Appl. Microbiol., Volume 2, Issue 3 (September 2022) – 20 articles

Despite great efforts have been made worldwide, the coronavirus disease 19 (COVID-19) still has not a definitive cure, although the availability of different vaccines are slowing down the transmission and severity. It has been shown that surfactin, a cyclic lipopeptide produced by Bacillus subtilis, is a molecule able to counteract both SARS-CoV-1, MERS-CoV and HCoV-229E coronaviruses. In this study the potential antiviral activity of surfactin against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested in vitro in a cellular model of infection. Our results show that 2 h treatment with surfactin is able to reduce SARS-CoV-2 infectivity on Vero E6 cells both at 24 h and after 7 days from viral inoculation, probably impairing the viral membrane integrity. Moreover, surfactin, at the concentrations used in our experimental settings, is not cytotoxic. We suggest surfactin as a new potential molecule against SARS-CoV-2, to be employed at least as a disinfectant. Full article

Biochar, derived from the pyrolysis of plant materials has the potential to enhance plant growth in soilless media. Howevetar, little is known about the impact of biochar amendments to soilless growth media, microbial community composition, and fate of chemical constituents in the media. In this study, different concentrations of biochar were added to soilless media and microbial composition, and chemical constituents were analyzed using metagenomics and gamma spectroscopy techniques, respectively. Across treatments, carboxyl-C, phenolic-C, and aromatic-C were the main carbon sources that influenced microbial community composition. Flavobacterium (39.7%), was the predominantly bacteria genus, followed by Acidibacter (12.2%), Terrimonas (10.1%), Cytophaga (7.5%), Ferruginibacter (6.0%), Lacunisphaera (5.9%), Cellvibrio (5.8%), Opitutus (4.8%), Mucilaginibacter (4.0%) and Bryobacter (4.0%). Negative relationships were found between Cytophaga and ²²⁶Ra (r = −0.84, p = 0.0047), ⁴⁰K (r = −0.82, p = 0.0069) and ¹³⁷Cs (r = −0.93, p = 0.0002). Similarly, Mucilaginibacter was negatively correlated with ²²⁶Ra (r = −0.83, p = 0.0054) and ¹³⁷Cs (r = −0.87, p = 0.0021). Overall, the data suggest that high % biochar amended samples have high radioactivity concentration levels. Some microorganisms have less presence in high radioactivity concentration levels. Full article

(1) Background: HPV infection can progress over the years to become cervical cancer. In this study, genotype and a normalized viral load were evaluated as surrogate markers of progression to cancer. (2) Methods: A total of 558 endocervical swabs were collected from 120 women (mean, 40.1 ± 11.8 years old). Seventy-eight of the women underwent clinical intervention (CI) to clear the infection during the course of the study, while forty-two did not (NCI). Normalized viral load (NVL) was calculated using a COBAS 4800 system. The INNOLIPA genotyping system was used to classify HPV which was neither type 16 or 18. (3) Results: The mean age of CI women was 41.1 ± 11.4 (22–68) years old and that of the NCI group was 37.7 ± 12.13 (23–65) (p: 0.104). HPV16 was present in 11 (25%) NCI and 30 (35.2%) CI patients, HPVα9non16 in 20 (45%) NCI and 34 (40%) CI, and HPVnonα9 in 13 (29.5%) NCI and 21 (24.7%) CI (p = 0.48). In NCI women there was an average NVL decrease of 0.95 log after two years and a further decrease of 2.35 log at the end of the third year. At the end of the study, 34 (80%) of the NCI patients were clear of HPV. However, NVL of CI women remained at around 5 log until intervention (p < 0.001). (4) Conclusions: Viral load decreased in NCI women at follow-up in the second year. In contrast, in CI women, their viral load did not fall over the follow-up period. This work thus demonstrates that a reduction in normalized viral load was associated with good evolution. Full article

