Flagellotropic phages: common yet diverse host interaction strategies.

Many bacteriophages (phages) interact with flagella and rely on bacterial motility for successful infection of their hosts. Yet, limited information is available on how phages have evolved to recognize and bind both flagella and subsequent surface receptors for phage DNA injection. Here, we present an update on the current knowledge of flagellotropic phages using a few well-studied phages as examples to unravel the molecular details of bacterial host recognition. We discuss the recent advances in the role of globular exposed flagellin domains and flagella glycosylation in phage binding to the flagella. In addition, we present diverse types of surface receptors and phage components responsible for the interaction with the host. Finally, we point to questions remaining to be answered and new approaches to study this unique group of phages.

Effect of dietary tall oil fatty acids and hydrolysed yeast in SNP2-positive and SNP2-negative piglets challenged with F4 enterotoxigenic Escherichia coli.

Reduction of post-weaning diarrhoea caused by ETEC is a principal objective in pig farming in terms of welfare benefits. This study determined the effects of genetic susceptibility and dietary strategies targeting inflammation and fimbriae adherence on F4-ETEC shedding and diarrhoea in weaned piglets in an experimental challenge model. A DNA marker test targeting single nucleotide polymorphism 2 (SNP2) identified piglets as heterozygous (SNP2+, susceptible) or homozygous (SNP2-, resistant) to developing F4ac-ETEC diarrhoea. A total of 50 piglets, 25 SNP2+ and 25 SNP2-, were weaned at 30 days of age and equally distributed to different treatments (n = 10): Positive control (PC): piglets fed with a negative control diet and provided with colistin via drinking water; Negative control (NC): piglets fed with a negative control diet; Tall oil fatty acids (TOFA): piglets fed with a negative control diet + 1.0 g TOFA/kg feed; Yeast hydrolysate (YH): piglets fed with a negative control diet + 1.5 g YH/kg feed derived from Saccharomyces cerevisiae; and Combination (COM): piglets fed with a negative control diet + 1.0 g TOFA and 1.5 g YH/kg feed. On day 10 post-weaning, all piglets were infected with F4-ETEC by oral administration. Piglets fed with PC, TOFA, YH or COM had a lower faecal shedding of F4-ETEC than NC piglets (P < 0.001), which was also shorter in duration for PC and TOFA piglets than for NC piglets (P < 0.001). Piglets in PC, TOFA, YH and COM had a shorter diarrhoea duration versus NC when classified as SNP2+ (P = 0.02). Furthermore, PC, TOFA and YH piglets grew more than NC and COM piglets in the initial post-inoculation period (P < 0.001). In addition, the level of faecal F4-ETEC shedding and the percentage of pigs that developed F4-ETEC diarrhoea (72 vs. 32%, P < 0.01) following infection were higher, and the duration of F4-ETEC diarrhoea longer (2.6 vs. 0.6 days, P < 0.001), in SNP2+ piglets than in SNP2- piglets, and led to reduced growth performance (P = 0.03). In conclusion, piglets fed with TOFA, YH or their combination, irrespective of their SNP2 status, are more resilient to F4-ETEC infection. Moreover, SNP2+ piglets show a higher level of F4-ETEC shedding and diarrhoea prevalence than SNP2- piglets, confirming an association between SNP2 and F4ac-ETEC susceptibility.

Complete association between CHCF1 genotype and enterotoxigenic Escherichia coli F4ab-associated post-weaning diarrhea in a pig challenge trial.

Enterotoxigenic E. coli (ETEC) susceptibility in pigs is highly influenced by their genotype. The aim of this study was to determine the association between CHCF1 genotype and ETEC F4ab susceptibility in experimentally infected pigs. We investigated ETEC diarrhea development in CHCF1 heterozygous susceptible (RS) (n = 12 pigs) compared to CHCF1 homozygous resistant (RR) (n = 12 pigs) for six days after ETEC F4ab challenge. Afterwards, we genotyped with MUC4 and MUC13 markers to relate performance in identifying ETEC F4ab diarrhea susceptible pigs. In the CHCF1 RS group, 12/12 pigs developed ETEC diarrhea compared with 0/12 pigs in the CHCF1 RR group. Weight gain was lower in CHCF1 RS pigs compared with RR pigs (mean ± SD: 208 ± 323 g and 987 ± 615 g, p = 0.0007). Further, the shedding of hemolytic E. coli was significantly higher in CHCF1 RS pigs from 2 to 6 days post inoculation and they shed the challenge strain for more days (mean ± SD: 3.5 ± 1.6 days versus 0.5 ± 0.5 days, p < 0.0001). Twelve pigs with ETEC diarrhea were misclassified as resistant with the MUC4 marker and four pigs without ETEC diarrhea were misclassified as susceptible with the MUC13 marker. We found complete association between CHCF1 genotype and ETEC diarrhea development in pigs from a herd with Danbred genetics. The CHCF1 marker was more likely to determine the true host susceptibility to ETEC F4ab than the other markers. The marker shows potential for improving reliability of PWD challenge models and potentially for use in breeding for ETEC F4ab/ac resistance.

