The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the ß(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Concentrations of Campylobacter spp., Escherichia coli, Enterococci, and Yersinia spp. in the Feces of Farmed Red Deer in New Zealand.

Intensive deer farming can cause environmental issues, mainly by its impact on soils and water quality. In particular, there is a risk to the microbial quality of water, as high quantities of suspended sediment and fecal bacteria can enter into water systems. The feces of farmed red deer (, = 206) from Canterbury and Southland, New Zealand, were analyzed with regard to the presence of spp., , enterococci, and spp.. Enterococci and were isolated from all samples, with mean concentrations of 4.5 × 10 (95% CI 3.5 × 10, 5.6 10) and 1.3 × 10 (95% CI 1.1 × 10, 1.5 × 10) per gram of dry feces, respectively. spp. were isolated from 27 fecal samples, giving an overall prevalence of 13.1%. isolation rates were variable within and between regions (Canterbury 7.95% [95% CI 2-14%], Southland 16.95% [95% CI 10-24%]). Five out of 42 composite samples were positive for , and one sample for The overall prevalence ranges on a per-animal basis were therefore 2.43 to 11.17% and 0.49 to 2.91%, respectively. This study is the first to quantify the concentration of spp. present in healthy deer farmed in New Zealand. Deer feces are a potential source of human campylobacteriosis, with all genotypes isolated also previously observed among human cases. The fecal outputs from deer should be regarded as potentially pathogenic to humans and therefore be appropriately managed.

Host range and in vitro lysis of Listeria monocytogenes seafood isolates by bacteriophages.

Listeria-infecting bacteriophages (listeriaphages) can be used to control Listeria monocytogenes in the food industry. However, the sensitivity of many of seafood-borne Listeria strains to phages has not been reported. This research investigated the host ranges of three listeriaphages (FWLLm1, FWLLm3 and FWLLm5) by the formation of lytic zones and plaques on host lawns and in vitro lysis kinetics of listeriaphage FWLLm3. The study also predicted the phage titres required to lyse host cells. The host ranges of the phages were determined using 50 L. monocytogenes strains, of which 48 were isolated from the seafood industry and two from clinical cases. Of the 50 strains, 36 were tested at 25 and 30 ℃ and the remainder (14) at 15 and 25 ℃. Based on the formation of either discrete plaques or lytic zones (host kill zones), the host ranges of FWLLm1, FWLLm3 and FWLLm5 were about 87%, 81% and 87%, respectively, at 25 ℃. Six L. monocytogenes strains from the seafood environment were insensitive to all three phages, while the other seafood strains (42) were phage-sensitive. The adsorption rate constant (k value) of listeriaphage FWLLm3 was between 1.2 × 10(-9) and 1.6 × 10(-9 )ml/min across four host strains in tryptic soy broth at 25 ℃. The cultures (at 3-4 log colony-forming unit (CFU/ml) were completely lysed (<1 log CFU/ml) when cultures were infected with FWLLm3 at > 8.7 log phage-forming units (PFU/ml) for 30 min. Re-growth of phage-infected cultures was not detected after 24 h. The effective empirical phage titre was similar to the calculated titre using a kinetic model. Results indicate the potential use of the three phages for controlling L. monocytogenes strains in seafood processing environments.

Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Nonbacterial Microflora in Wastewater Treatment Plants: an Underappreciated Potential Source of Pathogens.

