Publisher Correction: Genomic content typifying a prevalent clade of bovine mastitis-associated Escherichia coli.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibacterial Activities of Ga(III) against E. coli Are Substantially Impacted by Fe(III) Uptake Systems and Multidrug Resistance in Combination with Oxygen Levels.

The continued emergence and spread of antimicrobial resistance (AMR), particularly multidrug resistant (MDR) bacteria, are increasing threats driving the search for additional and alternative antimicrobial agents. The World Health Organization (WHO) has categorized bacterial risk levels and includes Escherichia coli among the highest priority, making this both a convenient model bacterium and a clinically highly relevant species on which to base investigations of antimicrobials. Among many compounds examined for use as antimicrobials, Ga(III) complexes have shown promise. Nonetheless, the spectrum of activities, susceptibility of bacterial species, mechanisms of antimicrobial action, and bacterial characteristics influencing antibacterial actions are far from being completely understood; these are important considerations for any implementation of an effective antibacterial agent. In this investigation, we show that an alteration in growth conditions to physiologically relevant lowered oxygen (anaerobic) conditions substantially increases the minimum inhibitory concentrations (MICs) of Ga(III) required to inhibit growth for 46 wild-type E. coli strains. Several studies have implicated a Trojan horse hypothesis wherein bacterial Fe uptake systems have been linked to the promotion of Ga(III) uptake and result in enhanced antibacterial activity. Our studies show that, conversely, the carriage of accessory Fe uptake systems (Fe_acc) significantly increased the concentrations of Ga(III) required for antibacterial action. Similarly, it is shown that MDR strains are more resistant to Ga(III). The increased tolerance of Fe_acc/MDR strains was apparent under anaerobic conditions. This phenomenon of heightened tolerance has not previously been shown although the mechanisms remain to be defined. Nonetheless, this further highlights the significant contributions of bacterial metabolism, fitness, and AMR characteristics and their implications in evaluating novel antimicrobials.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Substantial Extracellular Metabolic Differences Found Between Phylogenetically Closely Related Probiotic and Pathogenic Strains of Escherichia coli.

Since its first isolation a century ago, the gut inhabitant Escherichia coli strain Nissle 1917 has been shown to have probiotic activities; however, it is yet not fully elucidated which differential factors play key roles in its beneficial interactions with the host. To date, no metabolomics studies have been reported investigating the potential role of small molecules in functional strain differentiation of Nissle from its genetically close neighbors. Here, we present results of liquid chromatography coupled to high-resolution mass spectrometry characterization of extracellular metabolomes of E. coli strains as a proxy of their bioactivity potential. We found that phylogroup B2 strains exported a more diverse arsenal of metabolites than strains of other phylogroups. Zooming into the phylogroup B2 metabolome identified consistent substantial differences between metabolic output of E. coli Nissle and other strains, particularly in metabolites associated to the Argimine biosynthesis pathway. Nissle was found to release higher levels of Ornithine and Citrulline whilst depleting greater amounts of Arginine from the medium. Moreover, a novel Nissle-specific metabolite not reported before in bacteria, 5-(Carbamoylamino)-2-hydroxypentanoic acid (Citrulline/Arginic Acid related) was observed. Finally, Nissle, CFT073 and NCTC12241/ATCC25922 shared the excretion of N5-Acetylornithine, whereas other strains released N2-Acetylornithine or no N-Acetylornithine at all. Thus, we found substantial metabolic differences in phylogenetically very similar E. coli strains, an observation which suggests that it is justified to further investigate roles of small molecules as potential modulators of the gut environment by probiotic, commensal, and pathogenic strains, including E. coli Nissle 1917.

Author Correction: Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

The original version of this Article contained an error in the spelling of the author David Ruano-Gallego, which was incorrectly given as David R. Gallego. This has now been corrected in both the PDF and HTML versions of the Article.

Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence.

