Tree-Based Unrooted Phylogenetic Networks

Phylogenetic networks are a generalization of phylogenetic trees that are used to represent non-tree-like evolutionary histories that arise in organisms such as plants and bacteria, or uncertainty in evolutionary histories. An unrooted phylogenetic network on a non-empty, finite set X of taxa, or network, is a connected, simple graph in which every vertex has degree 1 or 3 and whose leaf set is X. It is called a phylogenetic tree if the underlying graph is a tree. In this paper we consider properties of tree-based networks, that is, networks that can be constructed by adding edges into a phylogenetic tree. We show that although they have some properties in common with their rooted analogues which have recently drawn much attention in the literature, they have some striking differences in terms of both their structural and computational properties. We expect that our results could eventually have applications to, for example, detecting horizontal gene transfer or hybridization which are important factors in the evolution of many organisms. Correction available at dx.doi.org/10.1007/s11538-018-0530-

Crystal structures of type-II inositol polyphosphate 5-phosphatase INPP5B with synthetic inositol polyphosphate surrogates reveal new mechanistic insights for the inositol 5-phosphatase family

The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3′,4,4′,5,5′-hexakisphosphate [BiPh­(3,3′,4,4′,5,5′)­P₆, IC₅₀ 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz­(1,2,3)­P₃] [one ring of BiPh­(3,3′,4,4′,5,5′)­P₆] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz­(1,2,4,5)­P₄, IC₅₀ = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz­(1,2,4,5)­P₄ and BiPh­(3,3′,4,4′,5,5′)­P₆ similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B–BiPh­(3,3′,4,4′,5,5′)­P₆ complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed “moving metal” mechanism

Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®Green Real-Time PCR Methods and High Resolution Melting Analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform

Enterohaemorrhagic Escherichia coli and Shigella dysenteriae type 1-induced haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) can be classified according to the aetiology of the different disorders from which it is composed. The most prevalent form is that induced by shigatoxin producing Escherichia coli (STEC) and, in some tropical regions, by Shigella dysenteriae type 1. STEC cause a zoonosis, are widely distributed in nature, enter the food chain in different ways, and show regional differences. Not all STEC are human pathogens. Enterohaemorrhagic E. coli usually cause attachment and effacing lesions in the intestine. This is not essential, but production of a shigatoxin (Stx) is. Because Stx are encoded by a bacteriophage, this property is transferable to naïve strains. Laboratory methods have improved by identifying STEC either via the toxin or its bacteriophage. Shigella dysenteriae type 1 produces shigatoxin, identical to Stx-1, but also has entero-invasive properties that enterohaemorrhagic Escherichia coli (EHEC) do not. Shigella patients risk bacteremia and benefit from early antibiotic treatment, unlike those with EHEC

Escherichia coli Bacteriocins: Antimicrobial Efficacy and Prevalence among Isolates from Patients with Bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin

Differential Gene Expression and Adherence of Escherichia coli O157:H7 In Vitro and in Ligated Pig Intestines

BACKGROUND: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. METHODOLOGY/PRINCIPAL FINDINGS: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15), TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48), Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. CONCLUSIONS/SIGNIFICANCE: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further

A Novel Escherichia coli O157:H7 Clone Causing a Major Hemolytic Uremic Syndrome Outbreak in China

An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks