IntroductionStaphylococcus aureus is a major opportunistic pathogen associated with dairy production systems, where it contributes to intramammary infections and may enter the food chain through contaminated milk. The increasing prevalence of antimicrobial-resistant strains underscores the need for alternative biocontrol strategies. Here, we report the isolation and comprehensive characterization of a novel lytic bacteriophage, vB_SauP-INT105, obtained from wastewater in Extremadura, Spain.Methods and resultsPlaque morphology revealed a distinctive three-zone halo pattern suggestive of phage-encoded exopolysaccharide depolymerase activity, confirmed by bioinformatic prediction of two candidate depolymerase-encoding ORFs. Electron microscopy showed a bacteriophage with an icosahedral head and a short non-contractile tail. The phage exhibited lytic activity against 86.5% of 37 dairy-associated S. aureus isolates tested. One-step growth analysis revealed a latency period of ~40 minutes and a burst size of ~23 PFU/cell. The phage showed robust stability across a wide range of temperatures and pH values relevant to dairy-processing conditions, retaining viability over 20 months at 4 °C. Genome sequencing revealed a 17.45 kb dsDNA genome encoding 20 ORFs, with no detectable genes associated with virulence, lysogeny, or antibiotic resistance. Phylogenetic analysis placed INT105 as a novel species within the Rosenblumvirus genus. Antibiofilm assays demonstrated significant reductions in viable cell counts in two S. aureus strains with contrasting biofilm phenotypes.ConclusionThese results establish INT105 as a genomically safe, environmentally robust and functionally effective phage with strong potential for biocontrol applications in dairy-production environments and broader food-safety and clinical contexts.

Motivation:Phage therapy is emerging as a promising alternative to antibiotics in biomedical research, highlighting the growing need for computational tools to rationally design effective phage cocktails. However, its clinical potential is often compromised by the evolution of heritable bacterial resistance, which is frequently exacerbated by repeated phage exposure. This can lead to broad-spectrum cross-resistance and reduced long-term efficacy. Existing approaches typically rely on host range matrices but often overlook viral interference and the complex, non-binary nature of virus-host interactions. Results:We present SocialViruses, a tool for designing optimized phage cocktails selecting up to twelve viruses and using two alternative algorithms. SocialViruses integrates quantitative host range infection and virus-virus interaction matrices to guide cocktail design. It produces a detailed report with key quality metrics and allows users to define multiple cocktails while minimizing viral interference and managing co-infection redundancy. Availability and implementation:SocialViruses is freely available as a Cytoscape application and can be downloaded from: https://apps.cytoscape.org/apps/SocialViruses.

The life cycle of most non-conventional yeasts, such as Torulaspora delbrueckii (Td), is not as well-understood as that of Saccharomyces cerevisiae (Sc). Td is generally assumed to be haploid, which detracts from some biotechnological properties compared to diploid Sc strains. We analyzed the life cycle of several Td wine strains and found that they were mainly diploid during exponential growth in rich medium. However, most cells became haploid in stationary phase, as observed for Sc haploid heterothallic strains. When transferred and incubated in nutrient-deficient media, these haploid cells became polymorphic, enlarged, and transitioned to diploid or polyploid states. The increased ploidy, that mainly results from supernumerary mitosis without cytokinesis, was followed by sporulation. A similar response was observed in yeasts that remained alive during the second fermentation of base wine for sparkling wine making, or during growth in ethanol-supplemented medium. This response was not observed in the Sc yeast populations under any of the experimental conditions assayed, which suggests that it is a specific adaptation of Td to the stressful fermentation conditions. This response allows Td yeasts to remain alive and metabolically active longer during wine fermentation. Consequently, we designed procedures to increase the cell size and ploidy of haploid Td strains. Td inocula with increased ploidy showed enhanced fermentation efficiency compared to haploid inocula of the same strains.