Wastewater treatment plants (WWTPs) receive and treat large volumes of domestic, industrial, and urban wastewater containing pathogenic and nonpathogenic microorganisms, chemical compounds, heavy metals, and other potentially hazardous substances. WWTPs play an essential role in preserving human, animal, and environmental health by removing many of these toxic and infectious agents, particularly biological hazards. Wastewater contains complex consortiums of bacterial, viral, archaeal, and eukaryotic species, and while bacteria in WWTP have been extensively studied, the temporal and spatial distribution of nonbacterial microflora (viruses, archaea, and eukaryotes) is less understood. In this study, we analyzed the viral, archaeal, and eukaryotic microflora in wastewater throughout a treatment plant (raw influent, effluent, oxidation pond water, and oxidation pond sediment) in Aotearoa (New Zealand) using Illumina shotgun metagenomic sequencing. Our results suggest a similar trend across many taxa, with an increase in relative abundance in oxidation pond samples compared to influent and effluent samples, except for archaea, which had the opposite trend. Additionally, some microbial families, such as Podoviridae bacteriophages and Apicomplexa alveolates, appeared largely unaffected by the treatment process, with their relative abundance remaining stable throughout. Several groups encompassing pathogenic species, such as Leishmania, Plasmodium, Toxoplasma, Apicomplexa, Cryptococcus, Botrytis, and Ustilago, were identified. If present, these potentially pathogenic species could be a threat to human and animal health and agricultural productivity; therefore, further investigation is warranted. These nonbacterial pathogens should be considered when assessing the potential for vector transmission, distribution of biosolids to land, and discharge of treated wastewater to waterways or land. IMPORTANCE Nonbacterial microflora in wastewater remain understudied compared to their bacterial counterparts despite their importance in the wastewater treatment process. In this study, we report the temporal and spatial distributions of DNA viruses, archaea, protozoa, and fungi in raw wastewater influent, effluent, oxidation pond water, and oxidation pond sediments by using shotgun metagenomic sequencing. Our study indicated the presence of groups of nonbacterial taxa which encompass pathogenic species that may have potential to cause disease in humans, animals, and agricultural crops. We also observed higher alpha diversity in viruses, archaea, and fungi in effluent samples than in influent samples. This suggests that the resident microflora in the wastewater treatment plant may be making a greater contribution to the diversity of taxa observed in wastewater effluent than previously thought. This study provides important insights to better understand the potential human, animal, and environmental health impacts of discharged treated wastewater.

Systematic review and meta-analysis: the efficiency of bacteriophages previously patented against pathogenic bacteria on food.

Food-borne diseases are a global public health issue with 1 in 10 people falling ill after eating contaminated food every year. In response, the food industry has implemented several new pathogen control strategies, such as biotechnological tools using the direct application of bacteriophages for biological control. We have undertaken a systematic review and meta-analysis that evaluated the efficiency of patented phages as a biological control for food-borne pathogens and determined the physical-chemical characteristics of the antimicrobial effect. Included and excluded criteria was developed. Included criteria: Phage patent files with an application in biological control on food and scientific articles and book chapters that used phages patented for food biological control. Excluded criteria: Patent documents, scientific articles, and book chapters that included phage therapy in humans, animals, and biological control on plants but did not have an application on food were not considered in our study. The systematic analysis identified 77 documents, 46 scientific articles, and 31 documents of patents and 23 articles was included in the meta-analysis. Listeria monocytogenes and Salmonella sp. comprised most of the targets identified in the screening, so that we focused on these strains to do the meta-analysis. There are a total of 383 and 192 experiments for Listeria and Salmonella phages for quantitative data analysis.Indexing databases for the bibliographic search (Scopus, Web of Science (WoS) and PubMed (Medline) were addressed by an automated script written in Python 3 Python Core Team (2015) and deposited on GitHub ( https://github.com/glenjasper ).A random-effects meta-analysis revealed (i) significant antimicrobial effect of Listeria phages in apple, apple juice, pear, and pear juice, (ii) significant antimicrobial effect of Salmonella phages in eggs, apple, and ready-to-eat chicken, (iii) no heterogeneity was identified in either meta-analysis, (iv) publication bias was detected for Listeria phages but not for Salmonella phages. (v) ListShield and Felix01 phages showed the best result for Listeria and Salmonella biological control, respectively, (vi) concentration of phage and bacteria, time and food had significant effect in the biological control of Listeria, (vii) temperature and time had a significant effect on the antimicrobial activity of Salmonella phages. The systematic review and meta-analyses to determine the efficiency of bacteriophages previously patented against pathogenic bacteria on dairy products, meat, fruits and vegetables. Besides, the discovering of key factors for efficacy, so that future applications of phage biotechnology in foods can be optimally deployed.

