Applied Microbiology

Benzophenone-3 (BP3) is an organic UV filter whose presence in the aquatic environment has been linked to detrimental developmental impacts in aquatic organisms such as coral and fish. The genus Rhodococcus has been extensively studied and is known for possessing large genomes housing genes for biodegradation of a wide range of compounds, including aromatic carbons. Here, we present the genome sequence of Rhodococcus sp. USK10, which was isolated from Chinese riverbank sediment and is capable of utilising BP3 as the sole carbon source, resulting in full BP3 mineralisation. The genome consisted of 9,870,030 bp in 3 replicons, a G+C content of 67.2%, and 9722 coding DNA sequences (CDSs). Annotation of the genome revealed that 179 of these CDSs are involved in the metabolism of aromatic carbons. The complete genome of Rhodococcus sp. USK10 is the first complete, annotated genome sequence of a Benzophenone-3-degrading bacterium. Through radiolabelling, it is also the first bacterium proven to mineralise Benzophenone-3. Due to the widespread environmental prevalence of Benzophenone-3, coupled with its adverse impact on aquatic organisms, this characterisation provides an integral first step in better understanding the environmentally relevant degradation pathway of the commonly used UV filter. Given USK10′s ability to completely mineralise Benzophenone-3, it could prove to be a suitable candidate for bioremediation application. Full article

Kombucha is a carbonated, slightly acidic beverage traditionally produced by the fermentation of sweetened tea by a symbiotic culture of bacteria and yeast (SCOBY). The microbial community of kombucha is a complex one, whose dynamics are still not fully understood; however, the emergence of culture-independent techniques has allowed a more comprehensive insight into kombucha microbiota. In recent times, advancements have been made towards the optimisation of the fermentation process, including the use of alternative substrates, defined starter cultures and the modification of fermentation parameters, with the aim of producing an innovative beverage that is improved in terms of its physiochemical, sensory and bioactive properties. The global kombucha market is rapidly increasing, with the rising popularity of the tea attributed in part to its purported health benefits, despite the lack of research in human subjects to substantiate such claims. Accordingly, the incidence of kombucha home-brewing has increased, meaning there is a requirement for individuals to recognise the potential hazards associated with fermentation and the relevant preventative measures to be undertaken to ensure the safe preparation of kombucha. The aim of this review is to provide an update regarding the current knowledge of kombucha production, microbiology, safety and marketing. Full article

We first sought to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA), on rumen fermentation and the resistance of DHA to degradation and biohydrogenation by rumen microbes through ex vivo fermentation experiments. Subsequently, we sought to quantify the diet-derived DHA content of milk and the impact of AURA on microbial composition and metabolism in a pilot feeding trial with rumen-cannulated dairy cows. To achieve our aims, rumen fluid from cannulated cows was used as inoculum, and the effect of AURA inclusion on fermentation ex vivo was examined. At doses corresponding to the amount of AURA recommended for commercial production animals, only ~10% of DHA was degraded or biohydrogenated by rumen microorganisms. The results show that feeding with AURA had no effect on either total bacterial density or short-chain fatty acid production. Real-time quantitative PCR analysis of the rumen fluid samples collected during a seven-week in vivo trial revealed that microbes related to lactic acid metabolism and methanogenesis were significantly suppressed by the AURA-supplemented diet. The DHA concentration in milk increased over 25-fold with the AURA-supplemented diet and dropped by 30–40% within one week of washout. The addition of A. limacinum biomass to dairy cow diets resulted in positive effects on rumen microbial composition with no adverse effect on fermentation activity. AURA-derived DHA was stable, with only modest degradation in the rumen, and was successfully deposited in milk. This is the first study to investigate the effect of supplementing the diet of dairy cows with a protist-based biomass, namely, on important rumen fermentation parameters and on DHA deposition in milk, using a combination of ex vivo and in vivo approaches. Full article