In population medical genetics, the study of autosomal recessive disorders in highly endogamous populations is a major topic where calculating the inbreeding and relationship coefficients on mating networks is crucial. However, a challenge arises when dealing with large and complex mating networks, making their traversal difficult during the calculation process. For this calculation, we propose using Iterative Level-0 (IL0) as a new and faster algorithm that traverses mating networks more efficiently. The purpose of this work is to explain in detail the IL0 algorithm and prove its superiority by comparing it with two algorithms based on the best-known algorithms in the area: Depth First Search (DFS) and Breadth First Search (BFS). A Cytoscape application has been developed to calculate the inbreeding and relationship coefficients of individuals composing any mating network. In this application, the IL0 proposal together with DFS-based and BFS-based algorithms have been implemented. Any user can access this freely available Cytoscape application (https://apps.cytoscape.org/apps/inbreeding) that allows the comparison between the IL0 proposal and the best-known algorithms (based on DFS and BFS). In addition, a diverse set of mating networks has been collected in terms of complexity (number of edges) and species (humans, primates, and dogs) for the experiments. The runtime obtained by the IL0, DFS-based, and BFS-based algorithms when calculating the inbreeding and relationship coefficients proved the improvement of IL0. In fact, a speedup study reflected that the IL0 algorithm is 7.60 to 127.50 times faster than DFS-based and BFS-based algorithms. Moreover, a scalability study found that the growth of the IL0 runtime has a linear dependence on the number of edges of the mating network, while the DFS-based and BFS-based runtimes have a quadratic dependence. Therefore, the IL0 algorithm can solve the problem of calculating the inbreeding and relationship coefficients many times faster (up to 127.50) than the two algorithms based on the famous DFS and BFS. Furthermore, our results demonstrate that IL0 scales much better as the complexity of mating networks increases.

BackgroundThis systematic review evaluates pneumolysin (PLY) as a target for new treatments against pneumococcal infections. Pneumolysin is one of the main virulence factors produced by all types of pneumococci. This toxin (53 kDa) is a highly conserved protein that binds to cholesterol in eukaryotic cells, forming pores that lead to cell destruction.MethodsThe databases consulted were MEDLINE, Web of Science, and Scopus. Articles were independently screened by title, abstract, and full text by two researchers, and using consensus to resolve any disagreements that occurred. Articles in other languages different from English, patents, cases report, notes, chapter books and reviews were excluded. Searches were restricted to the years 2000 to 2021. Methodological quality was evaluated using OHAT framework.ResultsForty-one articles describing the effects of different molecules that inhibit PLY were reviewed. Briefly, the inhibitory molecules found were classified into three main groups: those exerting a direct effect by binding and/or blocking PLY, those acting indirectly by preventing its effects on host cells, and those whose mechanisms are unknown. Although many molecules are proposed as toxin blockers, only some of them, such as antibiotics, peptides, sterols, and statins, have the probability of being implemented as clinical treatment. In contrast, for other molecules, there are limited studies that demonstrate efficacy in animal models with sufficient reliability.DiscussionMost of the studies reviewed has a good level of confidence. However, one of the limitations of this systematic review is the lack of homogeneity of the studies, what prevented to carry out a statistical comparison of the results or meta-analysis.ConclusionA panel of molecules blocking PLY activity are associated with the improvement of the inflammatory process triggered by the pneumococcal infection. Some molecules have already been used in humans for other purposes, so they could be safe for use in patients with pneumococcal infections. These patients might benefit from a second line treatment during the initial stages of the infection preventing acute respiratory distress syndrome and invasive pneumococcal diseases. Additional research using the presented set of compounds might further improve the clinical management of these patients.

The yeasts Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) may show a killer phenotype that is encoded in dsRNA M viruses (V-M), which require the helper activity of another dsRNA virus (V-LA or V-LBC) for replication. Recently, two TdV-LBCbarr genomes, which share sequence identity with ScV-LBC counterparts, were characterized by high-throughput sequencing (HTS). They also share some similar characteristics with Sc-LA viruses. This may explain why TdV-LBCbarr has helper capability to maintain M viruses, whereas ScV-LBC does not. We here analyze two stretches with low sequence identity (LIS I and LIS II) that were found in TdV-LBCbarr Gag-Pol proteins when comparing with the homologous regions of ScV-LBC. These stretches may result from successive nucleotide insertions or deletions (indels) that allow compensatory frameshift events required to maintain specific functions of the RNA-polymerase, while modifying other functions such as the ability to bind V-M (+)RNA for packaging. The presence of an additional frameshifting site in LIS I may ensure the synthesis of a certain amount of RNA-polymerase until the new compensatory indel appears. Additional 5′- and 3′-extra sequences were found beyond V-LBC canonical genomes. Most extra sequences showed high identity to some stretches of the canonical genomes and can form stem-loop structures. Further, the 3′-extra sequence of two ScV-LBC genomes contains rRNA stretches. The origin and possible functions of these extra sequences are here discussed.

