High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli

Background

Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.

Methodology/Principal Findings

E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m²) combination with different temperature (22, 28, 30, 32, and 37°C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.

Conclusions/Significance

These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.     

Comparative genomic analysis of a multiple antimicrobial resistant enterotoxigenic E. coli O157 lineage from Australian pigs

Background

Enterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7.

Results

O157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibiotic resistance genes but their association with mobile elements remains undetermined.

Conclusions

We present an analysis of the first draft genome sequences of two epidemiologically-unrelated, pathogenic ETEC O157. E. coli O157 SvETEC and E. coli O157:K88 734/3 belong to the ST23 complex and are phylogenetically distinct to EHEC O157 lineages that reside within the ST11 complex.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1382-y) contains supplementary material, which is available to authorized users.     

Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

Background

In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.

Methods

Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.

Results

Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.

Conclusions

MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.     

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment

Background

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Methodology/Principal Findings

The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg²⁺. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction.

Conclusions/Significance

Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.     

In Vivo and Ex Vivo Evaluations of Bacteriophages e11/2 and e4/1c for Use in the Control of Escherichia coli O157:H7

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10¹⁰ CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10¹¹ PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.Escherichia coli O157:H7 has become a worldwide public health concern since it was first identified as a human pathogen in 1982 (31). This pathogen has a very low infectious dose (approximately 10 cells) in humans, and symptoms of infection range from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and in some cases, death (22, 39). Ruminants are recognized as reservoirs for this pathogen and are the most common sources for food-borne outbreaks (8, 13, 25). It has been reported that the occurrence of E. coli O157:H7 in the feces and, in particular, the hide of cattle is a significant source of the pathogen on the carcass and in derived meat products (11, 12, 25). The control of this pathogen within the animal is difficult, because carriage in ruminants is asymptomatic and shedding can be intermittent and seasonal (12, 19).Research has highlighted the necessity for preharvest intervention strategies to control or reduce E. coli O157:H7 in the food chain (17, 18). Successful strategies to reduce the carriage of E. coli O157:H7 in ruminant animals could potentially reduce the risk of human exposure to this pathogen. There are currently no effective and reliable commercially available intervention strategies to control the carriage of E. coli O157:H7 in ruminants. However, research in this area is increasing, and numerous agents, such as vaccines, probiotics, and bacteriophages (phages), are being evaluated (15, 17, 18). The use of phages for the control of food-borne pathogens in the food chain is desirable, as they are natural, nontoxic viruses that target only specific bacteria (2) and are already being used in human and veterinary medicine, particularly prior to antibiotics (6, 14, 15, 30, 37). Many studies have investigated the use of different phages for the control of E. coli O157:H7 in various animals, including mice, calves, and sheep (4, 5, 35, 37, 41). Although the results between studies vary, some have reported the successful reduction of E. coli O157:H7 levels in animals (4), and one study has resulted in a U.S. patent (41). There are very few commercially available phage products to date, but research indicates promising outcomes for the use of phages for the control of E. coli O157:H7 within the food chain.The E. coli O157:H7-specific phages e11/2 and e4/1c were isolated from bovine slurry in a previous study (26) and have the potential to be used as biocontrol agents for E. coli O157:H7. Both phages have been found to be active against E. coli O157:H7 in a number of relevant test conditions involving different pHs, water activity, and temperatures (B. Coffey, L. Rivas, G. Duffy, A. Coffey, R. P. Ross, and O. McAuliffe, unpublished data). In addition, whole-genome sequencing revealed that neither phage encodes undesirable properties, such as virulence factors, that would hinder its use as a biocontrol agent for E. coli O157:H7 (B. Coffey, G. O''Flynn, A. Coffey, O. O''Sullivan, O. McAuliffe, and R. P. Ross, unpublished data). The objective of the present study was, first, to evaluate the effect of phages e4/1c and e11/2 against inoculated E. coli O157:H7 in an ex vivo model rumen system, and second, to assess the ability of a phage cocktail (e11/2 and e4/1c) to reduce the shedding of E. coli O157:H7 in experimentally inoculated cattle. Findings from ex vivo studies determined our phages to be effective against E. coli O157:H7 in a model rumen system; however, complete eradication of E. coli O157:H7 from cattle was not achieved.     

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

Background

Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.

Methods and Findings

Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.

Conclusions

The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.     

Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background

Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria.

Results

In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log₁₀ unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log₁₀ units.

Conclusion

The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.     

An Aptamer-Based Biosensor for Colorimetric Detection of Escherichia coli O157:H7

Background

An aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli) O157:H7.

Methodology/Principal Findings

The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10⁴∼ 10⁸ colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor.

Conclusions

The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.     

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

Background

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.

Results

Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.

Conclusions

SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-831) contains supplementary material, which is available to authorized users.     

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages

Background

Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution.

Results

Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named “core genome”, and the second group, containing mostly short ORFs without assigned functions was called “accessory genome”. Like in other phage groups, variable genes are confined to specific regions in the genome.

Conclusion

Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1146) contains supplementary material, which is available to authorized users.     

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity

Background

A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is “phage therapy”, the clinical application of bacteriophages to selectively kill bacteria.

Results

Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics.

Conclusions

The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1848-y) contains supplementary material, which is available to authorized users.     

Carriage of stx2a Differentiates Clinical and Bovine-Biased Strains of Escherichia coli O157

Background

Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.

Methods

Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.

Results

Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage - chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage – sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.