Genomic epidemiology and characterization of Staphylococcus aureus isolates from raw milk in Jiangsu, China: emerging broader host tropism strain clones ST59 and ST398.

Staphylococcus aureus is highly pathogenic and can cause disease in both humans and domestic animals. The aim of this study was to investigate the genomic epidemiology of S. aureus isolates from raw milk in Jiangsu Province, China, to identify predominant lineages and their associated genomic and phenotypic characteristics. In this study, we identified 117 S. aureus isolates collected from 1,062 samples in Jiangsu Province between 2021 and 2022. Based on whole-genome sequencing (WGS) data from 117 S. aureus isolates, molecular analyses indicated CC1-ST1 (26.50%, 31/117), CC97-ST97 (18.80%, 22/117), CC398-ST398 (10.26%, 12/117), CC8-ST630 (7.69%, 9/117) and CC59-ST59 (2.56%, 3/117) were the major lineages. The prevalence of mecA-positive strains was 11.11%. Four methicillin-resistant S. aureus (MRSA) lineages were found, including MRSA-ST59-t172 (n = 3), OS-MRSA-ST398-t011 (n = 1), MRSA-ST630-t2196 (n = 2) and OS-MRSA-ST630-t2196 (n = 7). Phenotypic resistance to penicillin (30.77%, 36/117), ciprofloxacin (17.09%, 20/117) and erythromycin (15.38%, 18/117) was observed which corresponded with resistance genotypes. All of the isolates could produce biofilms, and 38.46% (45/117) of isolates had invasion rates in mammary epithelial cells (MAC-T) of greater than 1%. Interestingly, most biofilm-producing and invading isolates harbored ebp-icaA-icaB-icaC-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map-can (27.35%, 32/117) and ebp-icaA-icaB-icaC-icaD-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map (33.33%, 39/117) adherence-associated gene patterns and belonged to lineages CC1 and CC97, respectively. Virulence factor assays showed that 47.01% of the isolates contained at least enterotoxin genes. Isolates harboring the immune evasion cluster (IEC) genes (sea, sak, chp, and scn) were predominantly categorized as STs 464, 398, and 59. IEC-positive ST398 and ST59 isolates contained a very high proportion of virulence genes located on prophages, whereas most IEC-negative ST398 clade isolates carried broad-spectrum drug resistance genes. Meanwhile, the IEC-positive ST398 clade showed a close genetic relationship with isolates from the pork supply chain and hospital surgical site infections. MRSA-ST59 strains showed the closest genetic relationship with an isolate from quick-frozen products. High-risk livestock-associated strains ST398 and MRSA-ST59 were detected in raw milk, indicating a potential public health risk of S. aureus transmission between livestock and humans. Our study highlights the necessity for S. aureus surveillance in the dairy industry.

Molecular Characterization of Salmonella Phage Wara Isolated from River Water in Brazil.

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture.

Isolation and lytic activity of the Listeria bacteriophage endolysin LysZ5 against Listeria monocytogenes in soya milk.

The endolysin gene (lysZ5) from the genome of the Listeria monocytogenes phage FWLLm3 was cloned in Escherichia coli and characterized. Comparative sequence analysis revealed that lysZ5 resembled the murein hydrolase ply511 encoded by L. monocytogenes phage A511. The encoded protein LysZ5 had a predicted molecular mass of 35.8 kDa and was expressed in E. coli as an N-terminal fusion protein of 41.5 kDa. Addition of purified fusion protein to lawns of indicator bacteria showed that LysZ5 could lyse L. monocytogenes, Listeria innocua and Listeria welshimeri, but not Staphylococcus aureus or Enterococcus faecalis. The purified protein was able to kill L. monocytogenes growing in soya milk, with the pathogen concentration reduced by more than 4 log10 CFU ml⁻¹ after 3 h incubation at 4 °C. As far as we know, this is the first report of a Listeria phage endolysin to control pathogens in soya milk and to demonstrate endolysin activity in foods at refrigeration temperatures. Moreover, LysZ5 may also be useful for biocontrol in other ready-to-eat foods.