While dietary supplements can have beneficial effects on the health of the intestine, these effects can come with unresolved issues in terms of therapeutic efficacy and mechanisms of action. In this study, the model probiotic Lacticaseibacillus rhamnosus GG ATCC 53103 and the anciently used dietary supplement Limnospira indica strain PCC 8005 were compared for their effects on murine intestinal ecology. Healthy male mice received either saline or suspensions of living cells of L. indica PCC 8005 or L. rhamnosus GG daily along a two-week intervention period, followed by a two-week washout period. Both bacteria-based solutions appeared able to transiently shift the microbial community, which were characterized by a higher relative abundance of members of the butyrate producing Lachnospiraceae and Porphyromonadaceae families. Full article

The production of lactic acid (LA) through the microbial conversion of agro-industrial residuals is an important process in the biotechnology industry. The growth kinetics of 30 strains of lactic acid bacteria (LAB) isolated from agro-industrial residues were determined and nine strains were selected for microbioreactor fermentation. Lactiplantibacillus pentosus_70-1 (1.662) and L. pentosus_19-2 (1.563) showed the highest OD₆₀₀ values, whereas the highest growth rates were observed for L. pentosus_19-2 (0.267 h⁻¹) and Weissella soli_31 (0.256 h⁻¹). The production of LA and acetic acid (AA), glucose consumption, and metabolic profiles were determined, without finding significant differences in the production of LA; however, W. soli_29 produced the highest amount of LA (20.833 gL⁻¹) and was able to metabolize most of the studied carbohydrates. Based on these results, W. soli_29 was chosen for a 20 h fermentation in a 7 L bioreactor using both standard medium and milk whey supplemented medium. W. soli_29 produced 16.27 gL⁻¹ and 7.21 gL⁻¹ of LA in each of these mediums, respectively. These results show the underlying potential of Weissella strains for biotechnological applications. Additional analysis which should contemplate different agro-industrial residues and other conditions in bioreactors must be carried out. Full article

The aim of this review was to investigate the effectiveness of the Human Oral Microbe Identification Microarray (HOMIM) in identifying and quantifying bacterial species of the oral microbiome in periodontal disease. The search for articles was conducted in CENTRAL, CINAHL, MEDLINE and EMBASE by two reviewers, and included articles published in English between January 1990 and December 2021. The selected articles were human observational studies in adults between 18 and 65 years, presenting specific predefined keywords. Articles were initially selected by title and abstract; articles that met the inclusion criteria were analyzed for methodological quality using a detailed checklist for quality assessment. Data were extracted and reported using the PRISMA tool. The study design, sample, follow-up period, collection and microbial analysis methods, statistical treatment, results and discussion were quality assessed and risk of bias was evaluated using the Cochrane Risk-of-Bias tool. A narrative synthesis approach was used to synthesize and interpret the extracted data. From the initial search, 2931 articles were retrieved; 51 of these were then selected after screening by title and abstract. Subsequently, 8 articles met the inclusion after full-text reading and were classed according to methodological quality as high (2), moderate (3) or low (3). Studies included in this review were of high and medium quality. Data from the Human Oral Microbe Identification Microarray (HOMIM) provide much more robust results, showing major shifts between periodontal health and periodontal disease. Compared to earlier techniques such as Denaturing Gradient Gel Electrophoresis (DGGE), HOMIM represents a more effective approach for quantification due to its high sensitivity; thus, it is able to identify a high prevalence of periodontal pathogens and novel species in low abundance. The literature provides moderate evidence that the Human Oral Microbe Identification Microarray (HOMIM) is more effective in identifying and quantifying bacterial species of the oral microbiome in periodontal disease, compared to earlier molecular and non-molecular methods such as Denaturing Gradient Gel Electrophoresis (DGGE) and a culture-based approach with phenotypic tests. Full article

Acute otitis media (AOM) constitutes a multifactorial disease, as several host and environmental factors contribute to its occurrence. Prevention of AOM represents one of the most important goals in pediatrics, both in developing countries, in which complications, mortality, and deafness remain possible consequences of the disease, compared to in developed countries, in which this condition has an important burden in terms of medical, social, and economical implications. The strategies for AOM prevention are based on reducing the burden of risk factors, through the application of behavioral, environmental, and therapeutic interventions. The introduction of culture-independent techniques has allowed high-throughput investigation of entire bacterial communities, providing novel insights into the pathogenesis of middle ear diseases through the identification of potential protective bacteria. The upper respiratory tract (URT) is a pivotal region in AOM pathogenesis, as it could act as a source of pathogens than of protective microorganisms for the middle ear (ME). Due to its direct connection with the external ambient, the URT is particularly exposed to the influence of environmental agents. The aim of this review was to evaluate AOM environmental risk factors and their impact on URT microbial communities, and to investigate AOM pathogenesis from the microbiota perspective. Full article

COVID-19 is caused by SARS-CoV-2 which has so far affected more than 500 million people worldwide and killed over 6 million as of 1 May 2022. The approved emergency-use vaccines were lifesaving in such a devastating pandemic. Inflammation-related pathways have been well documented to be upregulated in the case of SARS-CoV-2 in rodents, non-human primates and human samples. We reanalysed a previously published dataset to understand if certain molecular components of inflammation could be higher in infected samples. Mechanistically, viroporins are important players in the life cycle of SARS-CoV-2 and are primary to its pathogenesis. We studied the two prominent viroporins of SARS-CoV-2 (i) Orf3a and (ii) envelope (E) protein from a sequence and structural point of view. Orf3a is a cation-selective viral ion channel which has been shown to disrupt the endosomal pathways. E protein is one of the most conserved proteins among the SARS-CoV proteome which affects the ERGIC-related pathways. The aqueous medium through the viroporins mediates the non-selective translocation of cations, affecting ionic homeostasis in the host cellular compartments. We hypothesize a possible mechanistic approach whereby the ionic imbalance caused by viroporin action could potentially be one of the major pathogenic drivers leading to the increased inflammatory response in the host cell. Our results shed light into the transcriptomic, genomic and structural proteomics aspects of widely studied SARS-CoV-2 viroporins, which can be potentially leveraged for the development of antiviral therapeutics. Full article

The citrus industry is challenged by numerous arthropods, yet extensive research has not been conducted to determine the potential use of entomopathogenic fungi as endophytes in pest management strategies. Two inoculation methods (i.e., soil drench and foliar spray) using a suspension of Beauveria bassiana (strain PPRI 5339 contained in Velifer^®) containing 4 × 10⁷ conidia mL⁻¹ in 0.01% Tween 80 were conducted on three commercially available citrus rootstocks (i.e., ‘US-942’, ‘US-812’, ‘Swingle’). Seedlings were grown under greenhouse-controlled conditions over a 7-week observation period. Similarly, a third inoculation method (seed soaking) was conducted using seeds from the same three rootstocks. The fungus was re-isolated post-inoculation from ‘US-942’ and ‘US-812’ in the foliar spray and seed soaking treatments. In addition, the fungus was recovered from root tissue in the foliar-sprayed seedlings, suggesting possible systemic movement from leaves to roots. The fungus was not recovered from soil-drench-treated seedlings, nor from any of the ‘Swingle’ cultivars. This study assessed the potential of B. bassiana to endophytically colonize certain citrus rootstocks in planta. Full article

Chronic endometritis (CE) is an infectious disease of the uterine lining, which is characterized by endometrial stromal plasmacyte (ESPC) infiltration. CE is often seen in infertile women with a history of repeated implantation failure (RIF) following an in vitro fertilization-embryo transfer program, recurrent pregnancy loss, and unknown etiology. Oral antibiotic agents, such as doxycycline, metronidazole, ciprofloxacin, azithromycin, and moxifloxacin, have been prescribed and are effective in the treatment of CE. Multi-drug resistance (MDR), however, is an emerging issue, as in other medical fields. We report six cases of persistent MDR-CE in infertile women who were resistant to all the aforementioned antibiotic agents. The bacterial genera and microbial communities unique to persistent MDR-CE were not identified in their vaginal secretions and/or endometrial fluid. Oral lincomycin administration (14 days, 1500 mg/day) was effective in the eradication of ESPCs in these women. In the embryo transfer cycles following histopathologic confirmation of cure (elimination of ESPCs) of persistent MDR-CE, three out of them had a successful live birth. Full article

Outbreaks of Zika virus (ZIKV) have resulted in a call by global health advocates for increased surveillance and research with aggressive measures to combat ZIKV infections. There is no licensed ZIKV vaccine yet available, but a number of vaccine candidates are in development. Advancement of promising vaccine candidates to licensure may rely upon the development and use of well-characterized preclinical models developed based on the essential elements of an animal model as outlined in the U.S. FDA “Product Development Under the Animal Rule: Guidance for Industry”. Further, in the absence of adequate clinical cases to support a more traditional approval pathway based on clinical efficacy, regulatory approval could be based upon human safety data and use of a well-characterized animal model to evaluate vaccine efficacy. This report summarizes a statistical analysis that characterizes the progression of ZIKV infection in Rhesus macaques (RMs) with respect to viral load using available data on twenty-six (26) RMs from three (3) studies that were exposed to ZIKV and were not immunized with a ZIKV vaccine. Progression of infection was characterized by time to detection of viral RNA in serum (RT-qPCR) or time to positive viremia (plaque assay). Viral RNA was detected via RT-qPCR as early as day 1 post-infection and was undetectable for all animals by day 7. Viremia also was indicated by plaque assay as early as day 1 and was undetectable for all animals by day 5. Viral RNA was detected in all animals following exposure, while viremia was not observed in all animals. No significant differences in viral loads measured by either RT-qPCR or plaque assay were observed across sex, age, or study. Neither sex nor age were significant predictors of either time to detection of viral RNA or time to positive viremia following exposure to ZIKV. Progression of viral load, which is studied since infection is largely asymptomatic in both RMs and humans, is similar in RMs and humans with positive presentation ranging from 1 to 7 days post-infection and clearance by day 10. Overall, due to consistency of the model across sexes and ages and similarity to the infection profile in humans, it is concluded that the RM model of ZIKV infection is a well-characterized model for use for evaluation of ZIKV countermeasures. Full article

The interest in hemp seed oil (HSO) and tea tree oil (TTO) in the medical and food industries is increasing. The current study compares their bioactivity to other plant oils, mainly focusing on hemp seed oils (HSOs) with various cannabidiol (CBD) contents. A DPPH assay was employed to evaluate the antioxidant activity. The antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis was evaluated using time–kill, minimum inhibition concentration (MIC), and Kirby–Bauer disk diffusion methods. Tea tree oil showed significantly higher antimicrobial activity against S. enteritidis compared to E. coli and S. aureus (p < 0.05). The antioxitant activity range (lowest to highest) was sesame < vetiver < rosehip < tea tree < organic hemp < pure hemp < 5% CBD < vitamin C. Tea tree oil and 5% CBD showed antioxidant activity at IC50 of 64.45 μg/mL and 11.21 μg/mL, respectively. The opposing antimicrobial and antioxidant results for TTO and HSO indicate that these activities arise from different components within the oil compositions. Full article

(1) Background: Bangladesh must has to spend a large amount of foreign currency to import commercial baker’s yeast every year. We could save money by finding a potential Saccharomyces cerevisiae from natural sources compatible with commercial baker’s yeast production. (2) Methods: Grapes, rice, pineapples were collected, processed, and inoculated on YMA plates and incubated at 30 °C for 48 h. Then 11 single morphologically well-formed colonies were isolated, purified, and identified, three as S. cerevisiae, three as S. rouxii, three as S. bisporus, and two as S. exigus based on standard cultural, morphological, and biochemical characteristics. Identified S. cerevisiae (designated as G2, P5 and R3) were then assessed for CO₂ production as a measure of their baking potential during bread production and compared with two commercial strains (designated as C1 and C2). (3) Results: Isolate-G2 produced the maximum of 1830 mm³ of gas, whereas C1, C2, R3, and P5 produced 1520, 1680, 770, and 610 mm³ gas, respectively. No strain produced H₂S which is associated with an off-flavor and unpleasant taste. These isolates showed maximum cell density at a pH range of 4–5.5 in 4–16% molasses broth at 30 °C after 4 days of incubation and maximum 4.75 × 10⁹, 7.9 × 10⁸, 1.472 × 10¹⁰, 2.08 × 10¹⁰ and 5.24 × 10⁹ CFU mL⁻¹ were produced by C1, C2, G2, P5 and R3, respectively. Isolate-G2 was found to have the most potential, whereas isolate-R3 and P5 have satisfactory potential. (4) Conclusions: G2 could be a good candidate for commercial trials. Full article