The microbiome revolution brought the realization that diet, health, and safety for humans in reality means diet, health, and safety for the human holobiont/superorganism. Eating healthier means much more than just feeding human cells. Our diet must also nourish the combination of our microbiome and our connected physiological systems (e.g., the microimmunosome). For this reason, there has been an interest in returning to ancestral “complete” unprocessed foods enriched in microbes, including raw milks. To contribute to this inevitable “nourishing the holobiont” trend, we introduce a systematic risk–benefit analysis tool (evidence mapping), which facilitates transdisciplinary state-of-the-science decisions that transcend single scientific disciplines. Our prior paper developed an evidence map (a type of risk–benefit mind map) for raw vs. processed/pasteurized human breast milk. In the present paper, we follow with a comprehensive evidence map and narrative for raw/natural vs. processed/pasteurized cow’s milk. Importantly, the evidence maps incorporate clinical data for both infectious and non-communicable diseases and allow the impact of modern agricultural, food management, and medical and veterinary monitoring outcomes to be captured. Additionally, we focus on the impact of raw milks (as “complete” foods) on the microimmunosome, the microbiome-systems biology unit that significantly determines risk of the world’s number one cause of human death, non-communicable diseases. Full article

Foodborne illness due to the consumption of contaminated products continues to be a serious public health issue. Bacteriophages might provide a natural and effective way to control and reduce the pathogenic bacterial population on food products. Researchers have conducted various experiments to prove their effectiveness against different pathogens and their ability to act as a natural intervention to control pathogen populations, especially in the food industry. In this study, a cocktail of bacteriophages (phages) was added to wash water in the presence of a high organic load along with commercially used sanitizers (chlorine or Sanidate 5.0) to study the efficacy of the phage–sanitizer combination in the challenge water. It was determined that in the absence of organic loads, the sanitizer by itself or the combination with phages significantly (p < 0.001) reduced the contamination by 3.00–5.00 log CFU/mL. In the presence of organic loads, the sanitizer by itself did not contribute to a significant reduction (p > 0.05) compared to the control. However, the sanitizer–phage combination led to a 3.00-log and 6.00-log reduction (p < 0.001) of the pathogen at the end of 3 and 6 h, respectively, in the presence of high organic loads. Therefore, utilizing a combination treatment (phage–sanitizer) might be one solution to reduce pathogen contamination in the food industry, especially the fresh produce industry, thus providing safe food for consumption. Full article

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced. Full article

Lactobacilli are Gram-positive aerotolerant organisms that comprise the largest genus of Lactic Acid Bacteria (LAB). Most lactobacilli are devoid of the antioxidant enzymes, superoxide dismutases, and catalases, required for protection against superoxide radicals and hydrogen peroxide (H₂O₂), respectively. However, some lactobacilli can accumulate millimolar concentrations of intracellular manganese and spare the need for superoxide dismutase, while others possess non-heme catalases. L. plantarum is associated with plant materials and plays an important role in fermented foods and gut microbiomes. Therefore, understanding the effects of the environment on the growth and survival of this organism is essential for its success in relevant industrial applications. In this report, we investigated the physiological role of Mn-catalase (MnKat) in Lactobacillus plantarum ATCC 14431. To this end, we compared the physiological and morphological properties of a ΔMnkat mutant strain and its isogenic parental strain L. plantarum ATCC 14431. Our data showed that the MnKat is critical for the growth of L. plantarum ATCC 14431 in the presence of oxygen and resistance to H₂O₂. The aerobic growth of the mutant in presence or absence of H₂O₂ was improved in the Mn-rich medium (APT) as compared to the growth in MRS medium. Furthermore, under aerobic conditions the mutant strain possessed atypical cellular morphology (i.e., shorter, and fatter). In conclusion, the MnKat of L. plantarum ATCC 14431 is important for aerobic growth, protection against H₂O₂, and maintenance of the rod-shaped cell morphology under aerobic conditions. Full article

(1) Background: Slightly acidic electrolyzed water (SAEW) is an effective and safe sterilizing solution. Its active component is hypochlorous acid (HOCl) which has been proved to exhibit a strong disinfectant activity. In this research we evaluated the effectiveness of SAEW in the removal of Methylobacterium mesophilicum, Rhodotorula mucilaginosa and Cladosporium cladosporioides, responsible for pink-colored biofilm and black mold in households. (2) Methods: Two concentrations of SAEW, 20 mg/L and 40 mg/L, were tested against M. mesophilicum, R. mucilaginosa and C. cladosporioides. In vitro experiments and mesh experiments were conducted to test the effectiveness of SAEW. (3) Results: The test results showed that 40 mg/L SAEW was effective in removing R. mucilaginosa and C. cladosporioides, with the population decreasing by approximately two orders of magnitude. For M. mesophilicum, resistance towards SAEW was observed; to obtain a 1.3 order of magnitude decrease in bacterial population, washing 5 times with 40 mg/L SAEW was necessary. Mesh experiments showed that SAEW can remove black mold; (4) Conclusions: Overall results indicated that SAEW was particularly effective for R. mucilaginosa and C. cladosporioides species commonly found in Japanese households. Full article

