Bacteria isolated from parasitic nematodes - a potential novel vector of pathogens?

Bacterial pathogens are ubiquitous in soil and water - concurrently so are free-living helminths that feed on bacteria. These helminths fall into two categories; the non-parasitic and the parasitic. The former have been the focus of previous work, finding that bacterial pathogens inside helminths are conferred survival advantages over and above bacteria alone in the environment, and that accidental ingestion of non-parasitic helminths can cause systemic infection in vertebrate hosts. Here, we determine the potential for bacteria to be associated with parasitic helminths. After culturing helminths from fecal samples obtained from livestock the external bacteria were removed. Two-hundred parasitic helminths from three different species were homogenised and the bacteria that were internal to the helminths were isolated and cultured. Eleven different bacterial isolates were found; of which eight were indentified. The bacteria identified included known human and cattle pathogens. We concluded that bacteria of livestock can be isolated in parasitic helminths and that this suggests a mechanism by which bacteria, pathogenic or otherwise, can be transmitted between individuals. The potential for helminths to play a role as pathogen vectors poses a potential livestock and human health risk. Further work is required to assess the epidemiological impact of this finding

<i>Campylobacter jejuni</i>transmission and colonisation in broiler chickens is inhibited by Faecal Microbiota Transplantation

ABSTRACT BACKGROUND Campylobacter jejuni, the most frequent cause of foodborne bacterial infection, is found on around 70% of retail chicken. As such there is a need for effective controls in chicken production. Microbial-based controls such as probiotics are attractive to the poultry industry, but of limited efficacy. Furthermore, as commercially-produced chickens have no maternal contact, their pioneer microbiome is likely to come from the hatchery environment. Early delivery of microbials that lead to a more ‘natural avian’ microbiome may, therefore, improve bird health and reduce susceptibility to C.jejuni colonisation. A faecal microbiota transplant (FMT) was used to transfer a mature cecal microbiome to newly-hatched broiler chicks and its effects on C.jejuni challenge assessed. We used both a seeder-bird infection model that mimics natural bird-to-bird infection alongside a direct-challenge model. We used a 16S rRNA gene sequencing-based approach to characterize the transplant material itself alongside changes to the chicken microbiome following FMT. RESULTS FMT changes the composition of the chicken intestinal microbiome. We observed little change in species richness following FMT compared to untreated samples, but there is an increase in phylogenetic diversity within those species. The most significant difference in the ceca is an increase in Lactobacilli, although not a major component of the transplant material, suggesting the FMT results in a change in the intestinal milieu as much as a direct change to the microbiome. Upon direct challenge, FMT resulted in lower initial intestinal colonisation with C.jejuni. More significantly, in a seeder-bird challenge of infection transmission, FMT reduced transmission and intestinal colonisation until common UK retail age of slaughter. In a repeat experiment, transmission was completely blocked following FMT treatment. Delayed FMT administration at 7 days of-age had limited effect on colonisation and transmission. CONCLUSIONS We show that transfer of a whole mature microbiome to newly-hatched chicks reduces transmission and colonisation of C.jejuni. This indicates that modification of the broiler chick microbiome can reduce intestinal colonisation of C.jejuni to levels projected to lead to lower the human infection rate. We believe these findings offer a way to identify key taxa or consortia that are effective in reducing C.jejuni colonisation and improving broiler gut health

Cytokine responses in birds challenged with the human food-borne pathogen Campylobacter jejuni implies a Th17 response

Development of process orientated understanding of cytokine interactions within the gastrointestinal tract during an immune response to pathogens requires experimentation and statistical modelling. The immune response against pathogen challenge depends on the specific threat to the host. Here, we show that broiler chickens mount a breed-dependent immune response to Campylobacter jejuni infection in the caeca by analysing experimental data using frequentist and Bayesian structural equation models (SEM). SEM provides a framework by which cytokine interdependencies, based on prior knowledge, can be tested. In both breeds important cytokines including pro-inflammatory interleukin (IL)-1β, , IL-4, IL-17A, interferon (IFN)-γ and anti-inflammatory IL-10 and transforming growth factor (TGF)-β4 were expressed post-challenge. The SEM revealed a putative regulatory pathway illustrating a T helper (Th)17 response and regulation of IL-10, which is breed-dependent. The prominence of the Th17 pathway indicates the cytokine response aims to limit the invasion or colonization of an extracellular bacterial pathogen but the time-dependent nature of the response differs between breeds

Genome-wide fitness analysis identifies genes required for in vitro growth and macrophage infection by African and global epidemic pathovariants of Salmonella enterica Enteritidis

Salmonella enterica Enteritidis is the second most common serovar associated with invasive non-typhoidal Salmonella (iNTS) disease in sub-Saharan Africa. Previously, genomic and phylogenetic characterization of S . enterica Enteritidis isolates from the human bloodstream led to the discovery of the Central/Eastern African clade (CEAC) and West African clade, which were distinct from the gastroenteritis-associated global epidemic clade (GEC). The African S . enterica Enteritidis clades have unique genetic signatures that include genomic degradation, novel prophage repertoires and multi-drug resistance, but the molecular basis for the enhanced propensity of African S . enterica Enteritidis to cause bloodstream infection is poorly understood. We used transposon insertion sequencing (TIS) to identify the genetic determinants of the GEC representative strain P125109 and the CEAC representative strain D7795 for growth in three in vitro conditions (LB or minimal NonSPI2 and InSPI2 growth media), and for survival and replication in RAW 264.7 murine macrophages. We identified 207 in vitro-required genes that were common to both S . enterica Enteritidis strains and also required by S . enterica Typhimurium, S . enterica Typhi and Escherichia coli , and 63 genes that were only required by individual S . enterica Enteritidis strains. Similar types of genes were required by both P125109 and D7795 for optimal growth in particular media. Screening the transposon libraries during macrophage infection identified 177 P125109 and 201 D7795 genes that contribute to bacterial survival and replication in mammalian cells. The majority of these genes have proven roles in Salmonella virulence. Our analysis uncovered candidate strain-specific macrophage fitness genes that could encode novel Salmonella virulence factors

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella

Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds ofSalmonellagenomes has revealed that ST313 is closely related to the ST19 group ofSTyphimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish AfricanSalmonellafrom ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis ofSalmonellaWe identified a SNP in the promoter of thepgtEgene that caused high expression of the PgtE virulence factor in AfricanS.Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by AfricanSTyphimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome

<i>Salmonella enterica</i>serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

AbstractInvasive non-typhoidalSalmonella(iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa.Salmonella entericaserovar Typhimurium ST313 causes the majority of iNTS in Malawi, and we performed an intensive comparative genomic analysis of 608 isolates obtained from fever surveillance at the Queen Elizabeth Hospital, Blantyre between 1996 and 2018. We discovered that following the upsurge of the well-characterisedS.Typhimurium ST313 lineage 2 from 1999 onwards, two new multidrug-resistant sublineages designated 2.2 and 2.3, emerged in Malawi in 2006 and 2008, respectively. The majority ofS.Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineage 2.2 or 2.3. To identify factors that characterised the emergence of the prevalent ST313 sublineage 2.2, we performed genomic and functional analysis of two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). Comparative genomic analysis showed that the chromosome of ST313 lineage 2 and sublineage 2.2 were broadly similar, only differing by 29 SNPs and small indels and a 3kb deletion in the Gifsy-2 prophage region that spanned thesseIpseudogene. Lineage 2 and sublineage 2.2 have unique plasmid profiles that were verified by long read sequencing. The transcriptome was initially explored in 15 infection-relevant conditions and within macrophages. Differential gene expression was subsequently investigated in depth in the four most importantin vitrogrowth conditions. We identified up-regulation of SPI2 genes in non-inducing conditions, and down-regulation of flagellar genes in D37712, compared to D23580. Following phenotypic confirmation of transcriptional differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed-growth in minimal media. We speculate that this competitive advantage is contributing to the continuing presence of sublineage 2.2 in Malawi.</jats:p

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.This work was funded by Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk) grant BB/K004514/1. D.P.W. was funded by a Wellcome Trust (https://wellcome.ac.uk) Infection and Immunity PhD rotation studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Nutritional determinants of bacterial intracellular replication: parasitation of host cell glucose metabolism by pathogenic listeria

Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis, a highly fatal food-borne infection. After entry into host cells, L. monocytogenes escapes from the phagocytic vacuole and replicates actively in the cytosol. Although the replication phase is essential for the establishment and survival of the pathogen within the host and its further transmission to new hosts, investigations into the microbial pathogenesis have largely neglected this phase of the infection process and therefore little is known about the nutritional determinants of microbial growth in vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Use of Galleria mellonella as a Model for Insect Vector Transmission of the Foodborne Pathogen Campylobacter jejuni in Broiler Chickens: A Pilot Study