Metagenomics Approaches for Improving Food Safety: A Review.

ABSTRACT: Advancements in next-generation sequencing technology have dramatically reduced the cost and increased the ease of microbial whole genome sequencing. This approach is revolutionizing the identification and analysis of foodborne microbial pathogens, facilitating expedited detection and mitigation of foodborne outbreaks, improving public health outcomes, and limiting costly recalls. However, next-generation sequencing is still anchored in the traditional laboratory practice of the selection and culture of a single isolate. Metagenomic-based approaches, including metabarcoding and shotgun and long-read metagenomics, are part of the next disruptive revolution in food safety diagnostics and offer the potential to directly identify entire microbial communities in a single food, ingredient, or environmental sample. In this review, metagenomic-based approaches are introduced and placed within the context of conventional detection and diagnostic techniques, and essential considerations for undertaking metagenomic assays and data analysis are described. Recent applications of the use of metagenomics for food safety are discussed alongside current limitations and knowledge gaps and new opportunities arising from the use of this technology.

Evaluation of Biopolymer Materials and Synthesis Techniques to Develop a Rod-Shaped Biopolymer Surrogate for Legionella pneumophila.

Biopolymer microparticles have been developed for applications that require biocompatibility and biodegradability, such as drug delivery. In this study, we assessed the production of microparticles using carnauba wax, κ-carrageenan, alginate, and poly (lactic-co-glycolic acid) (PLGA) with the aim of developing a novel, DNA-tracer-loaded, biopolymer surrogate with a size, shape, surface charge, and relative hydrophobicity similar to stationary-phase Legionella pneumophila to mimic the bacteria's mobility and persistence in engineered water systems. We found that the type and concentration of biopolymer, reaction conditions, and synthesis methods affected the morphology, surface charge, relative hydrophobicity, and DNA tracer loading efficiency of the biopolymer microparticles produced. Carnauba wax, κ-carrageenan, and alginate (Protanal®, and low and medium viscosity) produced highly polydisperse microspheres. In contrast, PLGA and alginate-CaCO3 produced uniform microspheres and rod-shaped microparticles, respectively, with high DNA tracer loading efficiencies (PLGA 70% and alginate-CaCO3 95.2 ± 5.7%) and high reproducibilities. Their synthesis reproducibility was relatively high. The relative hydrophobicity of PLGA microspheres closely matched the cell surface hydrophobicity of L. pneumophila but not the bacterial morphology, whereas the polyelectrolyte layer-by-layer assembly was required to enhance the relative hydrophobicity of alginate-CaCO3 microparticles. Following this surface modification, alginate-CaCO3 microparticles represented the best match to L. pneumophila in size, morphology, surface charge, and relative hydrophobicity. This new biopolymer surrogate has the potential to be used as a mimic to study the mobility and persistence of L. pneumophila in water systems where the use of the pathogen is impractical and unsafe.

Bacteriophage-encoded lethal membrane disruptors: Advances in understanding and potential applications.

Holins and spanins are bacteriophage-encoded membrane proteins that control bacterial cell lysis in the final stage of the bacteriophage reproductive cycle. Due to their efficient mechanisms for lethal membrane disruption, these proteins are gaining interest in many fields, including the medical, food, biotechnological, and pharmaceutical fields. However, investigating these lethal proteins is challenging due to their toxicity in bacterial expression systems and the resultant low protein yields have hindered their analysis compared to other cell lytic proteins. Therefore, the structural and dynamic properties of holins and spanins in their native environment are not well-understood. In this article we describe recent advances in the classification, purification, and analysis of holin and spanin proteins, which are beginning to overcome the technical barriers to understanding these lethal membrane disrupting proteins, and through this, unlock many potential biotechnological applications.

