Appl. Microbiol., Volume 4, Issue 2 (June 2024) – 17 articles

Filamentous fungi exhibit unparalleled potential as cell factories for protein production, owing to their adeptness in protein secretion and remarkable proficiency in post-translational modifications. This review delineates the role of filamentous fungi in bio-input technology across different generations and explores their capacity to generate secondary metabolites. Our investigation highlights filamentous fungi as frontrunners in the production of bioactive compounds, emphasizing the imperative nature of elucidating their metabolic repertoire. Furthermore, we delve into common strategies for genetic transformation in filamentous fungi, elucidating the underlying principles, advantages, and drawbacks of each technique. Taking a forward-looking approach, we explore the prospects of genome engineering, particularly the CRISPR-Cas9 technique, as a means to propel protein secretion in filamentous fungi. Detailed examination of the protein secretion pathways in these fungi provides insights into their industrial applications. Notably, extensive research within the scientific community has focused on Aspergillus and Trichoderma species for the industrial production of proteins and enzymes. This review also presents practical examples of genetic engineering strategies aimed at augmenting enzyme secretion in filamentous fungi for various industrial applications. These findings underscore the potential of filamentous fungi as versatile platforms for protein production and highlight avenues for future research and technological advancement in this field. Full article

The Klebsiella pneumoniae ST307 clone, identified in the mid-1990s, has emerged as a global antimicrobial-resistant (AMR) high-risk clone, significantly contributing to the global health challenge also posed by other AMR K. pneumoniae lineages. The acquisition of a bla_CTX-M-15-carrying plasmid has facilitated its widespread dissemination. At Europe’s major transport hub for the movement of live animals, Frankfurt Airport, a shipment of 20 live leopard tortoises was sampled during German border control in 2014. Phylogenetic analysis (MLST) identified a K. pneumoniae ST307 strain, prompting further investigation. Our analysis revealed the presence of a ~193 kb plasmid carrying a broad range of AMR genes, including bla_CTX-M-15, bla_TEM-1B, bla_OXA-1, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id, and qnrB1. Additionally, mutations in the quinolone resistance-determining region in gyrA (S83I) and parC (S80I) were detected. Phenotypic testing demonstrated resistance of the isolate to the most common antimicrobials used in both human and veterinary medicine; exceptions included carbapenems and newer β-lactamase inhibitor combinations. Because the role of imported exotic animals in the dissemination of AMR genes is largely deficient, the present study fills yet missing mosaic pieces in the complete picture of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales. Full article

Although biofilms contribute to bacterial tolerance to desiccation and survival in low-moisture foods, the molecular mechanisms underlying biofilm formation have not been fully understood. This study created a mutant library from Salmonella Enteritidis using mini-Tn10 transposon mutagenesis. The biofilm-forming potential of acquired mutants was assessed before the genomic DNA of the mutants that formed significantly (p ≤ 0.05) less biofilm mass than their wildtype parent strain was extracted for deep DNA sequencing. The gene of each mutant interrupted by mini-Tn10 insertion was identified by aligning obtained sequencing data with the reference Genbank sequences using a BLAST search. Sixty-four mutant colonies were selected, and five mutants that formed the least amount of biofilm mass compared to the wildtype parent strain were selected for sequencing analysis. The results of the BLAST search revealed that the gene interrupted by mini-Tn10 in each mutant is responsible for the biosynthesis of aldehyde dehydrogenase (EutE), cysteine desulfurase (SufS or SufE), a transporter protein, porin OmpL, and a ribbon–helix–helix protein from the CopG family, respectively. Knock-off mutant construction is a possible approach to verify the potential of the identified genes to serve as targets of antimicrobial intervention to control Salmonella colonization on low-moisture foods and in their production environment. Full article

The endophytic fungal biodiversity of unique plants like Peperomia argyreia (Miq.) É. Morren (Piperaceae) has antimicrobial properties and can be employed for infection treatment. Fungal isolates were obtained from appropriately treated plant tissues cultured in solid media, characterized by morphology, and identified by molecular biology using ITS and NL primers. The antimicrobial properties of fungal extracts were analyzed by combining microdilution and bioautographic assays complemented with metabolic profiling by automated thin-layer chromatography and ¹H NMR techniques. Thirty-one filamentous fungi were isolated and characterized by ITS and/or D1/D2 region amplification of rDNA, identified as Thermothielavioides, Trichoderma, Cyphellophora, Cladosporium, Arcopilus, Plectosphaerella; Chaetomium, Sporothrix, Alboefibula, and Penicillium. Thermothielavioides spp. inhibited Staphylococcus aureus ATCC 25923; moreover, Penicillium westlingii P4 showed inhibitory activity on Ascochyta rabiei AR2. The bioactivity-guided fractionation of the EtOAc extract (MIC = 62.5 μg/mL) of P. westlingii P4 allowed the purification of citrinin as the main inhibitory compound (MIC = 62.5 μg/mL). Peperomia argyreia harbors a rich and diverse endophytic community able to produce bioactive molecules. Citrinin, with a minor influence of volatile compounds biosynthesized by P. westlingii P4, was responsible for the inhibition of A. rabiei AR2. Full article

