Salmonella enterica Serovar Typhimurium U288 is an emerging pathogen of pigs. The strain contains three plasmids of diverse origin that encode traits that are of concern for food security and safety, these include antibiotic resistant determinants, an array of functions that can modify cell physiology and permit genetic mobility. At 148,711?bp, pSTU288-1 appears to be a hybrid plasmid containing a conglomerate of genes found in pSLT of S. Typhimurium LT2, coupled with a mosaic of horizontally-acquired elements. Class I integron containing gene cassettes conferring resistance against clinically important antibiotics and compounds are present in pSTU288-1. A curious feature of the plasmid involves the deletion of two genes encoded in the Salmonella plasmid virulence operon (spvR and spvA) following the insertion of a tnpA IS26-like element coupled to a blaTEM gene. The spv operon is considered to be a major plasmid-encoded Salmonella virulence factor that is essential for the intracellular lifecycle. The loss of the positive regulator SpvR may impact on the pathogenesis of S. Typhimurium U288. A second 11,067?bp plasmid designated pSTU288-2 contains further antibiotic resistance determinants, as well as replication and mobilization genes. Finally, a small 4675?bp plasmid pSTU288-3 was identified containing mobilization genes and a pleD-like G-G-D/E-E-F conserved domain protein that modulate intracellular levels of cyclic di-GMP, and are associated with motile to sessile transitions in growth.
Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.
?-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae ?-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 ?mol min(-1) mg(-1) respectively against 4-nitrophenyl ?-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of ?-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.
Campylobacter jejuni strain PT14 is a clinical isolate previously used to propagate bacteriophages in the United Kingdom phage typing scheme. The strain has proven useful in the isolation of Campylobacter bacteriophages from several sources, and it functions as a model host in phage therapy experiments with poultry and poultry meat.
Salmonella enterica serovar Typhimurium U288 has firmly established itself within the United Kingdom pig production industry. The prevalence of this highly pathogenic multidrug-resistant serovar at such a critical point in the food chain is therefore of great concern. To enhance our understanding of this microorganism, whole-genome and plasmid sequencing was performed.
Most Campylobacter bacteriophages isolated to date have long contractile tails and belong to the family Myoviridae. Based on their morphology, genome size and endonuclease restriction profile, Campylobacter phages were originally divided into three groups. The recent genome sequencing of seven virulent campylophages reveal further details of the relationships between these phages at the genome organization level. This article details the morphological and genomic features among the campylophages, investigates their taxonomic position, and proposes the creation of two new genera, the "Cp220likevirus" and "Cp8unalikevirus" within a proposed subfamily, the "Eucampyvirinae"
Exposure to interferon results in the rapid transcriptional induction of genes, many of which function to create an antiviral environment in potential host cells. For the majority of adenoviruses, replication is unaffected by the actions of interferon. It has previously been shown, using non-gastrointestinal cells, that the species F human adenoviruses are sensitive to the action of interferon. Here, we have developed an enterocyte-like cell-culture model to re-evaluate this question, and determined the effects of interferon on species F adenovirus during infection of gastrointestinal cells. We show that species F adenovirus type 40 is sensitive to the effects of interferon in gastrointestinal-like cells, which may help to explain its fastidious growth in culture.
Whole genome sequencing of bacteriophages suitable for biocontrol of pathogens in food products is a pre-requisite to any phage-based intervention procedure. Trials involving the biosanitization of Salmonella Typhimurium in the pig production environment identified one such candidate, ?SH19.
Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P<0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of >1 log?? unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log?? 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (>99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.
Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ? 3) led to 1 to 3 log?? CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log?? CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.
It was noted that quantitative and qualitative differences occurred between the growth of Campylobacter in microaerobic atmospheres provided by a gas replacement jar and that in a modular atmosphere controlled system cabinet, despite the fact that oxygen levels were comparable. Hydrogen was, however, only present in the replacement mixture (3%). Investigations were therefore carried out to examine any accompanying physiological or transcriptional differences. Growth curves and motility studies using Campylobacter jejuni HPC5 showed that cultures growing in the cabinet were impaired, but only in the early stages of growth compared to growth in the jar. However, transcriptome studies highlighted profound changes in the transcript profiles of exponential cultures grown in the cabinet compared to the jar, including genes indicative of oxidative stress. Genes involved in detoxification, synthesis and modification of macromolecules, probable prophage genes and genes associated with inhibition of natural transformation showed relative increases in expression in the cabinet. Conversely, genes that function in energy metabolism, chaperones, heat shock and motility were increased in the jar, which was indicative of balanced growth. This work highlights the need to carefully annotate the different methods of atmosphere generation in the description of experiments in microarray databases; the assessment of these experimental details is crucial to overcome difficulties in comparing transcriptomic studies of campylobacter cultures between different laboratories.
Campylobacter jejuni activates the host transcription factor NF-kappaB that regulates the expression of a number of genes involved in the inflammatory response to bacterial infection. Signaling pathways leading to NF-kappaB by pathogens and/or their products include transmembrane Toll-like receptors (TLRs) and intracellular receptors nucleotide-binding oligomerization domain proteins (Nods). This study was carried out to investigate the role of TLRs (TLR2 and TLR4) and Nods (Nod1 and Nod2) receptors in mediating NF-kappaB activation by C. jejuni. By means of transfecting receptors/molecules under study and measuring reporter gene activity, NF-kappaB activation and subsequent cytokine production by live, heat-killed C. jejuni, or boiled cell extract (BCE) were observed in a range of tissue culture cell lines. This activation is reduced upon transfection of cells with the dominant negative versions (DNV) of TLR-adaptor molecule MyD88. NF-kappaB activation was observed to be augmented in cell lines transfected with TLR2, Nod1, and Nod2 but not with TLR4. Additionally, NF-kappaB activation by C. jejuni was observed to be independent of Nod1 and Nod2 in cells transfected with DNV of these receptors. NF-kappaB activation pathway by C. jejuni may represent a novel mechanism utilising unknown receptors up-regulated by yet to be characterized active component(s). To our knowledge, such observations have not been previously reported for C. jejuni or any other food-borne pathogen.