Use of a Novel DNA-Loaded Alginate-Calcium Carbonate Biopolymer Surrogate to Study the Engulfment of Legionella pneumophila by Acanthamoeba polyphaga in Water Systems.

The engulfment of Legionella pneumophila by free-living amoebae (FLA) in engineered water systems (EWS) enhances L. pneumophila persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between L. pneumophila and FLA in EWS are lacking. To address this, we have developed a biopolymer surrogate with a similar size, shape, surface charge, and hydrophobicity to those of stationary-phase L. pneumophila. Parallel experiments were conducted to observe the engulfment of L. pneumophila and the surrogate by Acanthamoeba polyphaga in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and L. pneumophila, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from A. polyphaga was also faster compared to that of L. pneumophila. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. L. pneumophila and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between L. pneumophila and FLA in EWS. IMPORTANCE The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers. Most importantly, the bacteria's adaptation to the intracellular environments of amoebae primes them for the infection of human macrophages, posing a significant public health risk in EWS. The significance of our study is that a newly developed L. pneumophila biopolymer surrogate can mimic the L. pneumophila engulfment process in A. polyphaga, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated L. pneumophila persistence in EWS and the associated public health risk management.

Degradation and adsorption of synthetic DNA water tracers in environmental matrices.

Synthetic DNA tracers are gaining interest as tools for tracking contamination pathways and hydraulic connections in surface water and groundwater systems. However, few quantitative data exist that describe DNA tracer degradation and adsorption in environmental matrices. We undertook laboratory experiments to quantify the degradation of multiple double-stranded DNA tracers in stream water, groundwater, and domestic and dairy-shed effluent, and adsorption to stream sediments, soils, coastal sand aquifer media and alluvial sandy gravel aquifer media. Faster DNA tracer degradation seemed to be associated with high bacterial concentrations in the liquid phase. Overall, the degradation of the 352 base pair (bp) DNA tracers in the aqueous phase was significantly (P = 0.018) slower than that of the 302 bp DNA tracers. Although the tracers' internal amplicon lengths were similar, the longer non-amplified flanking regions of the 352 bp tracers may better protect them from environmental degradation. Thermodynamic analysis suggests that longer flanking regions contribute to greater tracer stability. This finding may explain our previous field observations that 352 bp tracer mass reductions were often lower than 302 bp tracer mass reductions. The 2 sets of DNA tracers did not differ significantly regarding their adsorption to stream sediment-stream water or aquifer media-groundwater mixtures (P > 0.067), but the 352 bp tracers showed significantly less adsorption to soil-effluent mixtures than the 302 bp tracers (P = 0.005). The DNA tracers' adsorption to soil-effluent mixtures was comparatively less than their adsorption to the aquifer media-groundwater and stream sediment-stream water mixtures, suggesting that DNA tracers may compete with like-charged organic matter for adsorption sites. These findings provide insights into the fate of DNA tracers in the environment. The DNA tracers' degradation rate constants determined in this study for a range of environmental conditions could assist the design of future field investigations.

Hybrid Genomic Analysis of Salmonella enterica Serovar Enteritidis SE3 Isolated from Polluted Soil in Brazil.

In Brazil, Salmonella enterica serovar Enteritidis is a significant health threat. Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This approach yielded 10 contigs with 99.98% identity with S. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven phage defense systems, seven prophages and a heavy metal resistance gene were encoded in the genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3, revealed an open pangenome, with a core genome of 2137 genes. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. This approach enabled the identification of key resistance and virulence genes, and these data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil.

The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability.

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

Elastic Light Scatter Pattern Analysis for the Expedited Detection of Yersinia Species in Pork Mince: Proof of Concept.

