Interplay between genetic regulation of phosphate homeostasis and bacterial virulence.

International audienceBacterial pathogens, including those of humans, animals, and plants, encounter phosphate (Pi)-limiting or Pi-rich environments in the host, depending on the site of infection. The environmental Pi-concentration results in modulation of expression of the Pho regulon that allows bacteria to regulate phosphate assimilation pathways accordingly. In many cases, modulation of Pho regulon expression also results in concomitant changes in virulence phenotypes. Under Pi-limiting conditions, bacteria use the transcriptional-response regulator PhoB to translate the Pi starvation signal sensed by the bacterium into gene activation or repression. This regulator is employed not only for the maintenance of bacterial Pi homeostasis but also to differentially regulate virulence. The Pho regulon is therefore not only a regulatory circuit of phosphate homeostasis but also plays an important adaptive role in stress response and bacterial virulence. Here we focus on recent findings regarding the mechanisms of gene regulation that underlie the virulence responses to Pi stress in Vibrio cholerae, Pseudomonas spp., and pathogenic E. coli

PhoB activates Escherichia coli O157:H7 virulence factors in response to inorganic phosphate limitation.

International audienceEnterohemorrhagic Escherichia coli (EHEC), an emerging food- and water-borne hazard, is highly pathogenic to humans. In the environment, EHEC must survive phosphate (Pi) limitation. The response to such Pi starvation is an induction of the Pho regulon including the Pst system that senses Pi variation. The interplay between the virulence of EHEC, Pho-Pst system and environmental Pi remains unknown. To understand the effects of Pi deprivation on the molecular mechanisms involved in EHEC survival and virulence under Pho regulon control, we undertook transcriptome profiling of the EDL933 wild-type strain grown under high Pi and low Pi conditions and its isogenic ΔphoB mutant grown in low Pi conditions. The differentially expressed genes included 1067 Pi-dependent genes and 603 PhoB-dependent genes. Of these 131 genes were both Pi and PhoB-dependent. Differentially expressed genes that were selected included those involved in Pi homeostasis, cellular metabolism, acid stress, oxidative stress and RpoS-dependent stress responses. Differentially expressed virulence systems included the locus of enterocyte effacement (LEE) encoding the type-3 secretion system (T3SS) and its effectors, as well as BP-933W prophage encoded Shiga toxin 2 genes. Moreover, PhoB directly regulated LEE and stx2 gene expression through binding to specific Pho boxes. However, in Pi-rich medium, constitutive activation of the Pho regulon decreased LEE gene expression and reduced adherence to HeLa cells. Together, these findings reveal that EHEC has evolved a sophisticated response to Pi limitation involving multiple biochemical strategies that contribute to its ability to respond to variations in environmental Pi and to coordinating the virulence response

Shiga toxins decrease enterohaemorrhagic Escherichia coli survival within Acanthamoeba castellanii.

International audienceEnterohaemorrhagic Escherichia coli (EHEC) are zoonotic pathogens transmitted to humans through contaminated water or bovine products. One of the strategies used by pathogenic bacteria to survive in aquatic environments is using free-living amoebae as hosts. Acanthamoeba castellanii is an amoeba known to host several waterborne pathogens. This study investigates the survival of EHEC with A. castellanii, which could contribute to its spread and transmission to humans. We used a gentamicin protection assay as well as fluorescence and electron microscopy to monitor the intra-amoebae survival of EHEC O157:H7 over 24 h. The results showed that EHEC were able to survive within A. castellanii and that this survival was reduced by Shiga toxins (Stx) produced by EHEC. A toxic effect mediated by Stx was demonstrated by amoebae mortality and LDH release during co-culture of EHEC and amoeba. This work describes the ability of EHEC to survive within A. castellanii, and this host-pathogen interaction is partially controlled by the Stx. Thus, this ubiquitous amoeba could represent an environmental niche for EHEC survival and transmission

The ecological habitat and transmission of Escherichia coli O157:H7.

International audienceSince its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escherichia coli O157:H7 has emerged as an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. In the last decade, increases in E. coli O157:H7 outbreaks were associated with environmental contamination in water and through fresh produce such as green leaves or vegetables. Both intrinsic (genetic adaptation) and extrinsic factors may contribute and help E. coli O157:H7 to survive in adverse environments. This makes it even more difficult to detect and monitor food and water safety for public health surveillance. E. coli O157:H7 has evolved in behaviors and strategies to persist in the environment

Impact of Raised without Antibiotics Measures on Antimicrobial Resistance and Prevalence of Pathogens in Sow Barns

The growing concern over the emergence of antimicrobial resistance (AMR) in animal production as a result of extensive and inappropriate antibiotic use has prompted many swine farmers to raise their animals without antibiotics (RWA). In this study, the impact of implementing an RWA production approach in sow barns on actual on-farm antibiotic use, the emergence of AMR, and the abundance of pathogens was investigated. Over a 13-month period, fecal and nasopharynx samples were collected at 3-month intervals from sows raised in RWA barns and sows in conventional barns using antibiotics in accordance with the new regulations (non-RWA). Whole genome sequencing (WGS) was used to determine the prevalence of AMR and the presence of pathogens in those samples. Records of all drug use from the 13-month longitudinal study indicated a significant reduction in antimicrobial usage in sows from RWA barns compared to conventional non-RWA barns. Antifolates were commonly administered to non-RWA sows, whereas β-lactams were widely used to treat sows in RWA barns. Metagenomic analyses demonstrated an increased abundance of pathogenic Actinobacteria, Firmicutes, and Proteobacteria in the nasopharynx microbiome of RWA sows relative to non-RWA sows. However, WGS analyses revealed that the nasal microbiome of sows raised under RWA production exhibited a significant increase in the frequency of resistance genes coding for β-lactams, MDR, and tetracycline

Bio-informatic analyses of PhoB-binding sites.