Our understanding of the dynamics of genome stability versus gene flux within bacteriophage lineages is limited. Recently, there has been a renewed interest in the use of bacteriophages as therapeutic agents; a prerequisite for their use in such therapies is a thorough understanding of their genetic complement, genome stability and their ecology to avoid the dissemination or mobilisation of phage or bacterial virulence and toxin genes. Campylobacter, a food-borne pathogen, is one of the organisms for which the use of bacteriophage is being considered to reduce human exposure to this organism.
Members of the genus Campylobacter are frequently responsible for human enteric disease worldwide. Persistent Campylobacter contamination of poultry meat is a common problem that represents a significant food safety risk through the consumption of undercooked poultry meat or through cross-contamination of other foods during the preparation of poultry. Bacteriophage therapy is one possible means by which this colonization of poultry could be controlled, thus limiting the entry of Campylobacter into the human food chain. Previously group III phages with genome sizes of approximately 140 kb had been administered to Campylobacter jejuni-colonized poultry. The application of a group II Campylobacter phage, CP220, with a genome size of 197 kb is described here. Phage CP220 was administered to both C. jejuni- and C. coli-colonized birds. A 2-log CFU/g decline in cecal Campylobacter counts was observed after 48 h in birds colonized with C. jejuni HPC5 and administered with a single 7-log PFU dose of CP220. The incidence of phage resistance developing in Campylobacter-colonized chickens upon exposure to virulent phages was determined to be 2%, and the resistant types remained a minor component of the population. To achieve a similar reduction in Campylobacter numbers in C. coli OR12-colonized birds, a 9-log PFU dose of CP220 was required. Using phage to reduce Campylobacter colonization in poultry offers the prospect of a sustainable intervention measure that may limit the entry of these pathogens into the human food chain.
Campylobacter is considered to be the most common cause of bacterial diarrhoeal illness in the developed world. Many cases are thought to be acquired from consumption of undercooked poultry. The aim of this study was to compare the effect of the rate of cooling on the survival, at 4 degrees C and -20 degrees C, of Campylobacter coli and Campylobacter jejuni strains, inoculated on chicken skin from axenic culture or as mixed inoculums. Strains chilled in a domestic refrigerator varied in their tolerance to storage at 4 degrees C. Statistically significant differences between strains applied as axenic or mixed inoculums were observed for specific strain combinations using two-way ANOVA, including the enhanced survival of antibiotic resistant C. coli 99/367 at 4 degrees C. The use of rapid cooling (at -20 degrees C/min) enhanced the survival of all the Campylobacter strains chilled to 4 degrees C compared to standard refrigeration. Freezing to -20 degrees C reduced viable counts by 2.2-2.6 log10 CFU/cm(2) in 24 h. Rapid cooling to -20 degrees C (at -30 degrees C/min) enhanced the survival of C. coli 99/367 compared to freezing in a domestic freezer. Statistically significant interaction terms between specific strains were observed in mixed inoculums chilled to -20 degrees C by freezing in a domestic freezer and by rapid chilling to -20 degrees C. Rapid chilling of poultry, particularly for 4 degrees C storage may enhance survival of Campylobacter and although this is an issue that affects meat quality, it should be considered by poultry processors.
Campylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168.
Phage therapy is the use of bacteriophages as antimicrobial agents for the control of pathogenic and other problem bacteria. It has previously been argued that successful application of phage therapy requires a good understanding of the non-linear kinetics of phage-bacteria interactions. Here we combine experimental and modelling approaches to make a detailed examination of such kinetics for the important food-borne pathogen Campylobacter jejuni and a suitable virulent phage in an in vitro system. Phage-insensitive populations of C. jejuni arise readily, and as far as we are aware this is the first phage therapy study to test, against in vitro data, models for phage-bacteria interactions incorporating phage-insensitive or resistant bacteria. We find that even an apparently simplistic model fits the data surprisingly well, and we confirm that the so-called inundation and proliferation thresholds are likely to be of considerable practical importance to phage therapy. We fit the model to time series data in order to estimate thresholds and rate constants directly. A comparison of the fit for each culture reveals density-dependent features of phage infectivity that are worthy of further investigation. Our results illustrate how insight from empirical studies can be greatly enhanced by the use of kinetic models: such combined studies of in vitro systems are likely to be an essential precursor to building a meaningful picture of the kinetic properties of in vivo phage therapy.
Campylobacter jejuni is an important human food-borne intestinal pathogen, however relatively little is known about its mechanisms of pathogenesis or pathogen-host interactions. To monitor changes in gene expression and glycan binding of C. jejuni within a common avian host, an immunomagnetic separation technique (IMS) was utilised to directly isolate infecting C. jejuni 81116 from a chicken host. An average of 10(5) cells/g was re-isolated from chicken caecal samples by IMS technique. The in vivo passaged strains were used successfully in evaluation of carbohydrate binding through the use of a glycan array and were further suitable for transcriptome analysis. The glycan microarray analysis demonstrated differences in binding to negatively charged glycans of laboratory grown strains of C. jejuni compared with strains isolated after in vivo passage. The in vivo passaged strains showed marked up-regulation of chemotaxis receptors and toxin genes. The optimised Campylobacter IMS technique described in this study allowed isolation directly from an animal host. Changes in gene expression and glycan binding at an in vivo level can also be identified by using this method.
We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ?SH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.
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