Lactic Acid Bacteria isolated from traditional and innovative alheiras as potential biocontrol agents.

From a selection of seven traditional and 14 innovative alheiras, 491 lactic acid bacteria (LAB) were isolated and tested for their antimicrobial activity against several food-borne pathogens. Among these, six strains revealed antimicrobial activity through potential bacteriocin production against 14 Listeria monocytogenes strains, Enterococcus faecalis ATCC 29212, Clostridium sporogenes ESB050, and Clostridium perfringens ESB054. Through whole genome sequencing (WGS), these strains were identified as Lactiplantibacillus plantarum (2), Leuconostoc mesenteroides (1), and Pediococcus acidilactici (3). Furthermore, several orthologues of class II bacteriocins genes were identified, including Plantaricin E, Plantaricin F, Pediocin PA, Enterocin X, Leucocin A, and Coagulin A. No virulence or antibiotic resistance genes' orthologues were detected by WGS analysis. However, the selected LAB strains showed variable phenotypic patterns related to virulence genes and antibiotic resistance when assessed through classical methodologies. None of these strains demonstrated the production of biogenic amines, gelatinase or DNase. Additionally, no hemolytic activity or lipase enzyme production was observed. However, only Lpb. plantarum 9A3 was sensitive to all tested antibiotics and was thus chosen for further examination. The bacteriocins produced by Lpb. plantarum (9A3) exhibited stability across a broad range of conditions, including temperatures from 4 to 100 °C, pH values ranging from 2 to 8, exposure to surfactants and detergents (Tween 20 and 80, SDS, EDTA 0.1, 2 and 5 mM, urea and sodium deoxycholate), and enzymes (papain and catalase). Their maximum activity (AU/mL = 12,800) against four L. monocytogenes strains was observed between 21 and 36 h of growth of Lbp. plantarum 9A3, indicating a bacteriostatic mode of action. Therefore, this strain appears to be a robust candidate for potential application as a protective strain to be used in the food industry. Not only is it safe, but it also produces stable bacteriocins (harbouring genes encoding for the production of three) effectively inhibiting significant pathogens such as L. monocytogenes and C. perfringens.

Leishmanicidal and healing effects of 3ß,6ß,16ß-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo.

Treatment of cutaneous leishmaniasis depends on drugs that potentially cause serious side effects and resistance. Thus, topical therapies are attractive alternatives to the drugs currently used. 3ß, 6ß, 16ß-trihydroxylup-20 (29)-ene is a lupane triterpene isolated from Combretum leprosum Mart. leaves (CLF-1), with reports of in vitro antileishmanial effect against L. amazonensis and to promote lesion healing in animal model. Herein, we evaluated the in vitro and in vivo antileishmanial and healing effects of CLF-1 against L. braziliensis. CLF-1 treatment showed low toxicity in macrophages and significantly reduced parasite load in vitro. CLF-1 induced higher IL-12 and TNF-α production and more discrete IL-4 and IL-10 production. For in vivo evaluation, a CLF-1 cream formulation was prepared to treat hamsters infected with L. braziliensis. CLF-1 treatment was able to reduce parasite load of the infected skin and lymph node more efficiently than the conventional treatment. Histopathological analysis indicated a strong inflammatory response accompanied by an important healing response. Data from this study indicate that topical CLF-1 treatment was effective and non-toxic in L. braziliensis infected hamsters suggesting its potential for further development as a future therapeutic intervention.

Exploiting Potential Probiotic Lactic Acid Bacteria Isolated from Chlorella vulgaris Photobioreactors as Promising Vitamin B12 Producers.