<p>Sequence logos determined from 12 putative PhoB-binding sites in EDL933 are indicated (upper panel). Potential consensus sequences identified in the promoter regions of LEE 1, LEE 2 operons and upstream <i>stx2AB</i> genes are shown with their statistical scores and genomic positions (lower panel). Pho box prediction probabilities were determined using the matrix frequencies of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094285#pone.0094285.s006" target="_blank">Table S4</a> that were uploaded into the Gibbs software algorithm. <a href="http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html" target="_blank">http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html</a>.</p

Piglet Gut and in-Barn Manure from Farms on a Raised without Antibiotics Program Display Reduced Antimicrobial Resistance but an Increased Prevalence of Pathogens

In response to new stringent regulations in Canada regarding the use of antibiotics in animal production, many farms have implemented practices to produce animals that are raised without antibiotics (RWA) from birth to slaughter. This study aims to assess the impact of RWA production practices on reducing the actual total on-farm use of antibiotics, the occurrence of pathogens, and the prevalence of antimicrobial resistance (AMR). A 28-month longitudinal surveillance of farms that adopted the RWA program and conventional farms using antibiotics in accordance with the new regulations (non-RWA) was conducted by collecting fecal samples from 6-week-old pigs and composite manure from the barn over six time points and applying whole-genome sequencing (WGS) to assess the prevalence of AMR genes as well as the abundance of pathogens. Analysis of in-barn drug use records confirmed the decreased consumption of antibiotics in RWA barns compared to non-RWA barns. WGS analyses revealed that RWA barns had reduced the frequency of AMR genes in piglet feces and in-barn manure. However, metagenomic analyses showed that RWA barns had a significant increase in the frequency of pathogenic Firmicutes in fecal samples and pathogenic Proteobacteria in barn manure samples

Global analysis of differentially expressed genes in response to Pi starvation or phoB inactivation in EDL933 strain.

<p><b>A.</b> Classification of genes whose expression levels were altered in Pi-dependent and in PhoB-dependent manners. Left and right circles indicate the differentially expressed genes of wild-type and Δ<i>phoB</i>-mutant strains with expression levels that were altered over 2-fold under Pi limitation. Venn diagram: Group 1 includes 936 PhoB-independent genes that are differentially expressed under Pi limitation in the wild-type strain, but that did not change in the Δ<i>phoB</i> mutant. Group 3 includes 472 PhoB-dependent genes differentially expressed in the Δ<i>phoB</i> mutant but did not change under Pi-limitation in the wild-type strain. Group 2 included 131 PhoB-dependent Pi response genes that are differentially expressed under Pi limitation in the wild-type strain and between the wild-type and the Δ<i>phoB</i> strains. <b>B.</b> Functional classification of genes with altered expression in strain EDL933 grown in Pi-limited conditions compared to cells grown in Pi-rich conditions (Pi-dependent (white bars)) and EDL933 incubated in Pi-limited conditions compared to Δ<i>phoB</i> mutant cells grown in the Pi-limited conditions (PhoB-dependent (gray bars)).</p

Effect of Pi and PhoB on <i>stx2</i> gene expression and toxin production.

<p>Low Pi and PhoB increased transcription of <i>stx2</i> and its toxin production and of BP933W genes except for repressor gene <i>cI</i> that was repressed. <b>A</b>. Heat map of the expression levels of BP933W genes between wild-type EDL933 strain grown in low or high Pi conditions and between wild-type and Δ<i>phoB</i> strain grown in low Pi conditions. <b>B.</b> The expression levels of the BP933W genes <i>cI, cro</i> and <i>stx2AB</i> were analyzed by RT-qPCR in the wild-type strain and the Δ<i>phoB</i> mutant grown in low or high Pi media as indicated. The wild-type strain induced by mitomycin C (WT+MitC) was used as a positive control <b>C.</b> Fluorescence of the wild-type EDL933 strain carrying a chromosomal fusion reporting <i>stx2</i> transcription level grown in Pi+ or Pi- conditions. <b>D.</b> The production of Stx2 measured by ELISA in extra-cellular protein (ECP) and whole cell protein (WCP) fractions of the EDL933 wild-type strain grown in Pi+ or Pi- conditions and in the Δ<i>phoB</i> mutant and its complemented derivative. Asterisks represent the significant ANOVA <i>P value</i> (*<0.05, **<0.01, ***<0.001).</p

Genes among upregulated and downregulated PhoB-dependent Pi response genes.

<p>Genes among upregulated and downregulated PhoB-dependent Pi response genes.</p