Studies have shown that the production of reactive oxygen species (ROS) is triggered by bactericidal antibiotics, which contributes significantly to the killing of bacterial cells and increasing mutations in surviving cells. In this study, we hypothesized that exposure of Salmonella to sublethal concentrations of hypochlorite (NaOCl), a commonly used sanitizer in household and food industries increases mutation rates, leading to the development of antibiotic resistance. We found that a sublethal concentration (20 ppm) of NaOCl increased the mutation rates of S. typhimurium 14028s significantly (p < 0.05), which was prevented by the ROS scavenger thiourea, supporting that the increased mutation was due to NaOCl-triggered ROS production. We further found that the exposure of S. typhimurium 14028s to the same sublethal concentration of NaOCl increases resistance to kanamycin among the 3 antibiotics evaluated. The results of this study suggest that when NaOCl applied as a sanitizer fails to kill Salmonella due to diluted local concentrations or presence of organic materials, it can cause an adverse outcome of developing antibiotic resistance of the pathogen. Full article

This study involved the evaluation of the effect of vacuum pasteurization on physicochemical characteristics (pH, total soluble solids, titratable acidity, chroma, tone, IO, vitamin C, 5-hydroxymethylfurfural), microbiological properties (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, total coliforms, total mesophilic aerobes, molds and yeasts) and sensory characteristics of orange and carrot nectar. The thermal treatments were designed based on the thermal lethality of two heat-resistant microorganisms typical of the product (Neosartorya fischeri and Zygosaccaromyces bailii). The evaluation was carried out on raw nectar and pasteurized nectar. The shelf life was estimated to be 30 days (6 °C). The most favorable results were obtained by applying a heat treatment at 88 °C for 32.68 min, managing to retain 85.87% of vitamin C and a microbiological stability of 12 days (6 ± 0.6 °C) with regard to total mesophilic aerobes. Likewise, the tasters established that this treatment resulted in the best flavor, texture and acceptability characteristics. Full article

Partially hydrolyzed guar gum (PHGG) is a water-soluble, prebiotic fiber that is used in foods and supplements. The effects of PHGG and its role in gut health are still being studied. The purpose of this study was to evaluate changes in the gut microbiome composition of healthy individuals in response to low-dose PHGG supplementation compared with a low fiber diet. A randomized, double-blind, placebo-controlled crossover study was performed on 33 healthy subjects (17 males, 16 females). Each subject completed three 14-day treatment periods with a 2-week washout between each period. Treatments included supplementation with 3 g PHGG, 6 g PHGG, or a placebo. During all periods, the participants followed a low fiber diet (≤14 g/day). Stools were collected on days 0 and 14 of each period. Gut microbiome profiling was performed using 16S rRNA sequencing. Stools were assessed by investigators with the Bristol Stool Form Scale as a secondary outcome. Saliva cortisol was also measured as a secondary outcome. Supplementation of 3 g and 6 g PHGG significantly increased Verrucomicrobia on day 14 when compared to the placebo (p = 0.0066 and p = 0.0068, respectively). On the genus level, Akkermansia was significantly increased on day 14 with both the 3 g and 6 g PHGG doses (p = 0.0081 and p = 0.0083). Faecalibacterium was significantly decreased on day 14 with 3 g PHGG (p = 0.0054). Supplementing with low doses of PHGG has the potential to cause shifts in the gut microbiome composition. By increasing beneficial microbes, PHGG can improve the microbiome composition of healthy individuals and may play a role in the treatment of inflammatory gastrointestinal diseases. Full article

