Bacteriophages as Biotechnological Tools.

Bacteriophages are ubiquitous organisms that can be specific to one or multiple strains of hosts, in addition to being the most abundant entities on the planet. It is estimated that they exceed ten times the total number of bacteria. They are classified as temperate, which means that phages can integrate their genome into the host genome, originating a prophage that replicates with the host cell and may confer immunity against infection by the same type of phage; and lytics, those with greater biotechnological interest and are viruses that lyse the host cell at the end of its reproductive cycle. When lysogenic, they are capable of disseminating bacterial antibiotic resistance genes through horizontal gene transfer. When professionally lytic-that is, obligately lytic and not recently descended from a temperate ancestor-they become allies in bacterial control in ecological imbalance scenarios; these viruses have a biofilm-reducing capacity. Phage therapy has also been advocated by the scientific community, given the uniqueness of issues related to the control of microorganisms and biofilm production when compared to other commonly used techniques. The advantages of using bacteriophages appear as a viable and promising alternative. This review will provide updates on the landscape of phage applications for the biocontrol of pathogens in industrial settings and healthcare.

Plasmid-mediated Kluyvera-like arnBCADTEF operon confers colistin (hetero)resistance to Escherichia coli.

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB system and by still unknown mechanisms. This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to strains with mutations that confer colistin resistance. The expression of mcr genes negatively controls the transcription of the arnBCADTEF operon itself, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 62.1 Kb encoding an extra copy of the arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, called pArnT1, was cured by ethidium bromide and the cells lost resistance to colistin in parallel. Furthermore, a susceptible E. coli strain acquired heteroresistance after transformation with pArnT1 or pBAD24 carrying the Kluyvera-like arnBCADTEF operon, revealing it as a new colistin resistance determinant.

Risk assessment of enteric viruses along the food chain and in the population.

Food-borne microbial illness contributes up to one third of global disease burden. The largest category of food-borne illness is gastroenteritis, the majority of which is caused by enteric viruses. Viruses like these are transmitted to food either by waste-contaminated waters, or by handling and transfer during processing. An important tool for reducing or controlling food-borne microbial risk is risk analysis. This framework has been adopted globally to manage risks associated with microbial contamination in food. Several hundred microbial risk assessments (MRAs) have been published by different national and international organisations, for different food-hazard combinations. The use of MRAs in controlling and understanding virus risk has, to date, been limited, compared with the efforts made on bacterial pathogens. Given the large disease burden that viruses are responsible for, this disparity should be addressed. The main reasons for the relative lack of risk assessments are the difficulty in detecting and monitoring viruses compared with bacteria. This means less data on prevalence, concentration and inactivation, and allows viruses to remain silent contributors to global disease. There are also key conceptual differences between virus risk assessment and bacterial risk assessment. This project aimed to assess the current state of the art for food-borne virus risk assessment, then to progress the field further by using the data available to produce risk rankings and risk assessments. This was done by a combination of literature reviewing and various risk assessment tools. The result was an assessment of the overall evidence base in the literature, a semi-quantitative ranking comparison between the viruses and foods of most concern, and a survey of inactivation methods, leading to a quantitative ranking of the effectiveness of each in reducing and managing food-borne virus risk.

Characterization of Planktochlorella nurekis Extracts and Virucidal Activity against a Coronavirus Model, the Murine Coronavirus 3.

Certain members of the Coronaviridae family have emerged as zoonotic agents and have recently caused severe respiratory diseases in humans and animals, such as SARS, MERS, and, more recently, COVID-19. Antivirals (drugs and antiseptics) capable of controlling viruses at the site of infection are scarce. Microalgae from the Chlorellaceae family are sources of bioactive compounds with antioxidant, antiviral, and antitumor activity. In the present study, we aimed to evaluate various extracts from Planktochlorella nurekis in vitro against murine coronavirus-3 (MHV-3), which is an essential human coronavirus surrogate for laboratory assays. Methanol, hexane, and dichloromethane extracts of P. nurekis were tested in cells infected with MHV-3, and characterized by UV-vis spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), and the application of chemometrics through principal component analysis (PCA). All the extracts were highly efficient against MHV-3 (more than a 6 Log unit reduction), regardless of the solvent used or the concentration of the extract, but the dichloromethane extract was the most effective. Chemical characterization by spectrophotometry and NMR, with the aid of statistical analysis, showed that polyphenols, carbohydrates, and isoprene derivatives, such as terpenes and carotenoids have a more significant impact on the virucidal potential. Compounds identified by UPLC-MS were mainly lipids and only found in the dichloromethane extract. These results open new biotechnological possibilities to explore the biomass of P. nurekis; it is a natural extract and shows low cytotoxicity and an excellent antiviral effect, with low production costs, highlighting a promising potential for development and implementation of therapies against coronaviruses, such as SARS-CoV-2.