Lactic acid bacteria (LAB) have been documented as potential vitamin B12 producers and may constitute an exogenous source of cobalamin for the microalga Chlorella vulgaris, which has been described as being able to perform vitamin uptake. Hence, there is an interest in discovering novel B12-producing probiotic LAB. Therefore, the purpose of the current work was to perform a phenotype-genotype analysis of the vitamin B12 biosynthesis capacity of LAB isolated from C. vulgaris bioreactors, and investigate their probiotic potential. Among the selected strains, Lactococcus lactis E32, Levilactobacillus brevis G31, and Pediococcus pentosaceus L51 demonstrated vitamin B12 biosynthesis capacity, with the latter producing the highest (28.19 ± 2.27 pg mL-1). The genomic analysis confirmed the presence of pivotal genes involved in different steps of the biosynthetic pathway (hemL, cbiT, cobC, and cobD). Notably, P. pentosaceus L51 was the only strain harboring cobA, pduU, and pduV genes, which may provide evidence for the presence of the cobalamin operon. All strains demonstrated the capability to withstand harsh gastrointestinal conditions, although P. pentosaceus L51 was more resilient. The potential for de novo cobalamin biosynthesis and remarkable probiotic features highlighted that P. pentosaceus L51 may be considered the most promising candidate strain for developing high-content vitamin B12 formulations.

Multi-Drug Resistance in Bacterial Genomes-A Comprehensive Bioinformatic Analysis.

Antimicrobial resistance is presently one of the greatest threats to public health. The excessive and indiscriminate use of antibiotics imposes a continuous selective pressure that triggers the emergence of multi-drug resistance. We performed a large-scale analysis of closed bacterial genomes to identify multi-drug resistance considering the ResFinder antimicrobial classes. We found that more than 95% of the genomes harbor genes associated with resistance to disinfectants, glycopeptides, macrolides, and tetracyclines. On average, each genome encodes resistance to more than nine different classes of antimicrobial drugs. We found higher-than-expected co-occurrences of resistance genes in both plasmids and chromosomes for several classes of antibiotic resistance, including classes categorized as critical according to the World Health Organization (WHO). As a result of antibiotic-resistant priority pathogens, higher-than-expected co-occurrences appear in plasmids, increasing the potential for resistance dissemination. For the first time, co-occurrences of antibiotic resistance have been investigated for priority pathogens as defined by the WHO. For critically important pathogens, co-occurrences appear in plasmids, not in chromosomes, suggesting that the resistances may be epidemic and probably recent. These results hint at the need for new approaches to treating infections caused by critically important bacteria.

Plasmids Increase the Competitive Ability of Plasmid-Bearing Cells Even When Transconjugants Are Poor Donors, as Shown by Computer Simulations.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

The Impact of Non-Pathogenic Bacteria on the Spread of Virulence and Resistance Genes.

This review discusses the fate of antimicrobial resistance and virulence genes frequently present among microbiomes. A central concept in epidemiology is the mean number of hosts colonized by one infected host in a population of susceptible hosts: R0. It characterizes the disease's epidemic potential because the pathogen continues its propagation through susceptible hosts if it is above one. R0 is proportional to the average duration of infections, but non-pathogenic microorganisms do not cause host death, and hosts do not need to be rid of them. Therefore, commensal bacteria may colonize hosts for prolonged periods, including those harboring drug resistance or even a few virulence genes. Thus, their R0 is likely to be (much) greater than one, with peculiar consequences for the spread of virulence and resistance genes. For example, computer models that simulate the spread of these genes have shown that their diversities should correlate positively throughout microbiomes. Bioinformatics analysis with real data corroborates this expectation. Those simulations also anticipate that, contrary to the common wisdom, human's microbiomes with a higher diversity of both gene types are the ones that took antibiotics longer ago rather than recently. Here, we discuss the mechanisms and robustness behind these predictions and other public health consequences.

Estuarine Aquacultures at the Crossroads of Animal Production and Antibacterial Resistance: A Metagenomic Approach to the Resistome.