The low microbial flocculant yields and efficiencies limit their industrial applications. There is a need to identify bacteria with high bioflocculant production. The aim of this study was to isolate and identify a bioflocculant-producing bacterium from activated sludge wastewater and characterise its bioflocculant activity. The identification of the isolated bacterium was performed by 16S rRNA gene sequencing analysis. The optimal medium composition (carbon and nitrogen sources, cations and inoculum size) and culture conditions (temperature, pH, shaking speed and time) were evaluated by the one-factor-at-a-time method. The morphology, functional groups, crystallinity and pyrolysis profile of the bioflocculant were analysed using scanning electron microscope (SEM), Fourier transform infrared (FTIR) and thermogravimetric (TGA) analysis. The bacterium was identified as Proteus mirabilis AB 932526.1. Its optimal medium and culture conditions were: sucrose (20 g/L), yeast extract (1.2 g/L), MnCl₂ (1 g/L), pH 6, 30 °C, inoculation volume (3%), shaking speed (120 rpm) for 72 h of cultivation. SEM micrograph revealed the bioflocculant to be amorphous. FTIR analysis indicated the presence of hydroxyl, carboxyl and amino groups. The bioflocculant was completely pyrolyzed at temperatures above 800 °C. The bacterium has potential to produce bioflocculant of industrial importance. Full article

Streptomyces coelicolor A3(2) is used worldwide for genetic studies, and its complete genome sequence was published in 2002. However, as the whole genome of the type strain of S. coelicolor has not been analyzed, the relationship between S. coelicolor A3(2) and the type strain is not yet well known. To clarify differences in their biosynthetic potential, as well as their taxonomic positions, we sequenced whole genomes of S. coelicolor NBRC 12854^T and type strains of its closely related species—such as Streptomyces daghestanicus, Streptomyces hydrogenans, and Streptomyces violascens—via PacBio. Biosynthetic gene clusters for polyketides and non-ribosomal peptides were surveyed by antiSMASH, followed by bioinformatic analyses. Type strains of Streptomyces albidoflavus, S. coelicolor, S. daghestanicus, S. hydrogenans, and S. violascens shared the same 16S rDNA sequence, but S. coelicolor A3(2) did not. S. coelicolor A3(2) and S. coelicolor NBRC 12854^T can be classified as Streptomycesanthocyanicus and S. albidoflavus, respectively. In contrast, S. daghestanicus, S. hydrogenans, and S. violascens are independent species, despite their identical 16S rDNA sequences. S. coelicolor A3(2), S. coelicolor NBRC 12854^T, S. daghestanicus NBRC 12762^T, S. hydrogenans NBRC 13475^T, and S. violascens NBRC 12920^T each harbor specific polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) gene clusters in their genomes, whereas PKS and NRPS gene clusters are well conserved between S. coelicolor A3(2) and S. anthocyanicus JCM 5058^T, and between S. coelicolor NBRC 12854^T and S. albidoflavus DSM 40455^T, belonging to the same species. These results support our hypothesis that the repertoires of PKS and NRPS gene clusters are different between different species. Full article

The development of more sensitive methodologies, capable of quickly detecting and monitoring a microbial population present in a specific biological matrix, as well as performing to allow for the study of all its metabolic changes (e.g., during the formation of biofilm) to occur, is an essential requirement for both well-being and the food industry. Two techniques, in particular, have gained the attention of scientists: The first is “biospeckle”, an optical technique representing an innovative tool for applications in food quality, food safety, and nutraceuticals. With this technique, we can quickly evaluate and monitor the presence of bacteria (or their proliferation) in a solid or liquid biological matrix. In addition, the technique is helpful in quantifying and optimizing the correct storage time of the pro-biotics, if they are entrapped in matrices such as alginate and follow their survival rate in simulated gastro-intestinal conditions. A second technique with great chances is the “biofilm electrostatic test” (BET). BET undoubtedly represents a fast, simple, and highly reproducible tool suitable for admitting the evaluation of the in vitro bacterial capacity in order to adhere through an electrostatic interaction with a pyro-electrified carrier after only 2 h of incubation. BET could represent the way for a quick and standardized evaluation of bacterial resistance among biofilm-producing microorganisms through a fast evaluation of the potential presence of the biofilm. Full article