Exploiting 16S rRNA-based metagenomics to reveal neglected microorganisms associated with infertility in breeding bulls in Spanish extensive herds.

Bovine infectious infertility represents a problem due to the high impact on animal production and, in many cases, in public health. A lack of information on the characteristics of the bacterial population of the bovine reproductive system can hamper a comprehensive understanding of reproductive pathologies and the role that the microbiome could play. A metagenomic study based on the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed in 1029 preputial samples from bulls raised in an extensive regimen in Spain (944 from herds with low fertility rates -case group-, and 85 samples from reproductively healthy herds -control group-). The most representative phyla as well as the most 10 abundant bacterial families and their abundance did not show significant differences in both case and control groups. Similarly, the (alpha and beta) diversity of the bacterial populations was similar in both type of herds: the Shannon and Simpson indices show a high diversity of species, while the Bray-Curtis dissimilarity index did not show relevant differences in the bacterial communities. A deeper analysis of the operational taxonomic units showed the presence of one genera, Mycoplasma spp. significantly associated with fertility problems. Our study highlights the promising potential that the application of sequencing techniques (e.g. 16S rRNA-based metagenomics) possesses in examining bovine infertility, as they are able to reveal different pathogens that could go unnoticed using diagnostic approaches for only the main known pathogens.

Enteric viruses in food safety: New threats for an old problem.

Foodborne diseases are one of the most serious concerns in public health. It is estimated that around 600 million cases of gastroenteritis occur worldwide each year. At present, more than 200 food-borne diseases are known, which can cause from mild gastroenteritis to syndromes with a fatal outcome, with the added possibility of chronic complications. One of the major etiological agents in foodborne diseases are the food and waterborne viruses, which are attracting a great deal of attention to researchers, food hygienists and policy makers. Several aspects differentiate these pathogens from foodborne pathogenic bacteria: their high capacity for infection and preservation in food environments, and their difficulty for a correct and sensitive detection. In recent years, different initiatives have been carried out to prioritize research in the area of viruses in food, prioritizing different aspects of their detection, epidemiology and control. There is clear evidence that the existing data on their prevalence may be underestimated due to the lack of robust methods for their sensitive detection. It is also necessary to know exactly what the incidence is in the different stages of the food production chain, and particularly in that which is dedicated to the transformation of products of animal origin. Finally, it is also necessary to calibrate the current disinfection procedures in the food industry in order to reliably establish a quantitative evaluation of the viral risk in food.

Listeria monocytogenes survives better at lower storage temperatures in regular and low-salt soft and cured cheeses.

The behaviour of Listeria monocytogenes was investigated in soft pasteurized milk cheese elaborated with different salt concentrations (1.17 and 0.30% w/w) and in cured raw sheep milk cheese over storage up to 189 days at different isothermal conditions. Commercial 25-g cheese samples were inoculated with a 4-strain cocktail of L. monocytogenes (serovars 4b, 1/2a, 1/2b and 1/2c) at approximately 104 CFU/g. The inoculated samples were stored at 4 and 22 °C and withdrawn at proper intervals for L. monocytogenes enumeration. The prevalence of the different serovar strains of L. monocytogenes was characterized on soft cheese samples over storage at 4 °C using multiplex PCR. Salt reduction did not affect the survival of L. monocytogenes in soft cheeses and a maximum of 1-log reduction was observed in both regular and low-salt cheeses after 189 days of storage at 4 °C. The pathogen showed greater survival capacity in both soft and cured cheeses during storage at 4 °C compared to the storage at 22 °C, where more than 2.5 log reductions were computed. The fate of L. monocytogenes was described through a Weibull model fitted to survival data. The time required for a first tenfold reduction of the L. monocytogenes population (δ) at 4 °C is around 150 days in soft and 72 days in cured cheeses. At 22 °C, the estimated δ values are at least 60% lower in both cheese types. Among the four L. monocytogenes serovars present in the inoculated cocktail, the serovar 4b strain was the most sensitive to refrigerated storage, while the prevalence of serovar 1/2c strain increased over time in soft cheeses. Overall, the data obtained in this study help to deepen knowledge into factors affecting L. monocytogenes behaviour on cheeses and evidenced the variability between serovars in terms of survival capacity, which may be considered when performing microbial risk assessments.