There is growing pressure to find a way to eradicate or reduce the levels of foodborne pathogens such as Campylobacter in broiler chickens, whilst limiting the use of antimicrobials. For Campylobacter, there is currently no vaccine and on-farm biosecurity alone is insufficient to prevent colonization of broiler chicken flocks. Dipteran flies are proven carriers of Campylobacter and their entry into broiler houses may contribute to its transmission to broiler chickens. As there is currently no experimental vector transmission model for Campylobacter and chickens, we decided to examine experimentally whether Galleria mellonella could be used as vector to transmit Campylobacter to broiler chickens. More recently, the use of live insect feed has been proposed both for its nutritional qualities and improving bird welfare through the encouragement of natural foraging behaviours and it is unclear any risk this poses in terms of pathogen transmission. In this study, day-old chicks (n = 29) were obtained from a commercial hatchery. At three weeks of age, birds were split into 4 This groups; Group 1 was infected via oral gavage with 106 cells of C. jejuni-M1, Group 2 was fed Galleria mellonella infected with 106 cells of C. jejuni-M1, Group 3 was fed uninfected Galleria mellonella, whilst the remaining group was unchallenged. Cloacal swabs were taken at 2, 4, and 6 days post-infection (dpi) to follow transmission and at 8 dpi birds culled and C. jejuni load quantified in the caeca and liver. At 8 dpi, all birds in both the Campylobacter gavage group and those in the group fed the Campylobacter infected Galleria mellonella were Campylobacter positive, whereas those fed uninfected Galleria mellonella and the control group were all Campylobacter negative. The mean caecal Campylobacter load in the Campylobacter gavage group was 1.7 &times; 1010 per gram compared with 8.6 &times; 109 in the group fed the Campylobacter-infected Galleria mellonella. No liver positives were found in any of the groups. Our findings indicate that feeding broiler chickens with the vector Galleria mellonella infected with C. jejuni-M1 is sufficient to establish colonisation with C. jejuni. We propose that Galleria can be used as an easy and flexible model for vector transmission of foodborne pathogens in chicken

Use of Galleria mellonella as a Model for Insect Vector Transmission of the Foodborne Pathogen Campylobacter jejuni in Broiler Chickens: A Pilot Study

There is growing pressure to find a way to eradicate or reduce the levels of foodborne pathogens such as Campylobacter in broiler chickens, whilst limiting the use of antimicrobials. For Campylobacter, there is currently no vaccine and on-farm biosecurity alone is insufficient to prevent colonization of broiler chicken flocks. Dipteran flies are proven carriers of Campylobacter and their entry into broiler houses may contribute to its transmission to broiler chickens. As there is currently no experimental vector transmission model for Campylobacter and chickens, we decided to examine experimentally whether Galleria mellonella could be used as vector to transmit Campylobacter to broiler chickens. More recently, the use of live insect feed has been proposed both for its nutritional qualities and improving bird welfare through the encouragement of natural foraging behaviours and it is unclear any risk this poses in terms of pathogen transmission. In this study, day-old chicks (n = 29) were obtained from a commercial hatchery. At three weeks of age, birds were split into 4 This groups; Group 1 was infected via oral gavage with 106 cells of C. jejuni-M1, Group 2 was fed Galleria mellonella infected with 106 cells of C. jejuni-M1, Group 3 was fed uninfected Galleria mellonella, whilst the remaining group was unchallenged. Cloacal swabs were taken at 2, 4, and 6 days post-infection (dpi) to follow transmission and at 8 dpi birds culled and C. jejuni load quantified in the caeca and liver. At 8 dpi, all birds in both the Campylobacter gavage group and those in the group fed the Campylobacter infected Galleria mellonella were Campylobacter positive, whereas those fed uninfected Galleria mellonella and the control group were all Campylobacter negative. The mean caecal Campylobacter load in the Campylobacter gavage group was 1.7 × 1010 per gram compared with 8.6 × 109 in the group fed the Campylobacter-infected Galleria mellonella. No liver positives were found in any of the groups. Our findings indicate that feeding broiler chickens with the vector Galleria mellonella infected with C. jejuni-M1 is sufficient to establish colonisation with C. jejuni. We propose that Galleria can be used as an easy and flexible model for vector transmission of foodborne pathogens in chicken.</jats:p