It is recognized that the spread of antibiotic resistance (AR) genes among aquatic environments, including aquaculture and the human environment, can have detrimental effects on human and animal health and the ecosystem. Thus, when transmitted to the human microbiome or pathogens, resistance genes risk human health by compromising the eventual treatment of infections with antibiotic therapy. This study aimed to define the resistance profile of aquaculture farms and their potential risk for spreading. Twenty-four sediments from oyster and gilthead sea bream aquaculture farms located in three Portuguese river estuaries (17 sediments from Sado, 4 from Aveiro, and 3 from Lima) were studied by comparative metagenomic analysis. The computation of the diversity of genes conferring resistance per antibiotic class revealed a significant increase in aminoglycosides, beta-lactams, disinfectants, quinolones, and tetracyclines counts. In all geographic locations under study, the most diverse AR genes confer resistance to the macrolides, tetracyclines and oxazolidinones classes, all of which are medically important for human and animal therapies, as well as resistance to disinfectants. The diversity of mobile genetic elements correlated with the number of AR genes such as tetracyclines, suggesting that AR could be easily mobilized among bacterial genomes and microbiomes.

Genomic Analysis of Antibiotics Resistance in Pathogens.

The emergence of antibiotic-resistant pathogens currently represents a serious threat to public health and the economy worldwide [...].

Are Virulence and Antibiotic Resistance Genes Linked? A Comprehensive Analysis of Bacterial Chromosomes and Plasmids.

Although pathogenic bacteria are the targets of antibiotics, these drugs also affect hundreds of commensal or mutualistic species. Moreover, the use of antibiotics is not only restricted to the treatment of infections but is also largely applied in agriculture and in prophylaxis. During this work, we tested the hypothesis that there is a correlation between the number and the genomic location of antibiotic resistance (AR) genes and virulence factor (VF) genes. We performed a comprehensive study of 16,632 reference bacterial genomes in which we identified and counted all orthologues of AR and VF genes in each of the locations: chromosomes, plasmids, or in both locations of the same genome. We found that, on a global scale, no correlation emerges. However, some categories of AR and VF genes co-occur preferentially, and in the mobilome, which supports the hypothesis that some bacterial pathogens are under selective pressure to be resistant to specific antibiotics, a fact that can jeopardize antimicrobial therapy for some human-threatening diseases.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids.

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a 'weapon' to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Bacterial persistence is essential for susceptible cell survival in indirect resistance, mainly for lower cell densities.

Antibiotic-susceptible bacteria may survive bactericidal antibiotics if other co-inhabiting bacteria detoxify the medium through antibiotic degradation or modification, a phenomenon denominated as indirect resistance. However, it is unclear how susceptible cells survive while the medium is still toxic. One explanation relies on the speed of detoxification, and another, non-exclusive explanation, relies on persistence, a state of bacterial dormancy where cells with low metabolic activity and growth rates are phenotypically tolerant to antibiotics and other cytotoxic substances. Here we simulated the fate of susceptible cells in laboratory experiments in the context of indirect resistance to understand whether persistence is necessary to explain the survival of susceptible cells. Depending on the strain and experimental conditions, the decay of persister populations may follow an exponential or a power-law distribution. Therefore, we studied the impact of both distributions in the simulations. Moreover, we studied the impact of considering that persister cells have a mechanism to sense the presence of a toxic substance-a mechanism that would enable cells to leave the dormant state when the medium becomes nontoxic. The simulations show that surviving susceptible cells under indirect resistance may originate both from persister and non-persister populations if the density of detoxifying cells is high. However, persistence was necessary when the initial density of detoxifying cells was low, although persister cells remained in that dormancy state for just a few hours. Finally, the results of our simulations are consistent both with exponential and power-law decay of the persistence population. Whether indirect resistance involves persistence should impact antibiotic treatments.

Metagenomics and Other Omics Approaches to Bacterial Communities and Antimicrobial Resistance Assessment in Aquacultures.

The shortage of wild fishery resources and the rising demand for human nutrition has driven a great expansion in aquaculture during the last decades in terms of production and economic value. As such, sustainable aquaculture production is one of the main priorities of the European Union's 2030 agenda. However, the intensification of seafood farming has resulted in higher risks of disease outbreaks and in the increased use of antimicrobials to control them. The selective pressure exerted by these drugs provides the ideal conditions for the emergence of antimicrobial resistance hotspots in aquaculture facilities. Omics technology is an umbrella term for modern technologies such as genomics, metagenomics, transcriptomics, proteomics, culturomics, and metabolomics. These techniques have received increasing recognition because of their potential to unravel novel mechanisms in biological science. Metagenomics allows the study of genomes in microbial communities contained within a certain environment. The potential uses of metagenomics in aquaculture environments include the study of microbial diversity, microbial functions, and antibiotic resistance genes. A snapshot of these high throughput technologies applied to microbial diversity and antimicrobial resistance studies in aquacultures will be presented in this review.

