Applied Microbiology

Fish microbiota studies have mostly addressed intestinal bacterial communities because of their role in fish physiology; however, the skin microbiota has been studied less despite its role as the first pathogen barrier in the host. DNA-based molecular techniques have contributed to improving our understanding of the skin microbiota, but this approach faces challenges, such as the low count of bacteria in healthy fish skin. To overcome this limitation, an RNA-based approach was developed to study the skin microbiota in salmonids, including tissue sampling, RNA extraction, and downstream procedures to obtain PCR amplicons for next-generation sequencing. The protocol originated in this work overcomes the limitations of low bacterial counts and is useful for describing active microbiota in fish skin. The application of the protocol to salmonids reared in both an experimental setting and on a farm revealed that the trout skin microbiota was dominated by bacteria from the phylum Proteobacteria (>65%). At the genus level, Piscirickettsia (46%) was highlighted as the most abundant in the experimental unit samples; in contrast, Pseudoalteromonas (26%), followed by Escherichia_Shigella (~25%), was the most abundant in farmed trout. Full article

The occurrence of mycotoxigenic species in peanuts is a major concern, and has been investigated in depth for many years. However, most studies focus on the occurrence of aflatoxigenic fungi, such as Aspergillus section Flavi. The present study aimed to clarify the occurrence of Aspergillus section Nigri, a group that harbors species capable of producing ochratoxin A (OTA), which has scarcely been investigated in peanuts. A total of 52 peanut samples, collected in the field and from storage, were analyzed. Aspergillus section Nigri was isolated from 64% and 100% of field and storage samples, respectively, and 218 strains were obtained. Based on the multiloci phylogeny of the CaM and BenA loci, six species of Aspergillus section Nigri were identified: A. brasiliensis, A. niger, A. neoniger, A. welwitschiae, A. costaricaensis, and A. japonicus. The incidence of ochratoxigenic strains was 5.0% (11/218), and only A. niger and A. welwitschiae were able to produce OTA. The presence of OTA in peanuts was found in 6 field and 8 storage samples, with levels ranging from 0.106 to 0.387 and 0.090 to 0.160 µg/kg, respectively. Full article

Today’s perceived excess of work versus rest and excessive stressful situations are one of the potential factors for an increasing number of gastrointestinal disorders. Although the prevalence of irritable bowel syndrome (IBS) varies widely, it is assumed that up to 15% of the general population may be affected. Symptoms resulting from IBS take the form of pain, including difficulties with bowel movements, bloating, and a feeling of overflowing in the intestines. These symptoms have a negative impact on patients’ quality of life, so any possible intervention supported by Evidence-Based Medicine should effectively contribute to their improvement. In the following review, the authors compile the current knowledge based on the findings of researchers from around the world. The topic of probiotic therapy is increasingly discussed in the field of lower gastrointestinal disorders, and thus the authors discover links between probiotic strains and the improvement of health quality in patients with IBS, taking into consideration the ways it affects patients’ mental health. As a result, a systematic review was prepared based on 60 items of recent literature, of which more than 80% were published in the last 5 years. Full article

Throughout their cancer treatments, around half of all patients will undergo irradiation that is accompanied by several side effects reducing their quality of life and leading to the interruption or extension of their treatment course. Pelvic irradiation leads to the triggering of mucositis and dysbiosis, further impairing the daily life of the patients. In this work, we address the ability of Limnospira indica strain PCC 8005 and Lacticaseibacillus rhamnosus GG ATCC 53103 in alleviating the above-mentioned side effects triggered by the local pelvic irradiation of 12 Gy in a mouse model. We found that the combinatorial administration of these food supplements was able to confer partial tight junction protection while the bacterial translocation towards the mesenteric lymph nodes was found to be identical between the saline sham-irradiated and supplemented irradiated group. Furthermore, the supplemented group did not present a significant shift in microbial composition following pelvic irradiation, indicating that the bacterial formulation was able to mitigate the dysbiosis induced by the latter treatment, as observed in the saline irradiated group. These very promising results will be further completed by investigating the mode of action and/or active molecules mediating the beneficial effects of both L. indica PCC 8005 and L. rhamnosus GG. Full article