Longitudinal study of the mcr-1 gene prevalence in Spanish food-producing pigs from 1998 to 2021 and its relationship with the use of polymyxins.

BACKGROUND: Resistance to colistin was an uncommon phenomenon traditionally linked to chromosome point mutations, but since the first description of a plasmid-mediated colistin-resistance in late 2015, transmissible resistance to colistin has become a Public Health concern. Despite colistin is considered as a human last resort antibiotic, it has been commonly used in swine industry to treat post-weaning diarrhoea in piglets. However, the progressively increase of colistin resistance during the last decade led to the Spanish Medicines and Healthcare Products Agency (AEMPS) to launch a strategic and voluntary plan aimed to reduce colistin consumption in pig production. Our longitudinal study (1998-2021) aimed to evaluate the trend of colistin resistance mediated through the mcr-1 mobile gene in Spanish food-producing pig population and compare it with published polymyxin sales data in veterinary medicine to assess their possible relationships. RESULTS: The first mcr-1 positive sample was observed in 2004, as all samples from 1998 and 2002 were mcr-1 PCR-negative. We observed a progressive increase of positive samples from 2004 to 2015, when mcr-1 detection reached its maximum peak (33/50; 66%). From 2017 (27/50; 54%) to 2021 (14/81; 17%) the trend became downward, reaching percentages significantly lower than the 2015 peak (p < 0.001). The abundance of mcr-1 gene in PCR-positive samples showed a similar trend reaching the highest levels in 2015 (median: 6.6 × 104 mcr-1 copies/mg of faeces), but decreased significantly from 2017 to 2019 (median 2.7 × 104, 1.2 × 103, 4.6 × 102 mcr-1 copies/mg of faeces for 2017, 2018 and 2019, respectively), and stabilizing in 2021 (1.6 × 102 mcr-1 copies/mg of faeces) with similar values than 2019. CONCLUSIONS: Our study showed the decreasing trend of colistin resistance associated to mcr-1 gene, after a previous increase from among 2004-2015, since the European Medicines Agency and AEMPS strategies were applied in 2016 to reduce colistin use in animals, suggesting a connection between polymyxin use and colistin resistance. Thus, these plans could have been effective in mcr-1 reduction, reaching lower levels than those detected in samples collected 17 years ago, when resistance to colistin was not yet a major concern.

Classical and Next-Generation Vaccine Platforms to SARS-CoV-2: Biotechnological Strategies and Genomic Variants.

Several coronaviruses (CoVs) have been identified as human pathogens, including the α-CoVs strains HCoV-229E and HCoV-NL63 and the ß-CoVs strains HCoV-HKU1 and HCoV-OC43. SARS-CoV, MERS-CoV, and SARS-CoV-2 are also classified as ß-coronavirus. New SARS-CoV-2 spike genomic variants are responsible for human-to-human and interspecies transmissibility, consequences of adaptations of strains from animals to humans. The receptor-binding domain (RBD) of SARS-CoV-2 binds to receptor ACE2 in humans and animal species with high affinity, suggesting there have been adaptive genomic variants. New genomic variants including the incorporation, replacement, or deletion of the amino acids at a variety of positions in the S protein have been documented and are associated with the emergence of new strains adapted to different hosts. Interactions between mutated residues and RBD have been demonstrated by structural modelling of variants including D614G, B.1.1.7, B1.351, P.1, P2; other genomic variants allow escape from antibodies generated by vaccines. Epidemiological and molecular tools are being used for real-time tracking of pathogen evolution and particularly new SARS-CoV-2 variants. COVID-19 vaccines obtained from classical and next-generation vaccine production platforms have entered clinicals trials. Biotechnology strategies of the first generation (attenuated and inactivated virus-CoronaVac, CoVaxin; BBIBP-CorV), second generation (replicating-incompetent vector vaccines-ChAdOx-1; Ad5-nCoV; Sputnik V; JNJ-78436735 vaccine-replicating-competent vector, protein subunits, virus-like particles-NVX-CoV2373 vaccine), and third generation (nucleic-acid vaccines-INO-4800 (DNA); mRNA-1273 and BNT 162b (RNA vaccines) have been used. Additionally, dendritic cells (LV-SMENP-DC) and artificial antigen-presenting (aAPC) cells modified with lentiviral vector have also been developed to inhibit viral activity. Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.

