Erika J. Lingohr

Enumeration of bacteriophages by double agar overlay plaque assay.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.

Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms.

Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages’ genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2–12 h of incubation.

Enumeration of bacteriophages using the small drop plaque assay system.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples.

Complete genomic sequence of bacteriophage felix o1.

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent.

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

BackgroundComparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail.ResultsHere we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1.ConclusionsBc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G.

A Shigella boydii bacteriophage which resembles Salmonella phage ViI

BackgroundLytic bacteriophages have been applied successfully to control the growth of various foodborne pathogens. Sequencing of their genomes is considered as an important preliminary step to ensure their safety prior to food applications.ResultsThe lytic bacteriophage, ΦSboM-AG3, targets the important foodborne pathogen, Shigella. It is morphologically similar to phage ViI of Salmonella enterica serovar Typhi and a series of phages of Acinetobacter calcoaceticus and Rhizobium meliloti. The complete genome of ΦSboM-AG3 was determined to be 158 kb and was terminally redundant and circularly permuted. Two hundred and sixteen open reading frames (ORFs) were identified and annotated, most of which displayed homology to proteins of Salmonella phage ViI. The genome also included four genes specifying tRNAs.ConclusionsThis is the first time that a Vi-specific phage for Shigella has been described. There is no evidence for the presence of virulence and lysogeny-associated genes. In conclusion, the genome analysis of ΦSboM-AG3 indicates that this phage can be safely used for biocontrol purposes.

Determination of bacteriophage genome size by pulsed-field gel electrophoresis.

Standard agarose gel electrophoresis is extensively used to resolve DNA fragments from 0.2 to 40-50 kb. Larger fragments of genomic DNA or whole viral genomes can only effectively be resolved by pulsed-field gel electrophoresis (PFGE), which extends the range of molecular separation from 200 bp to 12 Mb.

Rapid Genoserotyping Tool for Classification of Salmonella Serovars

ABSTRACT We have developed a Salmonella genoserotyping array (SGSA) which rapidly generates an antigenic formula consistent with the White-Kauffmann-Le Minor scheme, currently the gold standard for Salmonella serotyping. A set of 287 strains representative of 133 Salmonella serovars was assembled to validate the array and to test the array probes for accuracy, specificity, and reproducibility. Initially, 76 known serovars were utilized to validate the specificity and repeatability of the array probes and their expected probe patterns. The SGSA generated the correct serovar designations for 100% of the known subspecies I serovars tested in the validation panel and an antigenic formula consistent with that of the White-Kauffmann-Le Minor scheme for 97% of all known serovars tested. Once validated, the SGSA was assessed against a blind panel of 100 Salmonella enterica subsp. I samples serotyped using traditional methods. In summary, the SGSA correctly identified all of the blind samples as representing Salmonella and successfully identified 92% of the antigens found within the unknown samples. Antigen- and serovar-specific probes, in combination with a pepT PCR for confirmation of S. enterica subsp. Enteritidis determinations, generated an antigenic formula and/or a serovar designation consistent with the White-Kauffmann-Le Minor scheme for 87% of unknown samples tested with the SGSA. Future experiments are planned to test the specificity of the array probes with other Salmonella serovars to demonstrate the versatility and utility of this array as a public health tool in the identification of Salmonella.

The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5

BackgroundBacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs.MethodsWe examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set.ResultsPhage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.