The membrane-damaging activities of four phenolics chosen for their bactericidal activity against Staphylococcus aureus CNRZ3 were investigated: 5,7-dihydroxy-4-phenylcoumarin (DHPC), 5,8-dihydroxy-1,4-naphthoquinone (DHNQ), epigallocatechin gallate (EGCG) and isobutyl 4-hydroxybenzoate (IBHB). Staphylococcus aureus CNRZ3 cells, as well as model liposomes mimicking its membrane phospholipids composition, were treated with each phenolic at its minimal bactericidal concentration. Membrane integrity, intracellular pH and intracellular esterase activity were examined by flow cytometric analysis of S. aureus cells stained with propidium iodide and SYTO® 9, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, and 5(6)-carboxyfluorescein diacetate, respectively. While intracellular pH was affected by the foyr phenolics, only DHNQ and to a lesser extent EGCG, caused a loss of membrane integrity. Flow cytometric analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC/POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoglycerol) liposomes stained with Coumarin 6 (which penetrates the lipid bilayer) or 5-N(octadecanoyl)-amino-fluorescein (which binds to the liposome shell) suggested that only EGCG and DHNQ penetrated the bilayer of phospholipids of liposomes. Taken together, these findings support the hypothesis that EGCG and DHNQ bactericidal activity results from their accumulation in the phospholipid bilayer of S. aureus cells membrane causing its disruption.

In pharmaceutical studies, a course of bacteriology based on case studies provided by the teacher was transformed in a course based on a combination of student/teacher co-creation of cases and peer reviewing. Our objectives were to describe the perception of students about the new format and to assess the impact of changing on the learning outcomes. For teaching evaluation, we used a questionnaire and focus groups. The assessment of learning outcome was performed by comparing the students' scores in final tests with the previous and the revised course formats. The students embraced the creation of cases in small groups with the teacher. In addition, they reported a perception of weakened hierarchy between the teacher and themselves, an increase of their self-confidence and a better ability to transfer their learning to their professional activities in community pharmacies. Lastly, their opinion about the transferability of this format in other disciplines were divided.

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is one of the major virulence factors. However, we have found cap-positive and cap-negative isolates in porcine endocarditis. Here, we compared genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis. Cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in pigs after infection. Different mutations in whole-genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole-genome occurred independently. Since cap-negative isolates are rarely found in lesions of other diseases, these results suggest that endocarditis lesions may simply favored cap-negative mutants to survive the niches, leading to their persistence in the lesions.

Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol.

Streptococcus pyogenes is a Gram-positive human-specific pathogen that asymptomatically colonizes the human respiratory tract. The factors affecting the colonization to the host is not clearly understood. Adherence of the pathogen to host epithelial cell is the initial step for a successful colonization process. In the host, bacteria live in a polymicrobial community; thus, the signaling mediated between the bacteria plays a significant role in the colonization of the pathogen to the host. Thus, the effect of acyl-homoserine lactone, secreted by Gram-negative bacteria on the adhesion properties of S. pyogenes M3 strain was examined. N-(3-Oxododecanoyl)-L-homoserine lactone (Oxo-C12) increased the cell size as well as hydrophobicity of S. pyogenes. qPCR data revealed that the expression of sagA and hasA was negatively affected by Oxo-C12. Moreover, Oxo-C12 leads to changes in the morphological characteristic of S. pyogenes, further promoting adherence to host epithelia and biofilm formation on abiotic surface. The study demonstrates the role of Oxo-C12 as a factor that can promote virulence in S. pyogenes M3.

Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.

Microbial degradation of organic matter along the vertical profile of the water column is a major process driving the carbon cycle in the ocean. Pseudoalteromonas has been identified as a dominant genus in pelagic marine environments worldwide, playing important roles in the remineralization of organic carbon. However, the current understanding of Pseudoalteromonas was mainly based on shallow water populations or cultivated species. This study analyzed for the first time the structure, activity potential and ecotypes differentiation of Pseudoalteromonas in the water column of the New Britain Trench (NBT) down to 6000 m. Analysis on diversities of the 16S rRNA gene and their transcripts showed that Pseudoalteromonas was greatly enriched in deep-sea waters and showed high activity potentials. The deep-sea Pseudoalteromonas were significantly different from their shallow-water counterparts, suggesting an obvious ecotype division along with the vertical profile. Phylogenetic analysis on the 16S rRNA gene and hsp60 gene of 219 Pseudoalteromonas strains isolated from different depths further showed that the vertical ecotype division could even occur at the strain level, which might be a result of long-term adaptation to environmental conditions at different depths. The discovered depth-specific strains provide valuable models for further studies on adaptation, evolution and functions of the deep-sea Pseudoalteromonas.

Hepatitis E virus (HEV) is worldwide distributed and might cause acute or chronic hepatitis mainly in immunocompromised individuals. In previous studies we found a high prevalence of antibodies to HEV within blood donors in south Brazil and also within backyard-raised pigs. Here, we aimed to investigate the prevalence of anti-HEV antibody and HEV RNA within the general population from three major municipalities (Caxias do Sul, Passo Fundo and Santa Maria) in south Brazil. A total of 3000 blood samples were randomly obtained from clinical laboratories at each of the three municipality (n = 1000 each) to determine the presence of anti-HEV antibodies and HEV RNA. Overall, anti-HEV antibodies were detected in 574/1000 (57,4%) samples in Caxias do Sul, 655/1000 (65.5%) samples in Passo Fundo and 554/1000 (55.4%) samples in Santa Maria. The prevalence of HEV-positive samples increased steadily and significantly (P < 0,001) with age and was unusually higher within individual over 40 years. Despite of this, none of the pooled serum samples had detectable levels of HEV RNA. The high anti-HEV antibody prevalence suggests that the virus might be present on the environment and/or foodstuff and poses a permanent threat to immune-compromised individuals.

Taxol has been regarded as one of the most successful anti-cancer drugs identified from natural sources to date. Although Taxol is known to sensitize cells by stabilizing microtubules, its ability to cause DNA damage in peripheral blood lymphocytes and to induce oxidative stress and apoptosis indicates that Taxol may have other modes of cytotoxic action. This study focuses on identifying the additional targets of Taxol that may contribute to its multifaceted cell killing property, using Saccharomyces cerevisiae. We show that yeast oxidative stress response mutants (sod1Δ, tsa1Δ and cta1Δ) and DNA damage response mutants (mre11∆, sgs1∆ and sub1∆) are highly sensitive to Taxol. Our results also show that Taxol increases the level of reactive oxygen species (ROS) in yeast oxidative stress response mutant strains. Further, 4',6-Diamidino-2'-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining show that Taxol induces apoptotic features such as nuclear fragmentation and chromatin condensation in DNA repair mutants. On the whole, our results suggest that Taxol's cytotoxic property is attributed to its multifaceted mechanism of action. Yeast S. cerevisiae anti-oxidant and DNA repair gene mutants are sensitive to Taxol compared to wild-type, suggesting yeast model can be used to identify the genetic targets of anti-cancer drugs.