The novel coronavirus disease 2019 (COVID-19) pandemic has brought to light the role of environmental hygiene in controlling disease transmission. Healthcare facilities are hot spots for infectious pathogens where physical distancing and personal protective equipment (PPE) are not always sufficient to prevent disease transmission. Healthcare facilities need to consider adjunct strategies to prevent transmission of infectious pathogens. In combination with current infection control procedures, many healthcare facilities are incorporating ultraviolet (UV) disinfection into their routines. This review considers how pathogens are transmitted in healthcare facilities, the mechanism of UV microbial inactivation and the documented activity of UV against clinical pathogens. Emphasis is placed on the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) as well as multidrug resistant organisms (MDROs) that are commonly transmitted in healthcare facilities. The potential benefits and limitations of UV technologies are discussed to help inform healthcare workers, including clinical studies where UV technology is used in healthcare facilities. Full article

Senna alata (Linn) Roxb. plant is widely used to manage various infections in folkloric medicine. Methicillin-resistant Staphylococcus aureus (MRSA) infection continues to be a major global public health problem. This study aims to investigate the bioactive components of S. alata leaves active against MRSA. The leaves of S. alata were sequentially extracted and fractionated using standard methods and screened for activities against MRSA. The diethyl ether active thin layer chromatography (TLC) spot was subjected to infrared (IR) and gas chromatography-mass spectroscopic (GC-MS) studies. The aqueous extract and diethyl ether fraction of S. alata leaves elicited the highest activity against the MRSA. The GC-MS analysis of the fraction produced 15 eluates; only the sub-fraction 13 was effective. The TLC analysis of the sub-fraction 13 revealed three spots; only the second spot produced activity. The GC-MS result of the spot showed six peaks. The spectral results for peak 3 match the data from the IR study, suggestive of 9-octadecenoic acid methyl ester. Senna alata leaves possess bioactive compounds closely related to 9-octadecenoic acid methyl ester with potent antibacterial activity against MRSA. Full article

Manila Bay contributes significantly to the Philippines’ economy through industry, trade, tourism, and agriculture. However, microbiological contamination in the bay is putting it at risk in the present. Pathogen contamination of the water poses a serious threat to food safety, implicating public health. To address these concerns, the present study examined the prevalence of Salmonella in six different aquaculture commodities, Perna viridis (mussel), Crassostrea iridalei (oyster), Scylla serrata (crab), Penaeus spp. (shrimp), Oreochromis niloticus (tilapia), and Chanos chanos (milkfish), as well as in environmental water from growing areas. A tandem approach of culture-based and real-time PCR methods for the isolation and identification of Salmonella was carried out. To accurately identify the isolates, a real-time PCR Taqman assay based on the invA gene was used. Among the fishery resources examined, no positive samples were observed from C. chanos and O. niloticus. In addition, Salmonella was found in twenty (20) samples, representing 16.26% of all aquaculture commodities collected. Furthermore, Salmonella was found in 13.58% of the 81 environmental water samples examined. P. viridis had the highest prevalence of 55.55% out of 18 samples examined. Samples contaminated with Salmonella failed to meet the regulatory limits set by BFAR FAO 210 series 2001 and EC No. 2073/2005. In addition, it was observed that the sample matrix had a significant impact on the presence of Salmonella (p < 0.05). However, the spatial and temporal distribution of Salmonella did not vary greatly (p > 0.05). This study underscores the importance of imposing strict policies by regulatory bodies to prevent diseases, thus avoiding severe health implications. Full article

Microorganisms efficiently colonize the external and internal surfaces of the animal body establishing mutually beneficial interactions and forming site- and individual-specific microbiota. The degradation of complex polysaccharides in the animal gut, the production of useful compounds, protection against pathogenic microorganisms and contribution to the development of an efficient immune system are the main beneficial effects of a balanced microbiota. A dysbiosis, an imbalanced composition of the microbiota, has been associated with a large number of diseases from gastro-intestinal or urogenital disorders to allergies, cardiovascular and autoimmune diseases and even to the onset of certain cancers. A growing body of evidence has indicated that probiotic treatments, aimed at maintaining or rebalancing the microbiota, are useful to treat/prevent those illnesses. Lactic Acid Bacteria and Bifidobacteria are the most common microbes used in probiotic preparations; however, other bacteria and yeast cells are also widely used in commercial products. Here we focus on the use of bacterial spore formers as probiotics. Spore formers have been marketed as probiotics for over 50 years and are now extensively used for the treatment of intestinal disorders and as dietary supplements in humans, as growth promoters and competitive exclusion agents in animals. Full article