SARS-CoV-2 in Human Sewage and River Water from a Remote and Vulnerable Area as a Surveillance Tool in Brazil.

In the present study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was monitored in environmental samples from rural and vulnerable areas (a presidio, worker accommodation units, and river waters upstream and downstream of a rural community) from Minas Gerais State region, Southern Brazil, in August 2020. The sampling was performed prior to official declaration of the coronavirus disease (COVID-19) cases in those sites. SARS-CoV-2 RNA was detected in the presidio and workers accommodation units (3.0 × 104 virus genome copies (GC)/mL and 4.3 × 104 GC/mL of sewage, respectively). While SARS-CoV-2 was not detected in the river water upstream of the rural community, SARS-CoV-2 RNA was detected in downstream river waters (1.1 × 102 SARS-CoV-2 GC/mL). The results obtained in this study highlight the utility of SARS-CoV-2 monitoring in wastewater and human sewage as a non-invasive early warning tool to support health surveillance in vulnerable and remote areas, particularly in development countries.

Uses of Bacteriophages as Bacterial Control Tools and Environmental Safety Indicators.

Bacteriophages are bacterial-specific viruses and the most abundant biological form on Earth. Each bacterial species possesses one or multiple bacteriophages and the specificity of infection makes them a promising alternative for bacterial control and environmental safety, as a biotechnological tool against pathogenic bacteria, including those resistant to antibiotics. This application can be either directly into foods and food-related environments as biocontrol agents of biofilm formation. In addition, bacteriophages are used for microbial source-tracking and as fecal indicators. The present review will focus on the uses of bacteriophages like bacterial control tools, environmental safety indicators as well as on their contribution to bacterial control in human, animal, and environmental health.

Tularemia Outbreaks in Spain from 2007 to 2020 in Humans and Domestic and Wild Animals.

In this study, tularemia outbreaks associated with humans and several domestic and wild animals (Iberian hares, wild rabbits, voles, mice, grey shrews, sheep, dogs, foxes, wolves, ticks, and river crayfish) are reported in Spain from 2007 to 2020. Special attention was paid to the outbreaks in humans in 2007-2009 and 2014-2015, when the most important waves occurred. Moreover, positive rates of tularemia in lagomorphs were detected in 2007-2010, followed by negative results in 2011-2013, before again returning to positive rates in 2014 and in 2017 and in 2019-2020. Lagomorphs role in spreading Francisella tularensis in the epidemiological chain could not be discarded. F. tularensis is described for the first time infecting the shrew Crocidura russula worldwide, and it is also reported for the first time infecting wild rabbits (Oryctolagus cuniculus) in Spain. Serological positives higher than 0.4% were seen for sheep only from 2007-2009 and again in 2019, while serological rates greater than 1% were revealed in dogs in 2007-2008 and in wild canids in 2016. F. tularensis were detected in ticks in 2009, 2014-2015, 2017, and 2019. Lastly, negative results were achieved for river crayfish and also in environmental water samples from 2007 to 2020.

In Vitro Antimicrobial Susceptibilities of Francisella tularensis subsp. holarctica Isolates from Tularemia Outbreaks That Occurred from the End of the 20th Century to the 2020s in Spain.

A collection of 177 Francisella tularensis subsp. holarctica clinical isolates (29 from humans and 148 from animals, mainly hares and voles) was gathered from diverse tularemia outbreaks in the Castilla y León region (northwestern Spain) that occurred from the end of the 20th century to the 2020s. Along with four F. tularensis subsp. holarctica reference strains, all of these clinical isolates were tested using a broth microdilution method to determine their susceptibility to 22 antimicrobial agents, including ß-lactams, aminoglycosides and one member each of the tetracycline, glycylcycline, quinolone and sulphonamide classes. Many multi-resistance profiles were found among the tested isolates, but especially among those of human origin (all but two isolates showed resistance to at least 13 of 18 antimicrobial agents). Even so, all human isolates were susceptible to gentamicin and tobramycin, while more than 96% of animal isolates were susceptible to these two aminoglycosides. Ciprofloxacin showed activity against more than 92% of animal and human isolates. However, almost 21% of human isolates were resistant to tetracycline, and more than 65% were resistant to tigecycline. Finally, a quite similar activity to other F. tularensis subsp. holarctica isolates collected 20 years earlier in Spain was observed.

Salmonella enterica Serovar Enteritidis Control in Poultry Litter Mediated by Lytic Bacteriophage Isolated from Swine Manure.