The misuse and overuse of antibiotics have boosted the proliferation of multidrug-resistant (MDR) bacteria, which are considered a major public health issue in the twenty-first century. Phage therapy may be a promising way in the treatment of infections caused by MDR pathogens, without the side effects of the current available antimicrobials. Phage therapy is based on phage cocktails, that is, combinations of phages able to lyse the target bacteria. In this work, we present and explain in detail two innovative computational methods to design phage cocktails taking into account a given phage-bacteria infection network. One of the methods (Exhaustive Search) always generates the best possible phage cocktail, while the other method (Network Metrics) always keeps a very reduced runtime (a few milliseconds). Both methods have been included in a Cytoscape application that is available for any user. A complete experimental study has been performed, evaluating and comparing the biological quality, runtime, and the impact when additional phages are included in the cocktail.

Gas production and subsequent food spoilage from the appearance of fissures, cracks or holes are undesirable consequences of saprophytic E. coli growth in cheese. Hence, the development of E. coli growth inhibitors is of special interest in soft and raw milk cheese production. This study investigates the inhibitory effect of a cocktail of four selected coliphages on the growth of E. coli in cheese and its ability to inhibit cheese early blowing. The microbiological analysis showed that the evolution of total bacterial counts during cheese-making and ripening was comparable among control and phage inoculated cheeses. Nevertheless these results also revealed a reduction in E. coli growth at the beginning of fermentation with phage treatment. Additionally, image analysis of cheese slices showed a noteworthy reduction in the number of cheese eyes and the area occupied by gas holes throughout cheese ripening. Thus, the use of bacteriophages as biocontrol agents provides an effective approach to combat cheese early blowing without noticeably altering the microbiological or physicochemical parameters of these fermented foods.

The application of bacteriophages as antibacterial agents has many benefits in the "post-antibiotic age". To increase the number of successfully targeted bacterial strains, phage cocktails, instead of a single phage, are commonly formulated. Nevertheless, there is currently no consensus pipeline for phage cocktail development. Thus, although large cocktails increase the spectrum of activity, they could produce side effects such as the mobilization of virulence or antibiotic resistance genes. On the other hand, coinfection (simultaneous infection of one host cell by several phages) might reduce the potential for bacteria to evolve phage resistance, but some antagonistic interactions amongst phages might be detrimental for the outcome of phage cocktail application. With this in mind, we introduce here a new method, which considers the host range and each individual phage-host interaction, to design the phage mixtures that best suppress the target bacteria while minimizing the number of phages to restrict manufacturing costs. Additionally, putative phage-phage interactions in cocktails and phage-bacteria networks are compared as the understanding of the complex interactions amongst bacteriophages could be critical in the development of realistic phage therapy models in the future.

Background and objective: Phage therapy is a resurgent strategy used in medicine and the food industry to lyse bacteria that cause damage to health or spoil a food product. Frequently, phage-bacteria infection networks have a large size, making it impossible to manually study all possible phage cocktails. Thus, this article presents an R package called PhageCocktail to automatically design efficient phage cocktails from phage-bacteria infection networks. Methods: This R package includes four different methods for designing phage cocktails: ExhaustiveSearch, ExhaustivePhi, ClusteringSearch, and ClusteringPhi. These four methods are explained in detail and are evaluated using 13 empirical phage-bacteria infection networks. More specifically, runtime and expected success (fraction of lysed bacteria) are analyzed. Results: The four methods have variations in terms of runtime and quality of the results. ExhaustiveSearch always provides the best possible phage cocktail, but its runtime could be long. ExhaustivePhi only focuses on one cocktail size, the one estimated as the best; thus, its runtime is less than ExhaustiveSearch, but it can produce cocktails with more phages than necessary. ClusteringSearch and ClusteringPhi are very fast (generally, less than one millisecond), providing always immediate results due to clustering techniques, but their accuracies can be lower, yielding cocktails with lower expected successes. Conclusions: The larger the phage-bacteria infection network is, the more complex its analysis is. Thus, this tool eases this task for scientists and other users while designing phage cocktails of good quality. This R package includes four different methods; therefore, users may choose among them, considering their preferences in speed and accuracy of results. (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