Variant O89 O-Antigen of E. coli Is Associated With Group 1 Capsule Loci and Multidrug Resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain - E. coli 26561 - represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) - 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

Postgenomics Characterization of an Essential Genetic Determinant of Mammary Pathogenic Escherichia coli.

Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental ("dairy-farm" E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.IMPORTANCE Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

A population genomics approach to exploiting the accessory 'resistome' of Escherichia coli.

The emergence of antibiotic resistance is a defining challenge, and Escherichia coli is recognized as one of the leading species resistant to the antimicrobials used in human or veterinary medicine. Here, we analyse the distribution of 2172 antimicrobial-resistance (AMR) genes in 4022 E. coli to provide a population-level view of resistance in this species. By separating the resistance determinants into 'core' (those found in all strains) and 'accessory' (those variably present) determinants, we have found that, surprisingly, almost half of all E. coli do not encode any accessory resistance determinants. However, those strains that do encode accessory resistance are significantly more likely to be resistant to multiple antibiotic classes than would be expected by chance. Furthermore, by studying the available date of isolation for the E. coli genomes, we have visualized an expanding, highly interconnected network that describes how resistances to antimicrobials have co-associated within genomes over time. These data can be exploited to reveal antimicrobial combinations that are less likely to be found together, and so if used in combination may present an increased chance of suppressing the growth of bacteria and reduce the rate at which resistance factors are spread. Our study provides a complex picture of AMR in the E. coli population. Although the incidence of resistance to all studied antibiotic classes has increased dramatically over time, there exist combinations of antibiotics that could, in theory, attack the entirety of E. coli, effectively removing the possibility that discrete AMR genes will increase in frequency in the population.

Genomic content typifying a prevalent clade of bovine mastitis-associated Escherichia coli.

E. coli represents a heterogeneous population with capabilities to cause disease in several anatomical sites. Among sites that can be colonised is the bovine mammary gland (udder) and a distinct class of mammary pathogenic E. coli (MPEC) has been proposed. MPEC are the principle causative agents of bovine mastitis in well-managed dairy farms, costing producers in the European Union an estimated €2 billion per year. Despite the economic impact, and the threat this disease presents to small and medium sized dairy farmers, the factors which mediate the ability for E. coli to thrive in bovine mammary tissue remain poorly elucidated. Strains belonging to E. coli phylogroup A are most frequently isolated from mastitis. In this paper, we apply a population level genomic analysis to this group of E. coli to uncover genomic signatures of mammary infectivity. Through a robust statistical analysis, we show that not all strains of E. coli are equally likely to cause mastitis, and those that do possess specific gene content that may promote their adaptation and survival in the bovine udder. Through a pan-genomic analysis, we identify just three genetic loci which are ubiquitous in MPEC, but appear dispensable for E. coli from other niches.

Draft Genome Sequences of Enterohemorrhagic Escherichia coli Encoding Extended-Spectrum Beta-Lactamases.

Extended-spectrum beta-lactamases (ESBLs) have rarely been observed among Shiga toxigenic Escherichia coli (STEC), and, to our best knowledge, only three ESBL-positive isolates of the enterohemorrhagic E. coli (EHEC) subpathotype have been reported. Here, we present the first draft genome sequences of two ESBL-positive EHEC isolates belonging to serotypes O111:H8 and O151:H16.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis.

Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit-variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates.

The host metabolite D-serine contributes to bacterial niche specificity through gene selection.

Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host-pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an 'evolutionary incompatibility' between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity.

Genome-wide evaluation of the interplay between Caenorhabditis elegans and Yersinia pseudotuberculosis during in vivo biofilm formation.