Lactobacillus plantarum has beneficial effects on the reduction of symptoms of poor lactose digestion and hypercholesterolemia, removal of the duration and severity of diarrheal processes, improvement of the intestinal permeability barrier, prevention of some types of cancer by adsorption or inactivation of genotoxic agents, increased resistance to intestinal and extraintestinal infections, attenuation of inflammatory bowel disease, and prevention of allergies (especially food). On the other hand, carao (Cassia grandis) has shown remarkable nutritious content with influential dietary applications. As a result, this investigation aimed to explore the effect of Cassia grandis pulp on viability of Lactobacillus plantarum under gastrointestinal conditions, immunomodulatory capacity, and probiotic potential. Adding carao to the medium under different experimental conditions, including rich and minimal culture media and a gastrointestinal digestion process of skimmed milk, did not substantially affect Lactobacillus plantarum’s growth but prolonged its viability. The administration of Lactobacillus plantarum with carao in mice did not induce a proinflammatory response at a systemic level. Still, it did cause an increase in the production of immunoregulatory cytokines. Also, the viability of TSB broth was improved by adding carao. Carao improved the growth of acid tolerance, bile tolerance, growth in TSB broth, and NaCl resistance. According to the results, carao may enhance the characteristics of L. plantarum when enriching fermented dairy products. Full article

Industrial wastewaters display a complex and diverse range of physicochemical properties that are measured, studied, and treated by businesses and water service providers. Less frequently measured are the microbial communities in these wastes, despite possible implications for health, equipment maintenance, and the environment. This study aimed to assess the microbial communities of eighteen raw and discharge-ready wastewaters across eleven industrial sites to compare the microbial compositions of these wastewaters across different industry sectors, on-site treatment levels, and other wastewater components. The potential for variance in the biomethane yield, depending on microbial communities, was also measured. Using targeted sequencing, a unique taxonomy was identified, including genera linked to animals (Acetitomaculum, Lactobacillus, NK4A214, Prevotella, and Shuttleworthia), cooling water (Bosea, Legionella, Methyloversatilis, and Reyranella), and extreme conditions (Alkalibacillus, Geobacillus, Halorubrum, and Pyrobaculum). However, the compositions of the microbial communities were not found to be directly correlated to industry sector or on-site treatment levels, nor were they found to have a direct effect on the biomethane potential. However, the presence of certain individual taxa is linked to the methane yield and treatment status and may be explained in the context of physicochemical properties while serving as potential markers for identifying, improving, or developing on-site processes. Full article

Industrial residues with high concentrations of hexavalent chromium [Cr(VI)], characterized by an alkaline pH (between 9 and 13) and high salinity (around 100 psu), were used as a source for extremophilic chromium-resistant and -reducing microorganisms. An investigation of biodiversity through MiSeq showed the presence of 20 bacterial classes, with Bacilli (47%), Negativicutes (15%), Bacteriodia (8%), Gammaproteobacteria (7%) and Clostridia (5%) being the most abundant. The bioprospection allowed the cultivation of 87 heterotrophic bacterial colonies and 17 bacterial isolates at the end of the isolation, and screening procedures were obtained. The isolates were related to Cellulosimicrobium aquatile, C. funkei, Acinetobacter radioresistens, Staphylococcus equorum, S. epidermis, Brachybacterium paraconglometratum, Glutamicibacter creatinolyticus, Pseudomonas songnenensis, Microbacterium algeriense and Pantoea eucalypti, most of them being resistant to Cr(VI). Resistances of up to 400 ${\mathrm{m}\mathrm{g}.\mathrm{L}}^{-1}$ of chromate were obtained for four related strains (QReMLB55A, QRePRA55, QReMLB33A and QReMLB44C). The C. aquatile strain QReMLB55A and the P. songnenensis strain QReMLB33A were exposed to ${\mathrm{K}}_2{\mathrm{C}\mathrm{r}}_2{\mathrm{O}}_7$ (200 ${\mathrm{m}\mathrm{g}.\mathrm{L}}^{-1}$) under optimal conditions, diminishing 94% and 24% of the Cr(VI) in 6 days, respectively. These strains exhibited a high potential for chromium remediation biotechnologies. Full article

Gorgonzola is an Italian “erborinato” blue cheese from cow’s milk, bearing blue-green “parsley-like” spots due to the spread of Penicillium roqueforti mycelium. Due to its pH, water activity, and high nutrient content, as well as the environmental conditions required for its maturation, Gorgonzola constitutes an optimal ecological niche supporting the growth of both yeasts and filamentous fungi. Therefore, exploring the abundant mycobiota present in this peculiar habitat is of great interest regarding the search for new probiotic strains. The present investigation aimed to characterize the Gorgonzola mycobiota using both phenotypic (macroscopic and microscopic morphological analyses) and genotypic (DNA barcoding) analyses to find possible putative probiotic strains to be used in veterinary medicine in feed supplements. Among the different isolated filamentous fungi (Mucor and Penicillium) and yeasts (Yarrowia, Debaryomyces, Saccharomyces, and Sporobolomyces), we selected a strain of Saccharomyces cerevisiae var. boulardii. We tested its adaptation to thermal stress and its stability in feed matrices. The overall results highlight that the selected strain is stable for three months and can be considered as a possible candidate for use as a probiotic in veterinary feed supplements. Full article

Wastewater-based epidemiology (WBE) is useful for detecting pathogen prevalence and may serve to effectively monitor diseases across broad scales. WBE has been used throughout the COVID-19 pandemic to track disease burden through quantifying SARS-CoV-2 RNA present in wastewater. Aside from case load estimation, WBE is being used to assay viral genomic diversity and emerging potential SARS-CoV-2 variants. Here, we present a study in which we sequenced RNA extracted from sewage influent obtained from eight wastewater treatment plants representing 16 million people in Southern California from April 2020 to August 2021. We sequenced SARS-CoV-2 with two methods: Illumina Respiratory Virus-Enriched metatranscriptomic sequencing (N = 269), and QIAseq SARS-CoV-2-tiled amplicon sequencing (N = 95). We classified SARS-CoV-2 reads into lineages and sublineages that approximated named variants and identified single nucleotide variants (SNVs), of which many are putatively novel SNVs and SNVs of unknown potential function and prevalence. Through our retrospective study, we also show that several SARS-CoV-2 sublineages were detected in wastewater before clinical detection, which may assist in the prediction of future variants of concern. Lastly, we show that sublineage diversity was similar across Southern California and that diversity changed over time, indicating that WBE is effective across megaregions. As the COVID-19 pandemic moves to new phases, and SARS-CoV-2 variants emerge, monitoring wastewater is important to understand local- and population-level dynamics of the virus. These results will aid in our ability to monitor the evolutionary potential of SARS-CoV-2 and help understand circulating SNVs to further combat COVID-19. Full article

The liver plays a vital role in the defense against infections. Porphyromonas gingivalis (P. gingivalis), a dominant etiologic oral bacterium implicated in periodontal disease (PD), has been associated with various systemic diseases. This study aimed to investigate the influence of P. gingivalis on alcohol-associated liver diseases (ALD). Mice were fed a Lieber–DeCarli liquid diet containing 5% ethanol for 10 days after an initial adaptation period on a diet with lower ethanol content for 7 days. Two days before tissue sample collection, the mice were administered P. gingivalis strain W83 (Pg) through intraperitoneal injection (IP). Pair-fed mice with Pg infection (PF+Pg) exhibited an activated immune response to combat infections. However, alcohol-fed mice with Pg infection (AF+Pg) showed liver injury with noticeable abscess lesions and elevated serum alanine aminotransferase (ALT) levels. Additionally, these mice displayed liver infiltration of inflammatory monocytes and significant downregulation of proinflammatory cytokine gene expression levels; and AF+Pg mice also demonstrated increased intrahepatic neutrophil infiltration, as confirmed by chloroacetate esterase (CAE) staining, along with elevated gene expression levels of neutrophil cytosol factor 1 (Ncf1), neutrophilic inflammation driver lipocalin 2 (Lcn2), and complement component C5a receptor 1 (C5ar1), which are associated with neutrophilic inflammation. Interestingly, compared to PF+Pg mice, the livers of AF+Pg mice exhibited downregulation of gene expression levels of NADPH oxidase 2 (Cybb), the leukocyte adhesion molecule Cd18, and the Toll-like receptor adaptor Myd88. Consequently, impaired clearance of P. gingivalis and other bacteria in the liver, increased susceptibility to infections, and inflammation-associated hepatic necrotic cell death were observed in AF+Pg mice, which is likely to have facilitated immune cell infiltration and contributed to liver injury. Furthermore, in addition to the Srebf1/Fasn pathway induced by alcohol feeding, Pg infection also activated carbohydrate response element-binding protein (ChREBP) in AF+Pg mice. In summary, this study demonstrates that P. gingivalis infection, acting as a “second hit”, induces dysfunction of immune response and impairs the clearance of bacteria and infections in alcohol-sensitized livers. This process drives the development of liver injury. Full article

The extremophile Halobacterium salinarum is an aerobic archaeon that has adapted to thrive in high-salt environments such as salted fish, hypersaline lakes, and salterns. Halophiles have garnered significant interest due to their unique interactions with bacteriophages known as haloarchaeophages. Studies have identified and characterized prophages in halophilic archaea, such as Haloferax volcanii, Haloquadratum walsbyi, and Haloarcula marismortui. Still, an investigation has yet to be conducted into the presence of prophage elements on Halobacterium salinarum ATCC 33170. This is of particular interest to us as we are using this strain as a source of archaeol, as one of the components of our sulfated lactosyl archaeol (SLA) archaeosome adjuvant. Genomic contigs of strain 33170 were bioinformatically assessed for prophage-like features using BLAST, PHASTER, InterProScan, and PHYRE2. A 7 kb region encoding six genes was identified as an incomplete prophage, and the proteins were further analyzed, revealing high homology to proteins encoded by bacteria, archaea, and an IS200 transposon. Restricting the BLASTp database to viruses resulted in hits to both myo- and siphoviral proteins, which would be unusual for an intact prophage. Additionally, no known phage structural proteins were identified in the search, suggesting a low chance that H. salinarum ATCC 33170 harbors a latent prophage. Full article

The transformation of the food chain due to technological advances has had significant implications in regard to food safety. A noteworthy trend in this evolution relates to the emergence of new or previously unseen pathogens within products, thereby altering the landscape of foodborne illness epidemiology. The escalating frequency of these events underscores the need for a comprehensive re-evaluation of preventive strategies. The occurrence of novel species of bacteria, viruses, parasites, and unusual biotoxins from unexpected sources has challenged the previous limits that had been set to prevent foodborne illness outbreaks. The repercussions, ranging from detrimental effects on public health to economic burden, are influenced by a myriad of factors affecting the evolution of foodborne pathogens and emerging ailments. Among these factors are shifts in population demographics and behaviors, especially dietary patterns, as well as climate extremes, advances in more precise pathogen detection, microbial adaptation, evolving agricultural practices, and transformative changes within the food industry. This review critically examines the impact of technological metamorphosis along the food chain, encompassing production, processing, handling, packaging, storage, transportation, and industry demographics on the dynamics influencing the emergence of foodborne pathogens. Additionally, potential solutions to mitigate and manage this escalating issue are proposed. Full article

Traditional culture-based methods, though a “gold standard” for bacterial detection in various industrial sectors, do often not fulfill today’s high requirements regarding rapidity, on-site applicability, and cost-efficiency both during operation and evaluation. Here, the feasibility of using an adenosine triphosphate (ATP)-based assay for determining microbial contaminations in paints and cosmetics was investigated and compared with standard plate count techniques and dipslides. Therefore, we initially determined the level of sensitivity and assessed the accuracy and concordance among the different methods via spiking tests using a mix of frequently abundant bacterial species to simulate microbial contamination. Bioluminescence intensity was linearly proportional to log colony counts over five orders of magnitude (R² = 0.99), indicating a high level of sensitivity. Overall, the accuracy varied depending on the test specimen, most probably due to matrix-related quenching effects. Although the degree of conformity was consistently higher at target concentrations ≥ 10⁵ CFU·mL⁻¹, microbial contaminations were detectable down to 10³ CFU·mL⁻¹, thus meeting the high requirements of various industries. ATP-based results tended to be within an order of magnitude lower than the reference. However, bearing that in mind, the developed assay serves as a rapid, real-time alternative for routine quality control and hygiene monitoring. Full article

In this study, the green macroalgae Ulva rigida, which contains 34.9% carbohydrates, underwent treatment with commercial hydrolytic enzymes. This treatment yielded a hydrolysate that contained 23 ± 0.6 g·L⁻¹ of glucose, which was subsequently fermented with Saccharomyces cerevisiae. The fermentation process resulted in an ethanol concentration of 9.55 ± 0.20 g·L⁻¹. The optimal conditions for ethanol production by S. cerevisiae were identified as follows: non-sterilized conditions, an absence of enrichment, and using an inoculum size of 118 mg·L⁻¹. Under these conditions, the fermentation of the green macroalgal hydrolysate achieved a remarkable conversion efficiency of 80.78%. The ethanol o/t ratio, namely the ratios of the experimental to theoretical ethanol produced, for Scheffersomyces stipitis, Candida guilliermondii, Kluyveromyces marxianus, and S. cerevisiae after 48 h of fermentation were 52.25, 63.20, 70.49, and 82.87%, respectively. Furthermore, S. cerevisiae exhibited the best outcomes in terms of ethanol production (9.35 g·L⁻¹) and conversion efficiency (80.78%) after 24 h (optimal time) of fermentation. Full article