We report the use of bacteriophages for control of Salmonella Enteritidis in poultry production. Phage was isolated by the double-agar plate assay from agricultural waste samples, and one isolate, named SM1, was selected and propagated for application in poultry litter. Two experimental protocols were tested: single treatment and repeated treatment (re-application of phage SM1 after 6 h and 12 h). Each treatment cycle involved 25 g of poultry litter placed in plastic boxes and contaminated with 105 Colony Forming Units mL-1 (CFU mL-1) of S. Enteritidis, in independent duplicates. The contaminated litter was treated with 106 Plaque Forming Units mL-1 (PFU mL-1) of SM1 phage by dripping. Repeated application of phage SM1 reduced Salmonella counts by over 99.9%; the phage persisted in poultry litter for over 35 days. This study illustrates the application of SM1 treatment as a promising technology for bacterial control in production matrices that could allow safe and sustainable use of agricultural waste products as biofertilizers.

Detection of Enteric Viruses and Core Microbiome Analysis in Artisanal Colonial Salami-Type Dry-Fermented Sausages from Santa Catarina, Brazil.

Microbial fermentation plays an important role in the manufacturing of artisanal sausages and can have major effects on product quality and safety. We used metagenomics and culture-dependent methods to study the presence of Hepatitis E virus (HEV) and Rotavirus-A (RV-A), and fungal and bacterial communities, in artisanal Colonial salami-type dry-fermented sausages in Santa Catarina state, Brazil. Lactic acid bacteria (LAB) and yeast dominated the microbiome. Latilactobacillus sakei and Debaryomyces hansenii were ubiquitous and the most abundant species. The DNA of some foodborne pathogens was found in very low concentrations although viable cells of most of these species were undetectable by cultivation methods. The characteristics of the raw material and hygiene of the artisanal sausage manufacturing process resulted in high loads of beneficial microorganisms and the absence of HEV and RV-A viruses as determined by RT-qPCR assays. In conclusion, high LAB load in sausages was more relevant to preventing pathogen growth than the ripening time and/or physicochemical characteristics. However, the presence of Clostridium spp. and other pathogens in some samples must be taken into account for the development of future preservation methods; appropriate LAB starter cultures and health surveillance are required in the production process to prevent foodborne outbreaks.

Bioactive Compounds from Mangrove Endophytic Fungus and Their Uses for Microorganism Control.

Mangroves are ecosystems with unique characteristics due to the high salinity and amount of organic matter that house a rich biodiversity. Fungi have aroused much interest as they are an important natural source for the discovery of new bioactive compounds, with potential biotechnological and pharmacological interest. This review aims to highlight endophytic fungi isolated from mangrove plant species and the isolated bioactive compounds and their bioactivity against protozoa, bacteria and pathogenic viruses. Knowledge about this type of ecosystem is of great relevance for its preservation and as a source of new molecules for the control of pathogens that may be of importance for human, animal and environmental health.

Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota.

Colistin has a long story of safe use in animals for the treatment and prevention of certain bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants. In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin (600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day 15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1 that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4 animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria reach the gut.

Evaluation of PCR assays for Campylobacter fetus detection and discrimination between C. fetus subspecies in bovine preputial wash samples.

Campylobacter fetus is a zoonotic pathogen found in cattle, in which it is one of the main causes of infectious infertility. Most diagnostic laboratories use PCR as quick easy tool for C. fetus identification. However, there is no standardized PCR assay for C. fetus detection and subspecies differentiation, hindering the comparison of results. In this study, we evaluated selected PCR assays targeting the 16S rRNA, gyrB, cpn60, cstA, cdtB and nahE genes for C. fetus identification and ISCfe1, sapB2, parA and virB11 for subspecies differentiation. Analytical sensitivity and specificity were assessed for each PCR assay, and the assays were then tested on 289 bull preputial samples that had also been analysed by 16S rRNA barcode metagenomics. In total, 41 C. fetus-positive samples were included. The P12 PCR assay targeting the gyrB gene performed best, detecting the pathogen in 95.1% of positive samples. For the discrimination of C. fetus subspecies, we were able to identify a proportion (85.4%) of the C. fetus-positive samples correctly as C. fetus venerealis with at least one subspecies-specific PCR, but C. fetus fetus was not detected in any of the samples tested. Remarkably, C. fetus subspecies amplification was observed following PCR on some samples (33.1%) considered C. fetus-negative, highlighting the need for rigorous criteria for discriminating between C. fetus subspecies, to improve understanding of the role of the two C. fetus subspecies in the epidemiology and pathogenesis of bovine infectious infertility.