During the coronavirus pandemic, second-year students on the B.Sc. molecular biology and genetics degree at Istanbul Technical University sat an open-ended online exam for a microbiology course in which one of the compulsory questions asked how the course had helped them during the first phase of the pandemic (April-July 2020). Fifty of 69 students gave consent for their (anonymous) responses to be analysed in order to discern any key ways in which their knowledge had been applied. The aim of the study was to investigate whether taking an advanced microbiology course increases understanding of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic and has a positive impact on student behaviours with respect to public health practices. Findings were divided into four major themes: course content (information), application of course content to behavioural change (practice), professionalism and their 'audience' whilst at home in lockdown (family and friends). Social distancing, wearing face masks, and hand and surface hygiene were described as important behaviours, with this practice informed by their basic microbiology knowledge. This paper describes a scenario where rote assessment can be used to assess wider scientific literacy with respect to application in society, providing students with an opportunity to incorporate and apply their learning into real-life situations, whilst tutors can assess constructivist learning, conceptual understanding and impact on student behaviour.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Serratia marcescens SCH909 is a multidrug resistant strain isolated in 1988 harboring three class 1 integrons. We wondered if these integrons were retained over time and if there were other antimicrobial resistant determinants contributing to its multidrug resistant profile. Genomic analysis showed a fourth multidrug resistance integron, a Tn7 transposon with dfrA1-sat2-ybeA-ybfA-ybfB-ybgA gene cassettes in the variable region. Insertion sequences were involved in the genesis of novel composite transposons in the L4 subtype plasmid pSCH909, such as Tn6824 carrying an arsenic regulon and two head to head class 1 integrons surrounded by two complete IS1. Remarkably, a novel chromosomal genomic island, SmaR, was identified, closely related to Multiple Antimicrobial Resistance Regions (MARR), usually found in AbaR0-type and AbGRI2-0 from global clones of Acinetobacter baumannii, and in M-type plasmids circulating in Enterobacteriaceae. Maintenance studies showed that the three class 1 integrons were maintained over 1 month without antimicrobial pressure. Since S. marcescens is considered a relevant nosocomial pathogen that can have a wide range of niches - human, plant, animal, soil and inanimate surfaces, our findings support the ability of this species to capture, maintain and spread a broad variety of antimicrobial resistance elements.

Agglutinin-like sequence protein 3 (Als3) is a cell surface glycoprotein of Candida albicans that plays essential roles in the processes of adherence and biofilm formation in vitro. In this study, we focused on the contribution of Als3 to the structure and drug susceptibility of biofilms. The C. albicans wild-type (WT) strain DAY185, the als3Δ/Δ null strain and the als3Δ/Δ + pALS3 complemented strain were used. Colony-forming unit enumeration, crystal violet and cell surface hydrophobicity assays, scanning electron microscopy and confocal laser scanning microscopy coupled with analyses using COMSTAT software were performed to evaluate the biomass and architecture of the biofilms. The detailed architectural analysis showed a significant variation in the biofilm parameters of the als3Δ/Δ biofilms compared with those of the WT biofilms. Fluconazole, miconazole and amphotericin B were selected as the antifungal agents for the antimycotic susceptibility test, and increased susceptibility was found with the ALS3 deletion biofilms. A quantitative real-time polymerase chain reaction analysis showed downregulation of biofilm formation-related genes (ALS1, EFG1, HWP1 and CSH1) and drug resistance-related genes (ERG11, CDR1, CDR2 and MDR1) in the als3Δ/Δ biofilms. We concluded that Als3 contributes to biofilm formation by changing the biofilm architecture and is involved in the antifungal resistance of C. albicans biofilms.

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

Fermentation is one of if not the oldest food processing technique, yet it is still an emerging field when it comes to its numerous mechanisms of action and potential applications. The effect of microbial activity on the taste, bioavailability and preservation of the nutrients and the different food matrices has been deciphered by the insights of molecular microbiology. Among those roles of fermentation in the food chain, biopreservation remains the one most debated. Presumably because it has been underestimated for quite a while, and only considered - based on a food safety and technological approach - from the toxicological and chemical perspective. Biopreservation is not considered as a traditional use, where it has been by design - but forgotten - as the initial goal of fermentation. The 'modern' use of biopreservation is also slightly different from the traditional use, due mainly to changes in cooling of food and other ways of preservation, Extending shelf life is considered to be one of the properties of food additives, classifying - from our perspective - biopreservation wrongly and forgetting the role of fermentation and food cultures. The present review will summarize the current approaches of fermentation as a way to preserve and protect the food, considering the different way in which food cultures and this application could help tackle food waste as an additional control measure to ensure the safety of the food.