Parkinson’s disease is a debilitating multisystemic disorder affecting both the central and peripheral nervous systems. Accumulating evidence suggests a potential interaction between gut microbiota and the pathophysiology of the disease. As a result of the degradation of dopaminergic neurons, PD patients develop motor impairments such as tremors, rigidity, and slowness of movement. These motor features are preceded by gastrointestinal issues, including constipation. Given these gastrointestinal issues, the gut has emerged as a potential modulator of the neurodegenerative cascade of PD. Several studies have been carried out to broaden our understanding of the gut–microbiota–brain axis in PD. As a result, a decrease in short-chain fatty acid synthesizing bacteria has been observed in multiple studies. Some studies, on the other hand, have shown an enrichment of mucin- and levodopa-degrading microbes. In this review, we compiled the available evidence from the literature on the bidirectional communication between the gut microbiome system and the brain in PD. We also addressed the association between dysbiosis and the clinical symptoms of PD and host–drug metabolism. Finally, we touched on some of the therapeutic interventions that may restore eubiosis and modulate the gut structure to restrain disease progression. Full article

Background: Blastocystis hominis (B. hominis) is a ubiquitous parasite that has spread worldwide and is commonly present in human stool specimens. It was hypothesized that infection with B. hominis plays a role in the pathogenesis of autoimmune diseases in humans. The aim of this study is to test this hypothesis by investigating patients with autoimmune diseases. Patients with various types of autoimmune diseases with gastrointestinal symptoms were enrolled in this study as cases (n = 72) along with nongastrointestinal symptom patients as controls (n = 58). All participants in this study were subjected to history taking and were investigated for B. hominis infection via wet-mount microscopic stool examinations, staining with trichrome stain, and molecular-based tests applied to their fecal samples. Blood samples were also tested for complete blood counts. B. hominis were identified with specific PCR more in cases (12/72; 16.6%) than in controls (3/58; 5.2%), with a significant difference (p < 0.05). Significant decreases in white blood cell counts were demonstrated in systemic lupus erythematosus (SLE) and ulcerative colitis (UC) patients infected with B. hominis when compared to patients with nongastrointestinal symptoms (p-value < 0.05). Full article

Taking into account the worrying scenario of water scarcity, it is essential to enable more efficient technologies for wastewater treatment. Wastewater may be treated by conventional biological processes that remove pathogenic organisms, particulate and soluble organic compounds, and other components. However, secondary effluents from treatment plants may still contain toxic elements or high concentrations of inorganic nutrients (mainly nitrogen and phosphorus), which enable the growth of photosynthetic microorganisms in water bodies, resulting in eutrophication. In this context, cultivation of photosynthetic microorganisms in secondary wastewater from sewage treatment allows the removal of nutrients from such wastewater, reducing the possibility of eutrophication. Moreover, microalgal biomass, produced in this tertiary wastewater treatment, may be harvested by different methods with the potential for different applications, such as fertilizer and biofuel. Full article

Survival time, percentage mortality, and feeding behavior were evaluated for adults of the weevil Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) exposed to biopesticides by using a Petri dish plant-based bioassay system. Treatments consisted of three registered entomopathogenic fungal formulations (BotaniGard ES, PFR-97 20% WDG, and Met52 EC) and three biochemical formulations (AzaMax, Entrust, and PyGanic EC); a synthetic chemical insecticide (Sevin SL) and distilled water served as control treatments. Thirty adult M. undecimpustulatus undatus (ten per bioassay cage replicate, three replicates per treatment) were given cocoplum leaves sprayed to the point of runoff with a treatment. Survivorship of weevils was checked daily for 15 days. Five trials (15 total replicates per treatment) were performed. Survival times of weevils in all product treatments were shorter than those of weevils in the water control treatment. Percentages of beetle mortality in the Entrust and BotaniGard treatments were about two-fold higher than in the other treatments. Mean percentages of mycosis for beetles in the BotaniGard, PFR-97, and Met 52 treatments were 90%, 8%, and 5%, respectively. Weevils that consumed BotaniGard-treated leaves caused significantly more leaf damage than weevils that consumed leaves with PFR-97 or Met52. Weevils on leaves treated with Entrust, PyGanic, AzaMax, and Sevin consumed significantly less than those in the fungal and water only treatments. The high mortality rates caused by Entrust and BotaniGard suggest that populations of adult M. undecimpustulatus undatus might be well-managed using these two commercially available biopesticides. Additional testing is needed in the field to corroborate our results obtained under controlled laboratory conditions. Full article

The amyE gene encoding α-amylase from Bacillus subtilis 168 was fused to several genes, the products of which are membrane proteins to express AmyE extracellularly. Genes of CapA, a subunit of the capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto; YiaT^R232, the first 232 residues of YiaT, an outer membrane protein of Escherichia coli K-12; and PgsA, cytidine 5′-diphosphate-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase of B. subtilis subsp. natto, were used. The Escherichia coli strain harboring the plasmid carrying the pgsA-amyE fusion expressed a very high level of amylase not only on the extracellular surface of the cells but also in the medium and the intracellular space and solubilized steamed rice in two days. The N-terminal amino acid sequence of the amylase purified from the culture medium (Ser-Ala-Glu-Thr-Ala) indicated that it was cleaved at the signal peptide cleavage site of AmyE. The strain SH2204, transformed with pKN11 carrying speA (arginine decarboxylase gene), speB (agmatinase gene) and argA^{ATG Y19C} (N-acetylglutamate synthase gene) and pMAN63 carrying pgsA-amyE produced 1.2 mM putrescine from 5 mM arginine by adding 0.5 mM IPTG in eight days. Discarding steamed rice is not only a food loss, but also a waste of renewable, biogenerated resources due to the burning of it with petroleum, increasing carbon dioxide in the atmosphere. The E. coli strain developed in this study can help solve this problem because it can produce an important chemical using steamed rice as the sole carbon and energy source. Full article

This study investigated the effects of arbuscular mycorrhizal fungi (AMF) on the growth, phosphorus (P) uptake, and yield of maize in the presence or absence of tillage. The two-year field experiment was conducted in Kanagawa, Japan. Firstly, we investigated whether the presence of indigenous AMF communities in the roots, as determined by amplicon sequencing analysis, contributed to maize growth in Experiment 1, a 2-year field-based study. The findings revealed that the maize (Zea mays L.) in rotary tillage had higher P uptake, growth at the six-leaves collar stage, and yield, compared to no tillage. The AMF communities colonizing maize roots were altered by the presence or absence of tillage; specifically, tillage increased the dominance of the Gigasporaceae, whereas no tillage increased the dominance of the Acaulosporaceae. Based on these findings, we confirmed whether the inoculation of similar AMF strains, as analyzed in the field study of tillage practices on maize roots, produces growth-promoting effects for maize growth in a controlled pot experiment consistent with the results of the field experiment. For experiment 2, Dentiscutata cerradensis TK-1, Cetraspora pellucida SZ-3 (Gigasporaceae), Acaulospora morrowiae AP-5, and A. longula F-1 (Acaulosporaceae) were inoculated as AMF inocula for a pot experiment. The results showed that aboveground biomass did not change with any inoculum compared to the control. The P concentration in maize was higher for D. cerradensis TK-1 and C. pellucida SZ-3 inoculation than for the control. However, inoculation with A. morrowiae AP-5 and A. longula F-1 did not change P concentrations from the control. This indicates that D. cerradensis TK-1 and C. pellucida SZ-3 are more effective in promoting P uptake in maize than in A morrowiae AP-5 and A. longula F-1. Based on field and pot experiments, our findings suggest that tillage practices lead to alterations in the AMF communities that colonize the roots, and this shift may also contribute to changes in P uptake and crop growth. Full article

The selection of probiotic bacteria based on their beneficial characteristics does not necessarily mean they can be later scaled up and used for technological applications and formula design. Three probiotic strains—Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085, and Limosolactobacillus mucosae CRL2069, originally isolated from feedlot cattle feces—have demonstrated beneficial characteristics when used as in-feed probiotics. Therefore, the current study was conducted to develop a low-cost culture medium to optimize growth conditions to enhance biomass production. The study also sought to identify appropriate cryoprotective agents to sustain high functional cell numbers after freeze drying. A central composite design was applied to determine the optimal medium composition. This yielded a simplified, low-cost effective medium containing 3% molasses and industrial yeast extracts (0.5 to 2.5%) as carbon and nitrogen sources, which were added to a basal medium for each strain. Established production conditions at 37 °C, without agitation, and pH-controlled for the CRL2085 and CRL2069 strains, and free pH for the CRL2074 strain, allowed us to obtain biomass yields of 12.95, 18.20, and 12.25 g, respectively, at 24-h incubation, compared with the MRS medium. In addition, the cryoprotective effect of the selected agents was demonstrated to be strain-dependent. Thus, the highest viability (10⁹–10¹⁰ CFU/g), stability during 30-d storage, and survival rate (88–99%) were achieved when 10% MSG (monosodium glutamate), sucrose + fructose + trehalose + WPC (whey protein concentrate) + 10% MSG, and 1.2% WPC + 10% trehalose, were used for freeze drying CRL2074, CRL2085, and CRL2069, respectively. Moreover, the probiotic strains retained their probiotic functionality when hydrophobic characteristics were evaluated. These results highlight the need to perform strain-specific evaluation of the critical factors involved in the large-scale production of probiotic lactobacilli to sustain viability and stability after the freeze drying and storage processes. Full article

Microbiome data is high dimensional, sparse, compositional, and over-dispersed. Therefore, modeling microbiome data is very challenging and it is an active research area. Microbiome analysis has become a progressing area of research as microorganisms constitute a large part of life. Since many methods of microbiome data analysis have been presented, this review summarizes the challenges, methods used, and the advantages and disadvantages of those methods, to serve as an updated guide for those in the field. This review also compared different methods of analysis to progress the development of newer methods. Full article

Evidence of congenital transmission of Lyme disease from mother to fetus has been investigated since the 1980s. This study reports the results of a retrospective analysis of 31 studies described in 27 papers published in a 2018 review article. Analysis of these identified statistically probable transmission in 13 (42%) of the 31 studies and possible transmission in 2 (6%). Most studies included mothers who had been treated with antimicrobials. When no antimicrobials were used, 74% of the pregnancies had adverse outcomes. When oral antimicrobials were used, 29% of pregnancies had adverse outcomes. When intravenous (IV) antimicrobials were used, adverse outcomes dropped to 12% of the pregnancies. This is a six-fold reduction in risk compared with no antimicrobial treatment. Some studies did not define whether oral or IV antimicrobials were used. When these results were included, adverse outcomes were 30 times higher for untreated mothers. Adverse outcomes included deaths, heart anomalies, and preterm births. Full article

Cutaway peatlands in the midlands of the Republic of Ireland are rarely the focus of scientific studies. The soil quality and related microenvironment have been severely impacted by peat extraction. Returning them to a ‘near-natural state’ would require greater insights into this ecological niche. The current research took the initiative to study the microbiology of vast cutaway sites in the midlands of Ireland. Peat was collected over two seasons in January, February and April. Homogenised peat was aseptically cultured on a range of specific and non-specific culture media. Microbial enumeration, Gram staining and other microscopic observations of morphologically distinct microorganisms were performed. The total viable bacterial and fungal numbers were highest in February (1.33 × 10⁵ CFU ml⁻¹ and 5.93 × 10⁶ CFU ml⁻¹, respectively) and were lowest in April (1.14 × 10³ CFU ml⁻¹ and 5.57 × 10⁶ CFU ml⁻¹). Penicillium spp. and Trichoderma spp. were common in all the sites. The highest values of phosphate solubilisation index were recorded in peat collected in April (SI = 3.167 & 3.000). Overall, there is a statistically significant difference (p ≤ 0.0001) among the microbial numbers across the three months. This variation could be due to the temperature and pH differences across peat soil. Full article

A phenomenological model of planktonic microbial cultures, reported in previous papers, suggests that the whole growth progress seems planned by the microbial population since a pre-growth latency phase, during which the population level remains at its starting level. This model is in line with recent suggestions about the behavior of complex systems, as long as it allows for the gathering of the growth trends of a number of real batch cultures in a single master plot of reduced variables, in spite of their metabolic and physiological differences. One important issue of the model concerns the origin of the time scale for the microbes that can differ from that for the observer. The present paper reports some consequences of the model in view of its potential use in predictive microbiology and proposes an extension to the steady and decay phases of the culture evolution suggesting that, consistent with the assumptions about the growth phase, the decay occurs by a scan of the cell generation steps. This view leads to the conclusion that the steady phase between growth and decay trends actually corresponds to the loss of the oldest cell generations, which represents minor fractions of the microbial population. Such early decay is almost undetectable in a log scale, looking like a steady phase. To account for cases that show a broad maximum instead of an intermediate steady trend, a single continuous function, still related to the model, can describe the whole growth and decay trend of the microbial culture. Full article