The formation of an incapacitating biofilm on Caenorhabditis elegans by Yersinia pseudotuberculosis represents a tractable model for investigating the genetic basis for host-pathogen interplay during the biofilm-mediated infection of a living surface. Previously we established a role for quorum sensing (QS) and the master motility regulator, FlhDC, in biofilm formation by Y. pseudotuberculosis on C. elegans. To obtain further genome-wide insights, we used transcriptomic analysis to obtain comparative information on C. elegans in the presence and absence of biofilm and on wild-type Y. pseudotuberculosis and Y. pseudotuberculosis QS mutants. Infection of C. elegans with the wild-type Y. pseudotuberculosis resulted in the differential regulation of numerous genes, including a distinct subset of nematode C-lectin (clec) and fatty acid desaturase (fat) genes. Evaluation of the corresponding C. elegans clec-49 and fat-3 deletion mutants showed delayed biofilm formation and abolished biofilm formation, respectively. Transcriptomic analysis of Y. pseudotuberculosis revealed that genes located in both of the histidine utilization (hut) operons were upregulated in both QS and flhDC mutants. In addition, mutation of the regulatory gene hutC resulted in the loss of biofilm, increased expression of flhDC, and enhanced swimming motility. These data are consistent with the existence of a regulatory cascade in which the Hut pathway links QS and flhDC. This work also indicates that biofilm formation by Y. pseudotuberculosis on C. elegans is an interactive process during which the initial attachment/recognition of Yersinia to/by C. elegans is followed by bacterial growth and biofilm formation.

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species.

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole-genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype-host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.

Genomic characterisation of an endometrial pathogenic Escherichia coli strain reveals the acquisition of genetic elements associated with extra-intestinal pathogenicity.

BACKGROUND: Strains of Escherichia coli cause a wide variety of intestinal and extra-intestinal diseases in both humans and animals, and are also often found in healthy individuals or the environment. Broadly, a strong phylogenetic relationship exists that distinguishes most E. coli causing intestinal disease from those that cause extra-intestinal disease, however, isolates within a recently described subclass of Extra-Intestinal Pathogenic E. coli (ExPEC), termed endometrial pathogenic E. coli, tend to be phylogenetically distant from the vast majority of characterised ExPECs, and more closely related to human intestinal pathogens. In this work, we investigate the genetic basis for ExPEC infection in the prototypic endometrial pathogenic E. coli strain MS499. RESULTS: By investigating the genome of MS499 in comparison with a range of other E. coli sequences, we have discovered that this bacterium has acquired substantial lengths of DNA which encode factors more usually associated with ExPECs and less frequently found in the phylogroup relatives of MS499. Many of these acquired factors, including several iron acquisition systems and a virulence plasmid similar to that found in several ExPECs such as APEC O1 and the neonatal meningitis E. coli S88, play characterised roles in a variety of typical ExPEC infections and appear to have been acquired recently by the evolutionary lineage leading to MS499. CONCLUSIONS: Taking advantage of the phylogenetic relationship we describe between MS499 and several other closely related E. coli isolates from across the globe, we propose a step-wise evolution of a novel clade of sequence type 453 ExPECs within phylogroup B1, involving the recruitment of ExPEC virulence factors into the genome of an ancestrally non-extraintestinal E. coli, which has repurposed this lineage with the capacity to cause extraintestinal disease. These data reveal the genetic components which may be involved in this phenotype switching, and argue that horizontal gene exchange may be a key factor in the emergence of novel lineages of ExPECs.

Draft Genome Sequence of Escherichia coli MS499, Isolated from the Infected Uterus of a Postpartum Cow with Metritis.

Specific Escherichia coli strains associated with bovine postpartum uterine infection have recently been described. Many recognized virulence factors are absent in these strains; therefore, to define a prototypic strain, we report here the genome sequence of E. coli isolate MS499 from a cow with the postpartum disease metritis.

Draft Genome Sequence of Trueperella pyogenes, Isolated from the Infected Uterus of a Postpartum Cow with Metritis.

Trueperella pyogenes is a common commensal bacterium and an opportunistic pathogen associated with chronic purulent disease, particularly in ruminants. We report here the genome sequence of a T. pyogenes isolate from a severe case of bovine metritis. This is the first full record of a T. pyogenes genome.