Isolation of the pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis from foods typically rely on slow (10-21 day) "cold enrichment" protocols before confirmed results are obtained. We describe an approach that yields results in 39 h that combines an alternative enrichment method with culture on a non-selective medium, and subsequent identification of suspect colonies using elastic light scatter (ELS) analysis. A prototype database of ELS profiles from five Yersinia species and six other bacterial genera found in pork mince was established, and used to compare similar profiles of colonies obtained from enrichment cultures from pork mince samples seeded with representative strains of Y. enterocolitica and Y. pseudotuberculosis. The presumptive identification by ELS using computerised or visual analyses of 83/90 colonies in these experiments as the target species was confirmed by partial 16S rDNA sequencing. In addition to seeded cultures, our method recovered two naturally occurring Yersinia strains. Our results indicate that modified enrichment combined with ELS is a promising new approach for expedited detection of foodborne pathogenic yersiniae.

Potential to use ultraviolet-treated bacteriophages to control foodborne pathogens.

The use of replication-deficient UV-treated bacteriophages, or phages, presents an alternative to viable phages for food biocontrol applications. Nontransducing UV-treated phages, if used correctly, are unlikely to produce viable progeny phages, which might otherwise mediate undesirable horizontal gene transfer events. Phage T4 and Escherichia coli were used as a model system to examine this possibility. UV-treated phages were able to cause a reduction in the optical density of outer membrane-free cell suspensions and they also killed host cells under conditions not permitting their multiplication, that is, 24 degrees C for 2 h and 37 degrees C for 15 min. Host cell reductions were also demonstrated in broth and on meat at 5 degrees C when high concentrations of phages of 2.3 x 10(9) PFU mL(-1) and 1.8 x 10(8) PFU cm(-2), respectively, were used. At 24 degrees C and 37 degrees C, "lysis from without" was likely to be the mechanism responsible for the reduction in host cell concentrations, but at 5 degrees C this may not have been the case.

On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering.

Bacteriophage endolysins have the potential to be a long-term antibacterial replacement for antibiotics. The exogenous application of endolysins on some bacteria results in rapid cell lysis. The prospects for endolysins are furthered by the ability to engineer them; novel endolysins can be developed with optimised stability, specificity, and lytic function. But the success of endolysin engineering and application requires a comprehensive understanding of the relationship between the enzymes biochemical, biophysical and bacteriolytic properties. Here, we examine their catalytic mechanisms, opportunities for developing novel endolysins, and highlight areas where a better understanding would support their long-term success as antibacterial agents.

Water tracking in surface water, groundwater and soils using free and alginate-chitosan encapsulated synthetic DNA tracers.

Investigating contamination pathways and hydraulic connections in complex hydrological systems will benefit greatly from multi-tracer approaches. The use of non-toxic synthetic DNA tracers is promising, because unlimited numbers of tracers, each with a unique DNA identifier, could be used concurrently and detected at extremely low concentrations. This study aimed to develop multiple synthetic DNA tracers as free molecules and encapsulated within microparticles of biocompatible and biodegradable alginate and chitosan, and to validate their field utility in different systems. Experiments encompassing a wide range of conditions and flow rates (19 cm/day-39 km/day) were conducted in a stream, an alluvial gravel aquifer, a fine coastal sand aquifer, and in lysimeters containing undisturbed silt loam over gravels. The DNA tracers were identifiable in all field conditions investigated, and they were directly detectable in the stream at a distance of at least 1 km. The DNA tracers showed promise at tracking fast-flowing water in the stream, gravel aquifer and permeable soils, but were unsatisfactory at tracking slow-moving groundwater in the fine sand aquifer. In the surface water experiments, the microencapsulated DNA tracers' concentrations and mass recoveries were 1-3 orders of magnitude greater than those of the free DNA tracers, because encapsulation protected them from environmental stressors and they were more negatively charged. The opposite was observed in the gravel aquifer, probably due to microparticle filtration by the aquifer media. Although these new DNA tracers showed promise in proof-of-concept field validations, further work is needed before they can be used for large-scale investigations.