The fish gut microbiome plays an essential role in the host’s development and survival. Environmental factors can shape the gut microbiome and potentially mediate physiological performance. Seasonal environments that experience regular abiotic and biotic transitions likely drive variability in the gut microbiome. However, we know very little about how seasonal transitions interact with the gut microbiome, especially at northern latitudes where seasonality is exaggerated. Therefore, in this study, we analyzed the native gut microbiome of a flexible top predator fish species, the lake trout, across seasons in a boreal lake using 16S rRNA sequencing. Seasonal changes explained one third of the bacterial variance. The relative abundance of several bacterial taxa influenced the observed seasonal differences in the community; pathogenic Pseudomonas and Stenotrophomonas were most abundant in the spring and winter seasons, and chemoheterotrophic Macrococcus and Lelliottia were most abundant in the summer. The fall season was dominated by unclassified Clostridiaceae, potentially linked to a shift in lake trout foraging. Therefore, we present novel evidence that suggests seasonality is likely to shape the native gut microbiome of lake trout; however, the mechanistic links between the environment and the microbial diversity of the gut remain to be uncovered. Full article

The development of alternative (and free-of-antibiotics) antibacterial and antibiofilm agents is an important strategy to circumvent the resistance of bacteria to antibiotics. Herein, we explore the production of mixed oxides by incorporating silver nanoparticles in titanium dioxide as a silver concentration-dependent antibacterial agent that is further incorporated in Tilapia fish skin (a promising prototype of xenograft), integrating the antibacterial activity of mixed oxide into the intrinsic properties of Tilapia skin. The antibiofilm activity of samples prepared with high concentrations of silver (10 wt% of precursor AgNO₃) has been considered a good antibiofilm response. The influence of silver content is also observed with respect to the minimum bactericidal concentration, which is reduced to 3.13 mg/mL with a characteristic kill time in the order of 30 min that is associated with antibiofilm activity in biofilm-forming strains of Staphylococcus aureus. These results indicate that modified Tilapia fish skin acquires antibacterial behavior and can be explored for xenografts with prospective applications in the light-dependent actuation of TiO₂-based compounds. Full article

Novel benzoxazines were synthesized by microwave irradiation and tested for their potential binding affinity towards receptors of advanced glycation end products (RAGE). We found that the compound (2-(2-bromophenyl)-6-methyl-2,4-dihydro-1H-benzo[d][1,3]oxazine) (3i) is a lead inhibitor of RAGE. Further, our in silico prediction that benzoxazines dock towards the AGE binding region of RAGE suggests that these ligands could bind effectively at the hydrophobic pocket of the receptor and additionally form key interactions with Arg48 and Arg104, revealing its diversity in developing anti-RAGE drugs to treat AGE–RAGE-dominant disease conditions. Functionally, we herein report the anti-tuberculosis activity of small molecules which could be bioactive in the culture of mycobacterium tuberculosis. Full article

The commercial onshore aquaculture of the spiny lobster Panulirus ornatus, while in its infancy, has progressed rapidly from the enabling research that continues at the University of Tasmania. The development of lobster feeds, both fresh and manufactured, has been critical to the success of this emerging aquaculture sector. Fresh feeds derived from mussel represent the gold standard in terms of the growth performance of juvenile lobsters. Nonetheless, concerns regarding availability, sustainability, and potential biosecurity issues of fresh feeds highlight the importance of developing manufactured feeds for lobster aquaculture. Wild lobsters are assumed to have a balanced natural diet that allows for standard growth and development, and as such natural diets are often used as a reference for feed development. Similarly, the gut microbiota associated with a natural diet is assumed to reflect a healthy microbial assemblage. The aim of this study was to compare the microbiota of the hindgut and hepatopancreas of cultured P. ornatus fed with a commercial prawn pellet or mussel to that of wild spiny lobster juveniles. Gut samples were analysed using Oxford Nanopore 16S rRNA gene sequencing. Based on principal coordinate analysis, the gut bacteria of cultured lobsters were different from the wild juveniles. The core microbiota of the hindgut and hepatopancreas libraries were phyla Proteobacteria (Gamma, Alpha) and Bacteroidetes. Vibrio was the most dominant genus in both organs. The differences in bacterial relative abundance were mainly between cultured (pellet-, mussel-fed) and wild lobsters. In conclusion, bacteria in the cultured lobsters had significantly different profiles to that of the wild juveniles, indicating that current onshore aquaculture practices alter the gut microbiota. A number of different feeding and culture practices may be required if the aim of closed culture practices is to attain a gut microbiota in cultured animals that is representative of that found in wild spiny lobsters. Full article