In recent years, the spread of antibiotic-resistant bacteria and efforts to preserve food microbiota have induced renewed interest in phage therapy. Phage cocktails, instead of a single phage, are commonly used as antibacterial agents since the hosts are unlikely to become resistant to several phages simultaneously. While the spectrum of activity might increase with cocktail complexity, excessive phages could produce side effects, such as the horizontal transfer of genes that augment the fitness of host strains, dysbiosis or high manufacturing costs. Therefore, cocktail formulation represents a compromise between achieving substantial reduction in the bacterial loads and restricting its complexity. Despite the abovementioned points, the observed bacterial load reduction does not increase significantly with the size of phage cocktails, indicating the requirement for a systematic approach to their design. In this work, the information provided by host range matrices was analyzed after building phage-bacteria infection networks (PBINs). To this end, we conducted a meta-analysis of 35 host range matrices, including recently published studies and new datasets comprising Escherichia coli strains isolated during ripening of artisanal raw milk cheese and virulent coliphages from ewes’ feces. The nestedness temperature, which reflects the host range hierarchy of the phages, was determined from bipartite host range matrices using heuristic (Nestedness Temperature Calculator) and genetic (BinMatNest) algorithms. The latter optimizes matrix packing, leading to lower temperatures, i.e., it simplifies the identification of the phages with the broadest host range. The structure of infection networks suggests that generalist phages (and not specialist phages) tend to succeed in infecting less susceptible bacteria. A new metric (Φ), which considers some properties of the host range matrices (fill, temperature, and number of bacteria), is proposed as an estimator of phage cocktail size. To identify the best candidates, agglomerative hierarchical clustering using Ward’s method was implemented. Finally, a cocktail was formulated for the biocontrol of cheese-isolated E. coli, reducing bacterial counts by five orders of magnitude.

Bacteriophages are highly specific predators that drive bacterial diversity through coevolution while striking tradeoffs among preserving host populations for long-term exploitation and increasing their virulence, structural stability, or host range. Escherichia coli and other coliform bacteria present in the microbiota of milk and during early ripening of raw milk cheeses have been linked to the production of gas, manifested by the appearance of eyes, and the development of off-flavors; thus, they might cause early blowing and cheese spoilage. Here, we report the characterization of coliphages isolated from manure from small ruminant farms and E. coli strains isolated from goat and sheep raw milk cheese. Additionally, the virulence and host range of locally isolated and laboratory collection phages were determined by comparing the susceptibility of E. coli strains from different sources. In agreement with the high genetic diversity found within the species E. coli, clustering analysis of whole-cell protein revealed a total of 13 distinct profiles but none of the raw milk cheese isolates showed inhibition of growth by reference or water-isolated coliphages. Conversely, 10 newly isolated phages had a broad host range (i.e., able to lyse ≥50% of bacterial hosts tested), thus exhibiting utility for biocontrol and only one cheese-isolated E. coli strain was resistant to all the phages. Whereas there was a high positive correlation between bacterial susceptibility range and lysis intensity, the phages virulence decreased as range increased until reaching a plateau. These results suggest local gene-for-gene coevolution between hosts and phages with selective tradeoffs for both resistance and competitive ability of the bacteria and host-range extension and virulence of the phage populations. Hence, different phage cocktail formulations might be required when devising long-term and short-term biocontrol strategies.

Cell‐free extracts from 157 Lactococcus lactis strains isolated from Artisan cheese were screened by protein fingerprinting previous to their technological characterisation. The strains were classified according to their electrophoretic patterns into five groups. A set of strains representing the different clusters were selected to study their acidifying activity in milk. Time and rate feature points, as well as the shape of the acidification curves, resulted in six different fermentation kinetics, mostly consistent with the electrophoretic groups. Thus, selection of native strains as starter cultures based on their acidifying activity could be optimised by protein fingerprinting.

The aim of this study was to identify the main Enterobacteriaceae species responsible for early gas blowing during curdling and the first week of ripening in raw goats' milk cheese. Two batches of raw goats' milk cheese were selected. One of them showed early blowing within the first 24 h of cheese ripening while the other showed no alteration. Although initial levels of Enterobacteriaceae were similar in defective and non-defective cheese, their dynamics (growth and disappearance rates of the species detected) were different. Klebsiella oxytoca and Enterobacter cloacae were the main species in the defective curd, whereas Buttiauxela spp. was predominant in normal curd. Hafnia alvei was the prevailing isolated species for both normal and defective cheese throughout the ripening process. The highest gas production was rendered by K. oxytoca and H. alvei, mainly isolated from curd and cheese. However, other species relevant in milk or curd, like Pantoea ssp. or Buttiauxela spp. were considered as low gas producers. The analysis of digitalized images of cheese showed that most of the cheese eyes were formed before the first week of ripening, although this process continued during maturation.According to the species found in the defective and non-defective cheese, their proportions at different ripening stages, their ability to produce gas and eye formation, K. oxytoca might be considered the most likely responsible for early blowing in raw goats' milk cheeses; while H. alvei increased the eyes number in the later stages of the ripening period.