Microbes in the 21st century are understood as symbionts ‘completing’ the human ‘superorganism’ (Homo sapiens plus microbial partners-in-health). This paper addresses a significant paradox: despite the vast majority of our genes being microbial, the lack of routine safety testing for the microbiome has led to unintended collateral side effects from pharmaceuticals that can damage the microbiome and inhibit innate ‘colonization resistance’ against pathobionts. Examples are discussed in which a Microbiome First Medicine approach provides opportunities to ‘manage our microbes’ holistically, repair dysbiotic superorganisms, and restore health and resilience in the gut and throughout the body: namely, managing nosocomial infections for Clostridioides difficile and Staphylococcus aureus and managing the gut and neural systems (gut–brain axis) in autism spectrum disorder. We then introduce a risk analysis tool: the evidence map. This ‘mapping’ tool was recently applied by us to evaluate evidence for benefits, risks, and uncertainties pertaining to the breastmilk ecosystem. Here, we discuss the potential role of the evidence map as a risk analysis methodology to guide scientific and societal efforts to: (1) enhance ecosystem resilience, (2) ‘manage our microbes’, and (3) minimize the adverse effects of both acute and chronic diseases. Full article

This study aimed to quantify survival in Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium isolates on melon rind surface samples achieved by sanitizer treatment under three differing melon contamination and sanitization scenarios. Sanitizing treatments consisted of the plant-derived antimicrobial (PDA) essential oil component (EOC) geraniol (0.5 wt.%) entrapped in the polymeric surfactant Pluronic F-127 (GNP), 0.5 wt.% unencapsulated geraniol (UG), 200 mg/L hypochlorous acid at pH 7.0 (HOCl), and a sterile distilled water wash (CON). The experimental contamination and sanitization scenarios tested were: (1) pathogen inoculation preceded by treatment; (2) the pathogen was inoculated onto samples twice with sanitizing treatment applied in between inoculation events; or (3) pathogen inoculation followed by sanitizing treatment. Reductions in the numbers of surviving pathogens were dependent on the sanitizing treatment, the storage period, or the interaction of these effects. GNP treatment provided the greatest reductions in surviving pathogen counts on melon rinds, but these did not regularly statistically differ from those achieved by HOCl or UG treatment. GNP treatment provided the best pathogen control under differing conditions of pre- and/or post-harvest cross-contamination and can be applied to reduce the risk of pathogen transmission on melon rinds. Full article

The introduction of next generation sequencing techniques has enabled the characterization of the urinary tract microbiome, which resulted in the rejection of the long-held notion of urinary bladder sterility. Since the discovery and confirmation of the human bladder microbiome, an increasing number of studies have defined this microbial community and understand better its relationship to urinary pathologies. The composition of microbial communities in the urinary tract is linked to a variety of urinary diseases. The purpose of this review is to provide an overview of current information about the urinary microbiome and diseases as well as the development of novel treatment methods. Full article

With fast-growing polymerase chain reaction (PCR) technologies and various application methods, the technique has benefited science and medical fields. While having strengths and limitations on each technology, there are not many studies comparing the efficiency and specificity of PCR technologies. The objective of this review is to summarize a large amount of scattered information on PCR technologies focused on the two majorly used technologies: qPCR (quantitative polymerase chain reaction) and ddPCR (droplet-digital polymerase chain reaction). Here we analyze and compare the two methods for (1) efficiency, (2) range of detection and limitations under different disciplines and gene targets, (3) optimization, and (4) status on antibiotic resistance genes (ARGs) analysis. It has been identified that the range of detection and quantification limit varies depending on the PCR method and the type of sample. Careful optimization of target gene analysis is essential for building robust analysis for both qPCR and ddPCR. In our era where mutation of genes may lead to a pandemic of viral infectious disease or antibiotic resistance-induced health threats, this study hopes to set guidelines for meticulous detection, quantification, and analysis to help future prevention and protection of global health, the economy, and ecosystems. Full article