Editorial: Integrating Whole Genome Sequencing Into Source Attribution and Risk Assessment of Foodborne Bacterial Pathogens

Source attribution and microbial risk assessment have proved to be crucial to identify and prioritize food safety interventions as to effectively control the burden of human illnesses (Cassini et al., 2016; Mughini-Gras et al., 2018a, 2019). By comparing human cases and pathogen occurrences in selected animal, food, and environmental sources, microbial subtyping approaches were successfully applied to pinpoint the most important sources of Salmonella, Campylobacter, Shiga toxin-producing Escherichia coli, and Listeria monocytogenes (Hald et al., 2004; Mullner et al., 2009a,b; Barco et al., 2013; Nielsen et al., 2017; Mughini-Gras et al., 2018b; Cody et al., 2019). Microbial risk assessment has been applied to assess known or potential adverse health effects resulting from human exposure to food-borne hazards. Through a scientific structured approach (FAO and WHO, 2021), microbial risk assessment helps to identify and quantify the risk represented by specific foods and the critical points in these foods' production chains for microbial control (Cassini et al., 2016; FAO and WHO, 2021). For both source attribution and risk assessment, one key challenge has been to define the hazard in question: is the whole foodborne pathogen species a hazard, or only some of its subtypes? In this regard the choice of the subtyping method becomes crucial. In recent years, Whole Genome Sequencing (WGS) has represented a major benefit for more targeted approaches, no longer focused on the species/genus level but at the level of subtypes (Franz et al., 2016; Fritsch et al., 2018; EFSA Panel on Biological Hazards, 2019). Besides WGS, metagenomics showed potentialities in source attribution. In particular, this approach was useful in attributing the source of environmental contamination by comparing the abundances of source-specific genetic markers (i.e., resistome) in different reservoirs (Gupta et al., 2019). Therefore, this special issue focuses on traditional and novel source attribution approaches applied on molecular, WGS, and metagenomic data as well as on a fine-tuning genetic characterization of foodborne pathogens useful for hazard identification and characterization. In particular, one study compares the outputs of a modified Hald model, which was applied to different subtyping input data of S. enterica Typhimurium and its monophasic variant (Arnold et al.) whereas two studies proposed a novel network approach and a method based on the core-genome genetic distance to attribute human infections of S. enterica Typhimurium monophasic variant and S. enterica Derby using WGS as input data (Merlotti et al.; Sévellec et al.). Another study by Duarte et al. included the relative abundance of antimicrobial resistance (AMR) associated genes (resistome) as metagenomic input data in an AMR source attribution study. Finally, two studies were focused on the molecular and genomic characterization of human isolates of Campylobacter jejuni and C. coli from China and of Listeria monocytogenes isolates collected from ready-to-eat meat and processing environment from Poland (Zhang et al.; Kurpas et al.). Arnold et al. performed a source attribution study including the genomes of S. enterica Typhimurium and its monophasic variant of 596 human sources and 327 animal sources from England and Wales between 2014 and 2016. Data from Seven Loci Multi Locus Sequence Typing (7-loci MLST), core-genome MLST (cg-MLST), and SNP calling were compared as input data. By applying a modified Hald model, 60% of human genomes were attributed to pork. Comparing different input data, results highlighted MLST as the method with the lowest fit and the lowest discriminatory power. Merlotti et al. applied a network approach to 351 human and animal genomes of S. enterica Typhimurium and its monophasic variant collected from 2013 to 2014. Three datasets of whole-genome MLST (wgMLST), cgMLST, and SNPs were used as input data. Genomes were clustered based on their genetic similarities. Interestingly, a higher percentage of cluster coherence was reported for animal sources in comparison to country and year of isolation, suggesting animal sources as the major driver of cluster formation. The approach showed to be effective in attributing up to 97.2% of human genomes to animal sources represented in the dataset. Among these genomes, the majority (84%) was attributed to pigs/pork. No significant differences were highlighted by comparing the three different input datasets. Core genome analysis was the approach applied by Sévellec et al. to attribute human sporadic cases of S. enterica Derby that occurred in France in 2014–2015 to non-human reservoirs. The authors analyzed 299 S. enterica Derby genomes corresponding to all S. enterica Derby sporadic human cases registered in the time frame, along with 141 non-human genomes. Within the non-human genomes, three main genomic lineages were detected in France: ST39-ST40 and ST682 associated to pork and ST71 associated to poultry. Within human genomes, 94% of S. enterica Derby clustered within the three genetic groups associated with pork, identifying this animal reservoir as the major contributor of S. enterica Derby to sporadic human cases in France. Relative abundance of antimicrobial resistance genes in shotgun metagenomic data was chosen in an antimicrobial resistance source attribution study by Duarte et al.. Starting from the assumption that fecal resistomes are source related, authors compared the resistomes of pooled fecal samples of pigs, broilers, turkeys, and veal calves with the resistomes of individual fecal samples of humans occupationally exposed to livestock production. Five supervised random forest models were applied on a total of 479 observations. Among the four livestock species, the results indicated that pigs have the resistome composition closest to the composition of the human resistome suggesting that occupational exposure to AMR determinants was higher among workers exposed to pigs than workers of broiler farms. Zhang et al. characterized genetic diversity and antimicrobial resistance of 236 Campylobacter jejuni and C. coli isolates collected from 2,945 individual stool samples of hospitalized patients with diarrhea in Beijing from 2017 to 2018. MLST results confirmed the high genetic diversity among isolates as well as CC21 as the most common clonal complex of C. jejuni in diarrhea patients in China. Clonal complex CC828 was the most frequently identified among C. coli isolates. Regarding antimicrobial resistance, rates higher than 88% were identified for the antimicrobials nalidixic acid, ciprofloxacin, and tetracycline. Last but not least, Kurpas et al. genetically characterized 48 L. monocytogenes isolates of PCR-serogroup IIb and IVb collected from ready-to-eat food and food processing environments. Additionally, the authors compared them with public genomes collected from humans in Poland. Among food isolates, 65% belonged to CC1, CC2, and CC6 already described as hypervirulent strains in humans. The clonal complex CC5 was also identified; mostly collected from food processing environments and belonging to PCR-serogroup IIB. Genomes of this clonal complex showed mutations in the inlA gene and a deletion of 144 bp in the inlB gene suggesting them as hypovirulent. Based on these studies, we conclude that the application of NGS data, in particular source attribution models, shows great potential. The results are improved by becoming more specific and to the point, which is considered very valuable for the decision support process. Integrations with phenotypic tests will continue to be essential for confirmation of NGS predicted outcomes

Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration

Abstract Background In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (τ) only up to a certain threshold cell density. Below this cell concentration (≤ 100 -1,000 CFU mL-1 ; ≤ 27-270 CFU well-1) we observed an obvious increase in the τ scatter. Results Working with a food-borne E. coli isolate we found that τ values derived from two different microtiter platereader-based techniques (i.e., optical density with growth time {=OD[t]} fit to the sigmoidal Boltzmann equation or time to calculated 1/2-maximal OD {=tm} as a function of initial cell density {=tm[CI]}) were in excellent agreement with the same parameter acquired from total aerobic plate counting. Thus, using either Luria-Bertani (LB) or defined (MM) media at 37°C, τ ranged between 17-18 (LB) or 51-54 (MM) min. Making use of such OD[t] data we collected many observations of τ as a function of manifold initial or starting cell concentrations (CI). We noticed that τ appeared to be distributed in two populations (bimodal) at low CI. When CI ≤100 CFU mL-1 (stationary phase cells in LB), we found that about 48% of the observed τ values were normally distributed around a mean (μτ1) of 18 ± 0.68 min (± στ1) and 52% with μτ2 = 20 ± 2.5 min (n = 479). However, at higher starting cell densities (CI>100 CFU mL-1), the τ values were distributed unimodally (μτ = 18 ± 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal τ distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. Conclusion These data argue that there is a dependence of growth rate on starting cell density.</p

The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

Reversal of the ΔdegP Phenotypes by a Novel rpoE Allele of Escherichia coli

RseA sequesters RpoE (σE) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σE to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σE regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σE levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σE-dependent RybB::LacZ construct showed only a weak activation of the σE pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σE and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrAL222Q, it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σE-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σE-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σE levels

Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur

Antibiotic Stress, Genetic Response and Altered Permeability of E. coli

BACKGROUND: Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure