Applied Microbiology

This review presents an approach to the incorporation of cyanobacteria and microalgae in yogurts and explores their impact on the nutritional, rheological, sensory, and antioxidant qualities of these products. First, the yogurt market context and its relationship with nutritional quality are outlined, emphasizing the quest for functional foods that meet consumer demands for healthy and nutritious products. A discussion of the incorporation of cyanobacteria and microalgae, especially Spirulina platensis, in foods, particularly yogurt, is then presented, highlighting the nutritional and functional benefits that this type of biomass can provide to the final product. The fermentation process and the quantity of algae to be incorporated are discussed to understand their fundamental role in the characteristics of the final product. In addition, this article considers some challenges such as sensory and rheological changes in the product resulting from the interaction of milk, algal biomass, and the fermentation process. Addressing these challenges involves delineating how these interactions contribute to changes in the traditionally consumed product, while obtaining a pro- and prebiotic product is crucial for creating an innovative dairy product that diversifies the market for derived dairy products with increased functional properties. Full article

The emergence of antimicrobial resistance (AMR) in pathogens transmitted through food poses a significant threat to global public health, complicating infection treatment and increasing mortality rates. This review explores the role of resistome mapping as a crucial tool for understanding the transmission dynamics of antimicrobial resistance genes (ARGs) in foodborne pathogens, such as Salmonella, Escherichia coli, Listeria monocytogenes, and Campylobacter spp., as well as various techniques for resistome mapping, such as metagenomic sequencing, PCR-based methods, and whole-genome sequencing (WGS), highlighting the significance of horizontal gene transfer (HGT) as a key mechanism for ARG dissemination in foodborne pathogens. Additionally, we investigated the influence of agricultural practices and environmental factors on AMR development, highlighting the critical need for improved surveillance, antibiotic stewardship, and global collaboration to mitigate the spread of resistant ARGs through the food chain. The perceptions gained from resistome mapping play an essential role in developing effective approaches to address AMR and to ensure food safety. Full article

A conventional, group-specific PCR method was developed to identify each of the four previously defined major taxa (Myoviridae, Siphoviridae, Podoviridae and Microviridae) of somatic coliphages and used to classify isolates from sewage. Somatic coliphage infectivity detection, occurrence and survival in primary human sewage effluent was observed over time to further understand the presence and behavior of the groups of somatic coliphages at two environmental temperatures (4 and 25 °C). Over time, the taxonomic composition of the somatic coliphage population in sewage changed, with the Microviridae family becoming the most prevalent family in the sewage population after several weeks. Based on their persistence and prevalence in environmental waters, phages belonging to the Microviridae family provide supporting information on sewage contamination and possibly of human enteric viruses in sewage-contaminated water. Full article

This study explores the integration of genome sequencing and digital droplet polymerase chain reaction (ddPCR)-based methods for tracking the diversity of COVID-19 variants in wastewater. The research focuses on monitoring various Omicron subvariants during a period of significant viral evolution. Genome sequencing, particularly using Oxford Nanopore Technology (ONT), provides a detailed view of emerging variants, surpassing the limitations of PCR-based detection kits that rely on known sequences. Of the 43 samples analyzed, 39.5% showed matching results between the GT Molecular ddPCR kits and sequencing, though only 4% were exact matches. Some mismatches occurred due to newer subvariants like XBB and BQ.1, which the ddPCR kits could not detect. This emphasized the limitations of ddPCR kits, which rely on known variant sequences, while sequencing provides real-time data on emerging variants, offering a more comprehensive view of circulating strains. This study highlights the effectiveness of combining these methodologies to enhance early detection and inform public health strategies, especially in regions with limited clinical sequencing capabilities. Full article

V. lutrae is an emerging human pathogen attributed to increasing hospitalization cases in humans; however, its biology and epidemiology are under-explored. The present study explored V. lutrae recovery, prevalence, and biology. A two-step enrichment method (i.e., step 1, nourishment; step 2, heat, 80 °C, 20 min) and thiosulfate–citrate–bile salts–sucrose (TCBS) agar were employed for recovering V. lutrae in raw seafood. Bacterial colonies were streaked for purification before 16S rRNA bacterial identification. Confirmed V. lutrae isolates were analyzed for their culture-challenged turbidity and virulence. Of 41 bacterial isolates, 9 confirmed V. lutrae, including regular (33%; nourished 24 h) and heat-resistant (67%; nourished 48 h plus heating) isolates, were exclusively from yellow colonies (i.e., TCBS) and were exclusively recovered from nourished shrimp (78%) and crab (22%) only. The culture and virulence biology revealed that they could diversely tolerate salinity (i.e., 0–17.5% additional NaCl), pasteurization (63 °C, 8 h), oxygen availability, and antibiotic sensitivity (i.e., erythromycin, gentamicin, and vancomycin). Further, this pathogen exhibited no visible hemolytic and alkalization activities. Emerging foodborne pathogens could readily evade the established food safety regime. The present study reveals systematic investigation and diverse phenotypes of V. lutrae to enhance its detection and contribute to public health initiatives. Full article

With the anticipated population growth by 2050, the demand for high-quality protein for human consumption is set to rise. To enhance the sustainability of U.S. cattle production, producers and researchers have traditionally concentrated on improving the feed efficiency of steers through advancements in genetics, nutrition, and microbiome tools, resulting in a more marketable beef product. However, without successful pregnancies, there would be no marketable animals to feed. Despite extensive research on hormonal impacts on cattle physiology, including nutrition and reproduction, there is limited knowledge about how the rumen microbial environment is impacted by pregnancy and feed efficiency in female beef cattle. Understanding the rumen microbiome’s role in feed efficiency and its response to hormonal changes during pregnancy is crucial for advancing sustainable beef cattle production. Therefore, this review highlights the importance of understanding the microbial dynamics in the rumen of pregnant beef females. Although progress has been made, gaps remain in understanding how varying nutritional requirements throughout pregnancy affect the rumen microbiome, highlighting the need for continued research. Addressing these areas will lead to more efficient and sustainable cattle production practices, benefiting beef production and contributing to global food security. Full article

This study explores the relationship between filamentous fungi and dental caries in isolated indigenous communities in Siltepec, Chiapas, Mexico. A total of 37 oral swabs were collected, with 22 participants harboring filamentous fungi, primarily from the genus Cladosporium. Statistical analysis using Student’s t-test and the Mann–Whitney U test revealed a significant reduction in extensive and fully cavitated caries (p < 0.0001) in individuals with fungi, while those without fungi exhibited higher rates of dental decay. Participants with fungi had a higher prevalence of healthy teeth and incipient caries. The findings suggest that traditional maize-based diets, particularly fermented beverages like pozol, may promote the growth of beneficial fungi in the oral microbiome, offering a protective effect against dental caries through microbial competition and the alteration of the oral environment. These results underline the need for further research into the long-term impact of traditional diets on oral health and the potential use of natural substances, such as probiotics and plant-based antimicrobials, to maintain oral homeostasis and prevent caries. Full article

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium and also an opportunistic pathogen for both humans and animals, causing acute or chronic infections. It has been frequently detected in healthy and diseased reptiles, more commonly in captive ones. Since most studies are primarily on clinical isolates, the pathogenic potential of strains originating from wild animals is poorly explored. We isolated the strain P. aeruginosa PM1012 from the cloacal microbiota of a common wall lizard (Podarcis muralis Laurenti, 1768) from a free-living population. The effect of temperature, pH and salinity on its growth was evaluated. Antibiotic resistance, the expression of several virulence factors as some extracellular enzymes, pyocyanin production and biofilm formation were also assessed. Apart from intrinsic resistance, the newly isolated strain P. aeruginosa PM1012 presented an antibiotic susceptibility profile with a low resistance rate limited to meropenem and intermediate to ceftazidime and aztreonam. Protease, lipase and gelatinase secretion was detected. Strong pyocyanin production was observed in the optimal range of growth conditions. An excellent biofilm-forming capacity was manifested. Full article

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spreads primarily through respiratory droplets, aerosols, and contaminated surfaces. While high-traffic locations like hospitals and airports have been studied extensively, detecting significant virus levels in aerosols and on environmental surfaces, campus settings remain underexplored. This study focused on two crowded buildings at the University of North Carolina at Charlotte (UNCC). From December 2021 to March 2022, we collected 16 indoor air samples and 201 samples from high-touch surfaces. During the sampling timeframe, 44.82% of surface samples from the Student Union and 28% from the University Recreational Center (UREC) tested positive for the presence of SARS-CoV-2 RNA. Median and average viral RNA copies per swab were higher in UREC (273 and 475) than in Student Union (92 and 269). However, all air samples tested negative. Surface positivity in these high-traffic campus locations was directly correlated with COVID-19 clinical cases in Mecklenburg County. The campus COVID-19 cases, driven by the Omicron wave, peaked a week before the peak detection of surface contamination. These findings underscore the importance of surface hygiene measures and highlight environmental conditions as potential contributors to COVID-19 spread on campuses. Full article

The terrestrial biota is a rich source of biologically active substances whose anti-biofilm potential is not studied enough. The aim of this review is to outline a variety of terrestrial sources of antimicrobial agents with the ability to inhibit different stages of biofilm development, expecting to give some ideas for their utilization in improved anti-biofilm treatments. It provides an update for the last 5 years on anti-biofilm plant products and derivatives, essential oils, antimicrobial peptides, biosurfactants, etc., that are promising candidates for providing novel alternative approaches to combating multidrug-resistant biofilm-associated infections. Based on the reduction in bacterial adhesion to material and cell surfaces, the anti-adhesion strategy appears interesting for the prevention of bacterial attachment in combating a broad range of mono- and multispecies bacterial biofilms. So far, few studies have been carried out in this direction. Anti-biofilm coatings made by or containing biologically active products from terrestrial biota have scarcely been studied although they are of significant interest for a reduction in infections associated with medical devices. Combination therapy with commercial antibiotics and natural products is accepted now as a promising base for future advances in anti-biofilm treatment. In vivo testing and clinical trials are necessary for clinical application. Full article

Lignocellulosic biomass is contemplated to be an inexpensive and copious feedstock that can be used for numerous industrial applications. However, lignin forms the lignin sheath and provides a physical barrier to enzymatic hydrolysis. In addition, lignin physically blocks cellulase, preventing it from being combined with the substrate in a process known as non-productive binding. Therefore, the depletion of lignin is a crucial method for obtaining fermentable sugars from the lignocellulosic biomass. Different white-rot fungi secrete different sets of lignin-mineralizing enzymes and each fungus secretes one or more of the three enzymes essential for lignin degradation. Among efficient redox enzymes, versatile peroxidase is extensively studied for its ability to degrade aromatics without the need for a mediator or polyvalent catalytic site. However, the presence of versatile peroxidase in F. spp. has not been studied. This study was planned with the objective of screening and comparing the production of versatile peroxidase enzymes from F. spp. and a standard culture of Pleurotus ostreatus MTCC-142. These fungal strains were first screened on solid media containing tannic acid, malachite green, or bromocresol green. The potency index for the tannic acid, malachite green, and bromocresol green on the 16th day of incubation was reported to be 1.28, 1.07, 1.09, and 1.10, respectively. Versatile peroxidase production patterns were investigated under solid state fermentation conditions for a period of 25 days at different temperatures ranging from 10 to 35 °C. The highest versatile peroxidase activity (592 UL⁻¹) in F. sp. was observed at 30 °C after the 7th day of incubation. The molecular confirmation showed the presence of the vp gene in F. sp. along with Pleurotus ostreatus MTCC-142. The results determined that F. sp. possesses a versatile peroxidase enzyme and is able to degrade lignin efficiently, and thus it could be utilized as an alternative to other ligninolytic enzyme-producing fungi. Full article

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats. Full article

Synbiotics are mixtures of prebiotics and probiotics that enhance the activity of probiotic bacteria when co-administered to provide greater benefits to the host. Traditionally, the synbiotics that have been discovered enhance gut probiotic strains and are nutritionally complex molecules that survive digestive breakdown until they reach the later stages of the intestinal tract. Here, we screened and identified sugars or sugar substitutes as synbiotics for the oral probiotic strains Streptococcus salivarius BLIS K12 and BLIS M18. Using a modified deferred antagonism assay, we found that 0.5% (w/v) galactose and 2.5% (w/v) raffinose were the best candidates for use as synbiotics with BLIS K12 and M18, as they trigger enhanced antimicrobial activity against a range of bacteria representing species from the mouth, gut, and skin. Using reverse transcriptase quantitative PCR, we found that this enhanced antimicrobial activity was caused by the upregulation of the lantibiotic genes salA, salB, and sal9 in either K12 or M18. This led to the conclusion that either 2.5% (w/v) raffinose or 0.5% (w/v) galactose, respectively, are suitable synbiotics for use in conjunction with BLIS K12 and M18 to enhance probiotic performance. Full article

Various microorganisms, referred to as kuratsuki microorganisms, inhabit each sake brewery. Previously, kuratsuki yeasts had been used for sake production in each sake brewery. Kuratsuki lactic acid bacteria have been used to produce kimoto, a fermentation starter. Kuratsuki non-lactic acid bacteria were examined to evaluate their potential roles and effects in sake production. The addition of kuratsuki bacteria to the sake-making process can change the flavor and taste of the sake. This change was observed in both the coculture experiments between sake yeast and kuratsuki bacteria and the sake making tests with and without kuratsuki bacteria. The comprehensive gene expression analysis of sake yeast cocultured with kuratsuki bacteria showed that 1.2% of the yeast genes were upregulated and 1.0% were downregulated following the addition of kuratsuki bacteria. This indicates that the change in flavor and taste of sake due to the addition of kuratsuki bacteria was caused by the interaction between sake yeast and kuratsuki bacteria. To understand the implications of kuratsuki bacteria in sake production, it is essential to study the interactions between sake yeast and kuratsuki bacteria. Full article

The pesticide active ingredient azoxystrobin is widely used in agriculture and has negative effects for the environment and contained organisms. Bacterial strains have been reported to degrade azoxystrobin, but precise methodologies for producing and storing these strains as potential biotechnological products are lacking. The study focused on creating and optimising a non-sterile, small-scale microbial fermentation protocol to produce azoxystrobin-degrading products and to test their shelf life. By testing 14 variants and sampling at three production and two storage time points, the trial demonstrated the successful production and storage of microbial products capable of pesticide degradation. Various measurement parameters such as pH value and organic acids were used to monitor the quality of the microbial products during the production and storage. Further, we developed and validated qPCR assays to rapidly and specifically assess the concentration of the two azoxystrobin degrading strains, namely Bacillus subtilis strain MK101 and Rhodococcus fascians strain MK144. To ensure good specificity, the combination of two qPCR assays targeting two different genome regions was implemented for each strain. The study highlights the significant impact of media selection and bacterial inoculum quantity on the microbial product quality. Full article

Sclerotinia minor (S. minor) Jagger is a phytopathogenic fungus that causes lettuce drop, a serious problem in lettuce (Lactuca sativa L.) production. The control of this pathogen is challenging because of the resistance of sclerotia, which can survive in the soil under favorable conditions. In Bulgaria, the management of lettuce drop relies primarily on the strategic application of synthetic fungicides. To find alternative methods for disease management, four bacterial isolates were screened for antagonism against S. minor. This study reports the in vitro evaluation of the antifungal activity of Bacillus subtilis, Priestia megaterium, Bacillus safensis, and Bacillus mojavensis against S. minor. The molecular identification of the isolates involved in the activity was examined through 16s rRNA sequencing. Isolated bacterial strains produced indole-3-acetic acid (IAA) in a medium supplemented with 0.1% L-tryptophan. The ability of these strains to increase the mobility of phosphorus and zinc was elucidated. The production of siderophores was confirmed on CAS (Chrom azurol S) medium. The inhibitory action of the bacterial growth broth filtrates against S. minor was demonstrated, indicating the nature of the molecules involved. The evaluation of antifungal activity was carried out in vitro and in pot experiments. This study determined the effect of growth-promoting rhizobacteria on the development of lettuce. This research focuses mainly on the development of biocontrol strategies for the management of lettuce drop in greenhouses. Full article