The aim of this work was to identify the main species of Enterobacteriaceae present in two different varieties of raw ewe's milk cheese, to study their evolution during manufacturing and ripening, and to assess their potential to produce gas from lactose. Enterobacteriaceae activity may influence cheese ripening; it can even be responsible for some specific characteristics of artisanal cheeses. Nevertheles some of these microorganisms are potential pathogens. Enterobacteriaceae growth and disappearance were different between the soft and the semi-hard cheese variety, as well as the proportions of the species detected. Citrobacter freundii, Citrobacter braakii and Klebsiella oxytoca were the predominant species in milk and curd, whereas Hafnia alvei was predominant throughout ripening. The highest gas production was generated by Enterobacter nimipresuralis, C. braakii and Enterobacter cloacae strains, followed by Enterobacter aerogenes, H. alvei, K. oxytoca and C. freundii strains. According to the species found, their levels during the initial ripening stages and gas production ability, C. braakii, C. freundii, K. oxytoca and H. alvei can be considered as responsible for early blowing in soft and semi-hard ewe cheeses. The identification of the Enterobacteriaceae species most likely responsible for early blowing can lead to a better understanding of raw milk cheese ripening, and therefore, to the improvement of its quality. (C) 2016 Elsevier B.V. All rights reserved.

Objective To propose how to incorporate equity issues, using the GRADE approach, into the development and implementation of Colombian Clinical Practice Guidelines. Methodology This proposal was developed in four phases: 1. Included a literature review and the development of a preliminary proposal about how to include equity issues; 2. Involved an informal discussion to reach a consensus on improving the first proposal; 3. Was a survey of the researchers' acceptance levels of the proposal, and; 4. A final informal consensus was formed to adjust the proposal. Results A proposal on how to incorporate equity issues into the GRADE approach was developed. It places particular emphasis on the recognition of disadvantaged populations in the development and implementation of the suggested guideline. PROGRESS-Plus is recommended for use in exploring the various categories of disadvantaged people. The proposal suggests that evidence be rated differentially by giving higher ratings to studies that consider equity issues than those that do not. The proposal also suggests the inclusion of indicators to monitor the impacts of the implementation of CPGs on disadvantaged people. Conclusions A consideration of equity in the development and implementation of clinical practice guidelines and quality assessments of the evidence would achieve more in the participation of potential actors in the process and reflect on the effectiveness of the proposed interventions across all social groups.

BACKGROUND:The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified.RESULTS:In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively.CONCLUSIONS:The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Evolution is the pivotal principle that biologists use to understand life. However, due to its multidisciplinary nature, teaching Evolutionary Biology is a challenging task. A notorious difficulty in teaching and learning evolution is the coexistence of alternative theories such as those concerned with altruistic behavior. Here the implementation of the jigsaw classroom, a cooperative learning technique developed by Elliot Aronson in the 1970s, is applied to study the correlation between the levels of selection and social behavior. The setup is aimed at undergraduate students, but also intended for other levels. Briefly, the class is divided into groups of up to six students. In each jigsaw group, each student is assigned the role of a scientist who supports a different theory: R. Dawkins, C. Darwin, V. C. Wynne Edwards, W. D. Hamilton, R. Trivers and D. Sloan Wilson. The main endeavor of the groups is to reconcile the alternative theories. To increase the chance of students grasping the assigned theory, meetings of "experts"are organized. This teams of specialists consist of all the students representing the same scientist and must discuss and gather information about: 1) the postulates of the corresponding theory, 2) a practical example to which the theory can easily be applied and 3) strengths and weaknesses. Measures of attention to diversity and a sequence of activities are proposed.

The cell cycle of slowly growing bacteria consists of three phases that resemble the eukaryotic cell cycle: the pre initiation, replication and post-replication periods, termed B, C and D, respectively. However, bacteria are also capable of very rapid growth, whereas the replication period remains long and hence the B period disappears and a round of replication is triggered before the previous one is finished. Several frequently used methods yield an incomplete analysis of these rapidly growing bacterial cultures. However, DNA distributions obtained by flow cytometry can be used to fully unravel the bacterial cell cycle independently of the duration of each period. In the present work, we have compared the accuracy of the cell cycle parameters estimation in different cultures. Whereas, for a given DNA per cell content, the accuracy of the C period determination by flow cytometry is essentially independent of the cell cycle, the precision of the D period estimation is inversely correlated with the number of replication forks per chromosome. We have established a data analysis routine to estimate the accuracy of the cell cycle parameters determination.