COVID-19 Lockdowns May Reduce Resistance Genes Diversity in the Human Microbiome and the Need for Antibiotics.

Recently, much attention has been paid to the COVID-19 pandemic. Yet bacterial resistance to antibiotics remains a serious and unresolved public health problem that kills hundreds of thousands of people annually, being an insidious and silent pandemic. To contain the spreading of the SARS-CoV-2 virus, populations confined and tightened hygiene measures. We performed this study with computer simulations and by using mobility data of mobile phones from Google in the region of Lisbon, Portugal, comprising 3.7 million people during two different lockdown periods, scenarios of 40 and 60% mobility reduction. In the simulations, we assumed that the network of physical contact between people is that of a small world and computed the antibiotic resistance in human microbiomes after 180 days in the simulation. Our simulations show that reducing human contacts drives a reduction in the diversity of antibiotic resistance genes in human microbiomes. Kruskal-Wallis and Dunn's pairwise tests show very strong evidence (p < 0.000, adjusted using the Bonferroni correction) of a difference between the four confinement regimes. The proportion of variability in the ranked dependent variable accounted for by the confinement variable was η2 = 0.148, indicating a large effect of confinement on the diversity of antibiotic resistance. We have shown that confinement and hygienic measures, in addition to reducing the spread of pathogenic bacteria in a human network, also reduce resistance and the need to use antibiotics.

The Perfect Condition for the Rising of Superbugs: Person-to-Person Contact and Antibiotic Use Are the Key Factors Responsible for the Positive Correlation between Antibiotic Resistance Gene Diversity and Virulence Gene Diversity in Human Metagenomes.

Human metagenomes with a high diversity of virulence genes tend to have a high diversity of antibiotic-resistance genes and vice-versa. To understand this positive correlation, we simulated the transfer of these genes and bacterial pathogens in a community of interacting people that take antibiotics when infected by pathogens. Simulations show that people with higher diversity of virulence and resistance genes took antibiotics long ago, not recently. On the other extreme, we find people with low diversity of both gene types because they took antibiotics recently-while antibiotics select specific resistance genes, they also decrease gene diversity by eliminating bacteria. In general, the diversity of virulence and resistance genes becomes positively correlated whenever the transmission probability between people is higher than the probability of losing resistance genes. The positive correlation holds even under changes of several variables, such as the relative or total diversity of virulence and resistance genes, the contamination probability between individuals, the loss rate of resistance genes, or the social network type. Because the loss rate of resistance genes may be shallow, we conclude that the transmission between people and antibiotic usage are the leading causes for the positive correlation between virulence and antibiotic-resistance genes.

Harmless or Threatening? Interpreting the Results of Molecular Diagnosis in the Context of Virus-Host Relationships.

Molecular methods, established in the 1980s, expanded and delivered tools for the detection of vestigial quantities of nucleic acids in biological samples. Nucleotide sequencing of these molecules reveals the identity of the organism it belongs to. However, the implications of such detection are often misinterpreted as pathogenic, even in the absence of corroborating clinical evidence. This is particularly significant in the field of virology where the concepts of commensalism, and other benign or neutral relationships, are still very new. In this manuscript, we review some fundamental microbiological concepts including commensalism, mutualism, pathogenicity, and infection, giving special emphasis to their application in virology, in order to clarify the difference between detection and infection. We also propose a system for the correct attribution of terminology in this context.

Characterization of a Lactiplantibacillus plantarum R23 Isolated from Arugula by Whole-Genome Sequencing and Its Bacteriocin Production Ability.

