by C. albicans to efficiently colonize different niches of its human host.

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.

A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein's expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5' end of the ARO1 gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicansIMPORTANCE Investigating a protein's functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach-the insertion of poly-A repeats within the coding sequence of the gene-is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

Pseudomonas aeruginosa Alters Its Transcriptome Related to Carbon Metabolism and Virulence as a Possible Survival Strategy in Blood from Trauma Patients.

Trauma patients (TPs) are highly susceptible to infections, which often lead to sepsis. Among the numerous causative agents, Pseudomonas aeruginosa is especially important, as P. aeruginosa sepsis is often fatal. Understanding the mechanism of its pathogenesis in bloodstream infections is imperative; however, this mechanism has not been previously described. To examine the effect of trauma-induced changes in blood on the expression of P. aeruginosa genes, we grew strain UCBPP-PA14 (PA14) in blood samples from eight TPs and seven healthy volunteers (HVs). Compared with its growth in blood from HVs, the growth of PA14 in blood from TPs significantly altered the expression of 285 genes. Genes whose expression was significantly increased were related to carbon metabolism, especially malonate utilization and mannitol uptake, and efflux of heavy metals. Genes whose expression was significantly reduced included genes of the type VI secretion system, genes related to uptake and metabolism of amino acids, and genes related to biosynthesis and transport of the siderophores pyoverdine and pyochelin. These results suggest that during systemic infection in trauma patients, and to adapt to the trauma-induced changes in blood, P. aeruginosa adjusts positively and negatively the expression of numerous genes related to carbon metabolism and virulence, respectively. IMPORTANCE While a considerable body of knowledge regarding sepsis in trauma patients is available, the potential influence of trauma-induced changes in the blood of these patients on the pathogenesis of Pseudomonas aeruginosa is basically an unexplored area. Rather than using standard laboratory media, we grew P. aeruginosa in whole blood from either healthy volunteers or trauma patients. The specific changes in the P. aeruginosa transcriptome in response to growth in blood from trauma patients reflect the adaptation of this organism to the bloodstream environment. This knowledge is vital for understanding the strategies this pathogen uses to adapt and survive within the host during systemic infection. Such information will help researchers and clinicians to develop new approaches for treatment of sepsis caused by P. aeruginosa in trauma patients, especially in terms of recognizing the effects of specific therapies (e.g., iron, zinc, or mannitol) on the organism. Further, this information can most likely be extrapolated to all patients with P. aeruginosa septicemia.

Expression, purification, and characterization of a metagenomic thioesterase from activated sludge involved in the degradation of acylCoA-derivatives

Metagenomic libraries are a novel and powerful approach to seek for pathways involved in xenobiotic degradation, since this technique abolishes the need for cultivating microorganisms that otherwise would be overlooked if they cannot grow on standard laboratory media and conditions. In this paper, we describe the expression, purification and characterization of a novel metagenomic thioesterase which was described to be involved in phenylacetic acid degradation (A. Sánchez-Reyes, R. Batista-García, G. Valdés-García E. Ortiz, L. Perezgasga, A. Zárate-Romero, N. Pastor, J. L. Folch-Mallol, A Family 13 thioesterase isolated from an activated sludge metagenome: insights into aromatic compounds metabolism, Proteins 85 (2017) 1222–1237). According to similarity and phylogenetic analyses, the enzyme seems to belong to an Actinobacterium. Nevertheless, after a process of denaturation and refolding, the protein expressed in E. coli was obtained in an active form. New data concerning the substrate preferences for this enzyme are presented which suggest that this thioesterase could be involved in breaking the ester bond in the CoA-linear acyl derivatives of the phenylacetic acetic pathway.

Engineering mucus to study and influence the microbiome

Mucus is a 3D hydrogel that houses the majority of the human microbiome. The mucous environment plays an important role in the differentiation and behaviour of microbial phenotypes and enables the creation of spatial distributions. Dysregulation of mucus is further associated with various diseases. Therefore, mucus is the key ingredient to study the behaviour of commensal and pathogenic microbiota in vitro. Indeed, microorganisms cultured in mucus exhibit phenotypes substantially different from those exhibited in standard laboratory media. In this Review, we discuss the impact of mucus on the microbiome and examine the structure and glycosylation of mucins — the main building blocks of mucus. We investigate the impact of glycans on mucin function and highlight different approaches for the engineering of synthetic mucins, including synthesis of the backbone, the design of mucin-mimetic hydrogels and the engineering of glycans. Finally, mucin mimetics for 3D in vitro cell culture and for modulating microbial community structure and function are discussed. Mucus is a 3D hydrogel composed of mucins that houses the human microbiome. Mucus guides microbial cell fate and is involved in the suppression of pathogenic bacteria. In this Review, the authors discuss the design of synthetic mucins for the investigation of mucus–microbiome interactions and for applications in 3D in vitro cell culture.