Full-scale industrial phage trial targeting Salmonella on pork carcasses.

Phages have been suggested as promising biocontrol agents in food, but trials demonstrating the efficiency of phage treatment under industrial settings are missing. Here we performed a full-scale industrial trial to evaluate the efficacy of a commercial phage product to reduce the prevalence of naturally occurring Salmonella on pork carcasses. A total of 134 carcasses from potentially Salmonella positive finisher herds were chosen to be tested at the slaughterhouse based on the level of antibodies in the blood. During five consecutive runs, carcasses were directed into a cabin spraying phages, resulting in a dosage of approximately 2 × 107 phages per cm2 carcass surface. To evaluate the presence of Salmonella, a predefined area of one half of the carcass was swabbed before phage application and the other half 15 min after. A total of 268 samples were analysed by Real-Time PCR. Under these optimized test conditions, 14 carcasses were found positive before phage application, while only 3 carcasses were positive after. This work shows that phage application allows to achieve approximatively 79% reduction of Salmonella-positive carcasses and demonstrates that implementation of phage application in industrial settings can be used as an additional strategy to control foodborne pathogens.

Post-weaning diarrhea in pigs from a single Danish production herd was not associated with the pre-weaning fecal microbiota composition and diversity.

Introduction: The association between the porcine pre-weaning gut microbiota composition and diversity, and subsequent post-weaning diarrhea (PWD) susceptibility is currently being studied. In this longitudinal study, we examined the association between pre-weaning fecal microbiome composition and diversity, and PWD development in a Danish sow herd. Methods: Forty-five pigs were followed from birth until 7 days after weaning (post-natal day (PND) 33). At PND 33, the pigs were categorized as PWD cases or healthy controls based on fecal consistency. We compared their fecal microbiomes at PND 8, late lactation (PND 27) and 7 days post weaning (PND 33) using 16S rRNA V3 region high-throughput sequencing. At PND 27 and 33, we also weighed the pigs, assessed fecal shedding of hemolytic Escherichia coli by culture and characterized hemolytic isolates by ETEC virulence factors with PCR and by whole genome sequencing. Results: A total of 25 out of 45 pigs developed PWD and one Enterotoxigenic E. coli strain with F18:LT:EAST1 virotype was isolated from most pigs. At PND 33, we found differences in beta diversity between PWD and healthy pigs (R2 = 0.027, p = 0.009) and that body weight was associated with both alpha and beta diversity. Pre-weaning fecal microbiome diversity did not differ between PWD and healthy pigs and we found no significant, differentially abundant bacteria between them. Conclusion: In the production herd under study, pre-weaning fecal microbiome diversity and composition were not useful indicators of PWD susceptibility.

Pilot study on CHCF1 genotype in a pig challenge model for enterotoxigenic Escherichia coli F4ab/ac associated post-weaning diarrhea.

Host genotype is important for enterotoxigenic E. coli (ETEC) susceptibility. We conducted two trials to evaluate the effect of CHCF1 genotype on incidence of ETEC diarrhea. In trial 1 (n = 15 pigs), pigs were inoculated with 108 CFU or 1010 CFU doses of an ETEC F4ac strain. In trial 2 (n = 33 pigs), pigs were inoculated with ETEC F4ab or F4ac. Across trials, all inoculated pigs that developed ETEC diarrhea were CHCF1 heterozygous susceptible (6/6). No inoculated CHCF1 homozygous resistant pigs developed ETEC diarrhea (0/26). Susceptibility towards ETEC F4ac/ab infection might correspond with CHCF1 genotype.

Genome Sequences of 16 Escherichia coli Bacteriophages Isolated from Wastewater, Pond Water, Cow Manure, and Bird Feces.

Escherichia coli is a highly diverse bacterial species comprising both commensal and pathogenic strains. Here, we report complete genome sequences of 16 E. coli bacteriophages isolated from various environmental samples using the ECOR collection as isolation hosts.

Looking into the future of phage-based control of zoonotic pathogens in food and animal production.

Using bacteriophages (phages) to control zoonotic pathogens in food and animal production is a realistic and promising antimicrobial approach. Recent studies have demonstrated their efficacy and safety, yet bringing phage products on the market remains a challenge. Here we summarize the procedure for advancing phage applications from the laboratory to simplified model systems and testing in pilot scale, to farms and food industries. We highlight the most important contributions concerning phages in food matrices and animal guts, and propose directions for future research required to understand interactions in such complex systems. Finally, we propose a holistic approach combining a data repository with modelling, multi-omic techniques and data analysis to modernize phage-based control of zoonotic pathogens.

Bacteriophage Enumeration and Detection Methods.

Application of phages as alternative antimicrobials to combat pathogenic bacteria and their association to a healthy gut microbiome has prompted a need for precise methods for detection and enumeration of phage particles. There are many applicable methods, but care should be taken considering the measured object (infectious phage, whole phage particle or nucleic acid and proteins) and the concept behind the technique to avoid misinterpretations. While molecular methods cannot discriminate between viable and non-infectious phages, the traditional techniques for counting infectious phages can be time consuming and poorly reproducible. Here, we describe the methods currently used for phage detection and enumeration and highlight their advantages as well as their limitations. Finally, we provide insight on how to deal with complex samples, as well as future prospects in the field of phage quantification.

F4- and F18-Positive Enterotoxigenic Escherichia coli Isolates from Diarrhea of Postweaning Pigs: Genomic Characterization.

This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type.IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Phage S144, A New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

A Simple, Reliable, and Inexpensive Solution for Contact Color Measurement in Small Plant Samples.

Correct color measurement by contact-type color measuring devices requires that the sample surface fully covers the head of the device, so their use on small samples remains a challenge. Here, we propose to use cardboard adaptors on the two aperture masks (3 and 8 mm diameter measuring area) of a broadly used portable spectrophotometer. Adaptors in black and white to reduce the measuring area by 50% and 70% were applied in this study. Representatives of the family Campanulaceae have been used to test the methodology, given the occurrence of small leaves. Our results show that, following colorimetric criteria, the only setting providing indistinguishable colors according to the perception of the human eye is the use of a 50%-reducing adaptor on the 3-mm aperture. In addition, statistical analysis suggests the use of the white adaptor. Our contribution offers a sound measurement technique to gather ecological information from the color of leaves, petals, and other small samples.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.

The genera of bacteriophages and their receptors are the major determinants of host range.

The host range of phages is a key to understand their impact on bacterial ecology and evolution. Because of the complexity of phage-host interactions, the variables that determine the breadth of a phage host range remain poorly understood. Here, we propose a novel holistic approach to identify the host range determinants of a new collection of phages infecting Salmonella, isolated from animal, environmental and wastewater samples that were able to infect 58 of the 71 Salmonella strains in our collection. By using a set of statistic approaches (non-metric dimensional scaling, Bray-Curtis distance, PERMANOVA), we analysed phenotypic (host range on wild-type and receptor mutants) and genetic data (taxonomic assignment and receptor binding proteins) to evaluate the impact of isolation strain and niche, phage receptor and genus on the host range. Statistical analysis revealed that two phage characteristics influence the host range by explaining the most variance: the receptor by 45% and the genus by 51%. Interestingly, phage genus and receptor in combination explained 79% of the variance, establishing these characteristics as the major determinants of the host range. This study demonstrates the power and the novelty of applying statistical approaches to phenotypic and genetic data to investigate the ecology of phage-host interactions.

Surface colour: An overlooked aspect in the study of cyanobacterial biofilm formation.

Cyanobacteria can grow as biofilms, communities that colonize surfaces and that play a fundamental role in the ecology of many diverse habitats and in the conversion of industrial production to green platforms. Although biofilm growth is known to be significantly affected by several characteristics, the effect of colour surface is an overlooked aspect that has not yet been investigated. In this study, we describe the effect of colour hues (white, red, blue and black) on the growth of cyanobacterial biofilms on air-exposed substrates. We measured growth, architecture, pigment production and levels of ATP and reactive oxygen species in cyanobacterial biofilms formed on different coloured substrates. The study findings demonstrate, for the first time, that the colour of a surface affects biofilm formation at the air-solid interface (with more biomass accumulating on white and red substrates than on blue and black substrates) and also alters the biofilm architecture. In addition, the roles of chromatic adaptation, phototrophic cells and reactive oxygen species as intermediates between colour sensing and biofilm response are discussed. Our results support the importance of colour as a new factor that favours surface colonization by cyanobacteria and its contribution to biofilm formation.

Mini-review: Biofilm responses to oxidative stress.

Biofilms constitute the predominant microbial style of life in natural and engineered ecosystems. Facing harsh environmental conditions, microorganisms accumulate reactive oxygen species (ROS), potentially encountering a dangerous condition called oxidative stress. While high levels of oxidative stress are toxic, low levels act as a cue, triggering bacteria to activate effective scavenging mechanisms or to shift metabolic pathways. Although a complex and fragmentary picture results from current knowledge of the pathways activated in response to oxidative stress, three main responses are shown to be central: the existence of common regulators, the production of extracellular polymeric substances, and biofilm heterogeneity. An investigation into the mechanisms activated by biofilms in response to different oxidative stress levels could have important consequences from ecological and economic points of view, and could be exploited to propose alternative strategies to control microbial virulence and deterioration.

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.

Evaluation of zosteric acid for mitigating biofilm formation of Pseudomonas putida isolated from a membrane bioreactor system.

This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (-97%) and thickness (-50%), and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

A simple and reliable methodology to detect egg white in art samples.

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii.

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.