Lactiplantibacillus plantarum is one of the lactic acid bacteria species most used as probiotics and starter cultures in food production. Bacteriocin-producers Lpb. plantarum are also promising natural food preservatives. This study aimed to characterize Lpb. plantarum R23 and its bacteriocins (R23 bacteriocins). The genome sequence of Lpb. plantarum R23 was obtained by whole-genome sequencing (WGS) in an Illumina NovaSeq platform. The activity of Lpb. plantarum R23-produced bacteriocin against two Listeria monocytogenes strains (L7946 and L7947) was evaluated, and its molecular size was determined by tricine-SDS-PAGE. No virulence or antibiotic resistance genes were detected. Four 100% identical proteins to the class II bacteriocins (Plantaricin E, Plantaricin F, Pediocin PA-1 (Pediocin AcH), and Coagulin A) were found by WGS analysis. The small (<6.5 kDa) R23 bacteriocins were stable at different pH values (ranging from 2 to 8), temperatures (between 4 and 100 °C), detergents (all, except Triton X-100 and Triton X-114 at 0.01 g/mL), and enzymes (catalase and α-amylase), did not adsorb to the producer cells, had a bacteriostatic mode of action and their maximum activity (AU/mL = 12,800) against two L. monocytogenes strains occurred between 15 and 21 h of Lpb. plantarum R23 growth. Lactiplantibacillus plantarum R23 showed to be a promising bio-preservative culture because, besides being safe, it produces a stable bacteriocin or bacteriocins (harbors genes encoding for the production of four) inhibiting pathogens as L. monocytogenes. Further studies in different food matrices are required to confirm this hypothesis and its suitability as a future starter culture.

Development and validation of a framework for the assessment of school curricula on the presence of evolutionary concepts (FACE).

Evolution is a key concept of biology, fundamental to understand the world and address important societal problems, but research studies show that it is still not widely understood and accepted. Several factors are known to influence evolution acceptance and understanding, but little information is available regarding the impacts of the curriculum on these aspects. Very few curricula have been examined to assess the coverage of biological evolution. The available studies do not allow comparative analyses, due to the different methodologies employed by the authors. However, such an analysis would be useful for research purposes and for the development of appropriate educational policies to address the problem of a lack of evolution acceptance in some countries. In this paper we describe the steps through which we developed a valid and reliable instrument for curricula analysis known as FACE: "Framework to Assess the Coverage of biological Evolution by school curricula." This framework was developed based on the "Understanding Evolution Conceptual Framework" (UECF). After an initial pilot study, our framework was reformulated based on identified issues and experts' opinions. To generate validity and reliability evidence in support of the framework, it was applied to four European countries' curricula. For each country, a team of a minimum of two national and two foreign coders worked independently to assess the curriculum using this framework for content analysis. Reliability evidence was estimated using Krippendorf's alpha and resulted in appropriate values for coding the examined curricula. Some issues that coders faced during the analysis were discussed and, to ensure better reliability for future researchers, additional guidelines and one extra category were included in the framework. The final version of the framework includes six categories and 34 subcategories. FACE is a useful tool for the analysis and the comparison of curricula and school textbooks regarding the coverage of evolution, and such results can guide curricula development.

The Social Distancing Imposed To Contain COVID-19 Can Affect Our Microbiome: a Double-Edged Sword in Human Health.

Hygienic measures imposed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and contain COVID-19 have proven effective in controlling the pandemic. In this article, we argue that these measures could impact the human microbiome in two different and disparate ways, acting as a double-edged sword in human health. New lines of research have shown that the diversity of human intestinal and oropharyngeal microbiomes can shape pulmonary viral infection progression. Here, we suggest that the disruption in microbial sharing, as it is associated with dysbiosis (loss of bacterial diversity associated with an imbalance of the microbiota with deleterious consequences for the host), may worsen the prognosis of COVID-19 disease. In addition, social detachment can also decrease the rate of transmission of antibiotic-resistant bacteria. Therefore, it seems crucial to perform new studies combining the pandemic control of COVID-19 with the diversity of the human microbiome.