Fermentation is a traditional technique used to increase nutrients, flavonoids, vitamins, minerals, and the flavor of raw materials. In this study, adlay bran was fermented by Lactobacillus brevis MJM60390 (FAB), and the anti-melanogenic effect was investigated. The results demonstrated that FAB significantly suppressed melanin accumulation in mouse melanogenic B16F10 cells, and the activity was higher than non-fermented adlay bran (NFAB). The molecular mechanism study showed that FAB inhibited melanin synthesis by suppressing the gene expression of melanocortin 1 receptor (Mc1r), melanocyte-inducing transcription factor (Mitf), tyrosinase (Tyr), tyrosinase-related protein-1 (Trp-1), and tyrosinase-related protein-2 (Trp-2) genes. Western blotting analysis showed that FAB strongly decreased the expression of Tyr, Trp-1, and Trp-2 compared to NFAB. Furthermore, phenolic compounds such as gallic acid, p-coumaric acid, ferulic acid, and sinapic acid, which are known for their anti-melanogenic effects, were significantly increased in FAB compared with NFAB. These findings suggest that FAB holds great potential as an anti-melanogenic agent and can be used for the development of whitening cosmetics. Full article

The aim of this study was to evaluate the performance of two of the most commonly used commercial kits for soil DNA extraction regarding the values of the taxonomic diversity of prokaryotes and community composition of saffron (Crocus sativus) cultivated fields. The impact of the QIAGEN-DNeasy PowerSoil Kit (MO) and Macherey-Nagel™ NucleoSpin™ Soil (MN) kit was tested on the soil of an Italian western alpine experimental site located in Saint Christophe (Aosta Valley, AO). Nine biological replicas of bulk soil were collected and analyzed independently with the two kits. 16S rRNA metabarcoding was applied to characterize soil microbial communities. We first noticed that both DNA extraction kits yielded nearly the same number of OTUs: 1284 and 1268 for MN and MO, respectively. Both kits did not differ in the alpha diversity of the samples, while they had an influence on the beta diversity. The comparative analysis of the microbial community composition displayed differences in microbial community structure depending on which kit was used. These differences were especially highlighted at Phylum and Class levels. On the other hand, the fact that, from a functional point of view, our approach did not highlight any differences allows us to state that the results obtained with the two extraction kits are comparable and interchangeable. Based on these results and those in the literature, we could undoubtedly recommend both commercial kits, especially if the soil target microorganisms are prokaryotes and the study focuses on agricultural sites. Full article

Influenza virus is a seasonal respiratory pathogen that produces global pandemics by genome reassortments. This rapid evolution creates difficulty in producing vaccines. Although several anti-influenza drugs have been developed, acquisition of rapid drug resistance by viruses is common. Therefore, it is important to develop novel therapeutic and prophylactic strategies. In this study, we evaluated the antiviral effects of a microalgae Coccomyxa sp. KJ (IPOD FERM BP-22254) extract in a BALB/c mouse model of influenza. Oral administration of dry algal powder (5 mg/day or 20 mg/day) before infection with influenza A virus (IFV) suppressed viral proliferation in the lungs and bronchoalveolar lavage fluid (BALF). It also exhibited stimulatory effects on systemic and local production of neutralizing antibodies. These results suggest that this powder is a promising candidate for the therapeutic and prophylactic management of influenza. Full article

A culture-independent mycobiome survey in Haplic Cambisol under Korean pine in a long-term field experiment in the Russian Far East was conducted using sequence analysis of the ITS region amplified with ITS3/ITS4 primers using the metagenomic DNA as a matrix. Overall 758 fungal OTUs were identified, representing 15 phyla, 47 classes, 104 orders, 183 families, and 258 genera. More OTUs represented the Ascomycota phylum (513) than Basidiomycota (113), with both phyla together comprising 95% of the relative abundance. The Leotiomycetes class was ultimately prevailing; apparently contributing significantly to the organic matter decomposition and microbial biomass in soil, as shown by a PCA. Only two dominant OTUs (Pseudogymnoascus sp. and Hyaloscyphaceae, both Ascomycota) were common in the studied samples. The presented high mycobiome diversity in soil under the monospecies artificial forest, where Korean pine had been the sole edificator for forty years, allows concluding that plant chemistry diversity is the main factor shaping the soil mycobiome in such an environment. The obtained data provide a reference for further studies of soil mycobiota, especially under Korean pine with its aesthetic, as well as nut-producing, potential. The results can be helpful in the targeted creating of a soil mycobiome beneficial for pines in afforestation and remediation contexts. Full article

Standardised molecular methods are available for the detection of norovirus from water and specific food items. Detection of norovirus from stool samples also relies on molecular methods, but differences exist between nucleic acid extraction, reverse transcription, and amplification strategies recommended by the ISO 15216-1:2017, and those employed in clinical laboratories. Here, we conduct a direct comparison of two methods for the detection and quantitation of norovirus from a stool sample and from artificially contaminated swabs. We also compare use of linear dsDNA standards as recommended in ISO 15216:2017 against an in vitro-transcribed single-stranded RNA (ssRNA) for estimation of norovirus genome copy number. Our results show that the two methods have comparable sensitivity for the detection of norovirus RNA from a clinical sample or swab. The use of a ssRNA standard revealed that quantitation performed against a linear dsDNA standard consistently underestimated the genome copy numbers by 1.5 to 2 log due to the relative inefficiency of the reverse transcription step. This has important implications for the estimation of the sensitivity of norovirus detection methods, comparability of results across sites, and assessment of viral loads that may be clinically significant or estimated to constitute infectious doses. Full article

Foodborne pathogens pose risks to populations all over the world. Pathogens can be used as bioterrorism agents, causing an outbreak that affects many individuals through the consumption of a commonly affected food or beverage. A PCR assay can be used to identify pathogens through their unique melting points using a high-resolution melt assay. Assays can be used to detect the bacteria individually or from a mixture using species-specific primers. An assay was developed to detect and identify three pathogens that routinely cause multistate foodborne outbreaks, as documented by the U.S. Centers for Disease Control and Prevention, Campylobacter jejuni (C. jejuni), Escherichia coli (E. coli), and Salmonella enterica (S. enterica), in single bacterium assays and a multiplex. The primers were targeted to specific and unique gene sequences of each pathogen, including cadF, yedN, and hilA, respectively. Each pathogen was identified by its unique melting temperature in single assays: 78.10 ± 0.58 °C for C. jejuni, 81.96 ± 0.42 °C for E. coli, and 87.55 ± 0.37 °C for S. enterica. The multiplex successfully detected and identified all three of the pathogens with the distinctly separated melt peaks. The PCR high-resolution melt assay also proved to be specific, reproducible, fast, and sensitive in experiments. Full article

A consideration of the outer structures of Gram-positive and Gram-negative bacteria, and of the surface contaminants present on Cu-containing substrates, has led to the identification of Cu₂O as a potent antimicrobial. In the presence of adsorbed water, the hydrated form ionizes to Cu^I-O⁻, which is capable of degrading the protective polysaccharide layer of the outer lipopolysaccharide membranes of Gram-negative bacteria; it is equally capable of attacking the peptidoglycan lattices present in both Gram-positive and Gram-negative bacteria. This Perspective underlines the importance of Cu^I-O⁻ in the early stages of contact killing, and points to information, still lacking, that would optimize contact killing and lead to broader applications in the therapeutic management of bacterial infections. Full article