Conclusions

Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.     

Phylogenetic and complementation analysis of a single-stranded DNA binding protein family from lactococcal phages indicates a non-bacterial origin

Background

The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14_bIL67-like proteins) have been identified and characterized structurally and biochemically.

Methodology/Principal Findings

This study focused on the determination of phylogenetic relationships between Orf14_bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14_bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14_bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli.

Conclusions/Significance

Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages.     

Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages

Background

Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.

Results

P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.

Conclusions

This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users.     

Light Scattering Sensor for Direct Identification of Colonies of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145 and O157

Background

Shiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates.

Methodology/Principal Findings

Initially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10–12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h.

Conclusions

BARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in less than 24 h.     

Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae

Background

Spounavirinae viruses have received an increasing interest as tools for the control of harmful bacteria due to their relatively broad host range and strictly virulent phenotype.

Results

In this study, we collected and analyzed the complete genome sequences of 61 published phages, either ICTV-classified or candidate members of the Spounavirinae subfamily of the Myoviridae. A set of comparative analyses identified a distinct, recently proposed Bastille-like phage group within the Spounavirinae. More importantly, type 1 thymidylate synthase (TS1) and dihydrofolate reductase (DHFR) genes were shown to be unique for the members of the proposed Bastille-like phage group, and are suitable as molecular markers. We also show that the members of this group encode beta-lactamase and/or sporulation-related SpoIIIE homologs, possibly questioning their suitability as biocontrol agents.

Conclusions

We confirm the creation of a new genus—the “Bastille-like group”—in Spounavirinae, and propose that the presence of TS1- and DHFR-encoding genes could serve as signatures for the new Bastille-like group. In addition, the presence of metallo-beta-lactamase and/or SpoIIIE homologs in all members of Bastille-like group phages makes questionable their suitability for use in biocontrol.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1757-0) contains supplementary material, which is available to authorized users.     

Down-Regulation of Platelet Surface CD47 Expression in Escherichia coli O157:H7 Infection-Induced Thrombocytopenia

Background

Platelet depletion is a key feature of hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC) infection. The mechanism underlying STEC-induced platelet depletion, however, is not completely understood.

Methodology/Principal Findings

Here we demonstrated for the first time that platelet surface expression of CD47 was significantly decreased in C57BL6 mice treated with concentrated culture filtrates (CCF) from STEC O157:H7. STEC O157:H7 CCF treatment also led to a sharp drop of platelet counts. The reduction of cell surface CD47 was specific for platelets but not for neutrophil, monocytes and red blood cells. Down-regulation of platelet surface CD47 was also observed in isolated human platelets treated with O157:H7 CCF. Platelet surface CD47 reduction by O157:H7 CCF could be blocked by anti-TLR4 antibody but not anti-CD62 antibody. Down-regulation of platelet surface CD47 was positively correlated with platelet activation and phagocytosis by human monocyte-derived macrophages. Furthermore, the enhanced phagocytosis process of O157:H7 CCF-treated platelets was abolished by addition of soluble CD47 recombinants.

Conclusions/Significance

Our results suggest that platelet CD47 down-regulation may be a novel mechanism underneath STEC-induced platelet depletion, and that the interactions between CD47 and its receptor, signal regulatory protein α (SIRPα), play an essential role in modulating platelet homeostasis.     

Prevalence and Impact of Bacteriophages on the Presence of Escherichia coli O157:H7 in Feedlot Cattle and Their Environment

The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as “phage”) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P < 0.05) in May. Overall recovery did not differ (P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r = −0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.Bacteriophages are the most abundant biological entities on earth. An estimated 10³⁰ marine bacteriophages are harbored in the ocean, and they significantly influence microbial communities and function (27). As resistance is an increasing challenge in antimicrobial therapy, the antimicrobial nature of bacteriophages is being more intensively studied (13, 15). Bacteriophages naturally inhabit the mammalian gastrointestinal tract (1, 8), and Escherichia coli-infecting bacteriophages are commonly isolated from sewage, hospital wastewater, and fecal samples from humans and animals (3). Ruminants have been shown to shed up to 10⁷ bacteriophage per gram of feces (6), and in humans multiple types of bacteriophage exhibiting activity against E. coli have been isolated from a single fecal sample (7).E. coli O157:H7 is an important zoonotic bacterium carried asymptomatically by cattle and readily isolated from manure, manure slurry, and drinking water in dairies and feedlots (11, 24, 30). Additionally, E. coli O157:H7 shedding by cattle has a seasonal pattern, peaking in the summer months (2, 25). Bacteriophage strains that infect E. coli O157:H7 have also been isolated from animal feces and have shown lytic activity against this bacterium in vivo and in vitro (5, 23, 28, 31). In recent studies, such phages were shown to be widely distributed in cattle and in feces on the pen floor within feedlots (4, 18). However, the relationships between the presence of E. coli O157:H7-infecting bacteriophage in cattle and their environment and the shedding of this bacterium by cattle are largely undefined. Consequently, the aims of the present study were (i) to determine the prevalence of endemic E. coli O157:H7-infecting bacteriophage (referred to as “phage”) in feedlots over a 7-month period and (ii) to compare the presence of phage to the occurrence of E. coli O157:H7 in cattle and their environment.     

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains

Background

Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.

Results

The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage {"type":"entrez-protein","attrs":{"text":"P13374","term_id":"138227","term_text":"P13374"}}P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.

Conclusions

At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.