Composting is an environmentally friendly process, turning animal waste into fertilizer. Chicken litter compost (CLC) improves soil properties, increasing crop yields. However, the CLC effect on the soil microbiome is understudied. This study aimed to compare bacteriobiome diversity in fallow arable Chernozem with and without CLC addition in a field experiment in the Novosibirsk region, Russia, using 16S rRNA gene metabarcoding. Pseudomonadota, Actinomycetota and Acidobacteriota were the most OTU-rich phyla, together accounting for >50% of the total number of sequence reads. CLC-related shifts in the bacteriobiome structure occurred at all taxonomic levels: the Bacillota abundance was 10-fold increased due to increased Bacilli, both being indicator taxa for the CLC-soil. The main Actinomycetota classes were the indicators for the CLC-soil (Actinobacteria) and no-CLC soil (Thermoleophilia, represented Gaiella). Both Bacillota and Actinomycetota phyla were the ultimate constituents of the CLC added, persisting in the soil for five months of fallowing. The no-CLC soil indicator phyla were Acidobacteriota (represented by Acidobacteria_Group3) and Verrucomicrobiota. Future metabarcoding studies of chicken litter application in agricultural soils, including cropped studies, should address the soil microbiome at the species/strain levels in more detail, as well as how it is affected by specific crops, preferably accompanied by a direct methodology revealing the microbiota functions. Full article

A purposely naïve and semi-empirical model allows for the reproduction of the phenomenological behavior of any real microbial culture by adjusting the values of three parameters, which have a biological meaning only for a virtual microbial culture that mimics the behavior of the real ones. Any genomic, biochemical, and physical peculiarity (microbial species, physiological condition, pH, water activity, temperature, etc.) that distinguishes one real culture from another is “translated” to an effect of the degree of progress of the population density and cell age in the virtual culture. The model leads to a self-consistent description of the growth curve, which looks like the result of an autocorrelation effect. This explains why, in spite of genomic and physiologic differences, all the growth curves show a sigmoid trend. The traditional growth curve and the subsequent exponential decay in the log(N)-vs-t plot can be replaced by straight-line trends when referring to the degree of progress of the population density of the virtual culture. Full article

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria frequently linked to burn wound infections. These bacteria can grow as biofilms, which increases their level of drug resistance to current antibiotics. The purpose of the present study is to analyze the effect of biofilm formation, phage and phage cocktail action on single species and dual species biofilms I, e the coexistence of Gram positive (S. aureus) and Gram negative (P. aeruginosa). To this scenario, we employed multi-drug resistant bacteria (P. aeruginosa and S. aureus at 10⁹ CFU/µL) biofilm as single and in combination of both Gram-positive and Gram-negative bacterial biofilms of 24 h grown with respective phage (10⁹ PFU/µL) and phage cocktail (10⁹ PFU/µL) at 4 h of incubation under static conditions. The bacteriolytic activity of phages vB_SAnS_SADP1 and vB_PAnP_PADP4 on 24-h-old biofilms of P. aeruginosa (0.761 ± 0.031) and S. aureus (0.856 ± 0.055), both alone and in combination (0.67 ± 0.02), was the focus of this investigation. The structural organization of biofilms in single- or dual-species combinations under in vitro conditions was validated by scanning and confocal laser scanning microscopy investigations. After 24 h of incubation, single-species biofilms are denser and more resilient whereas dual species biofilms are more loosely associated. Loose association of dual-species biofilm under scanning electron microscopic images at the same conditions, indicated the interspecies -competition of the Gram-positive and Gram-negative bacteria and dual-species biofilms (0.67 ± 0.02) have weak associations and are readily impacted by phage and a phage cocktail (0.16 ± 0.02). Dual-species biofilms were more readily impacted in in vitro settings. Full article