Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May-July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.     

The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing

Phagocytosis is a primary defense program orchestrated by monocytes/macrophages. Unregulated phagocytosis can lead to pathological conditions. In the current study we have demonstrated that Wnt5a stimulates phagocytosis through PI3 kinase–Rac1 and lipid-raft-dependent processes. Wnt5a-mediated augmentation in phagocytosis is suppressed by blocking expression of the putative Wnt5a receptor Frizzled 5. Enhanced phagocytosis of bacteria by Wnt5a–Fz5 signaling increases the secretion of proinflammatory cytokines, but not the bacterial killing rate. Furthermore, a small molecule inhibitor of Wnt production, IWP-2, which reduces secretion of functionally active Wnt5a, not only suppresses both phagocytosis and the secretion of proinflammatory cytokines but also accelerates the bacterial killing rate.     

Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Voltage-dependent potassium channels (Kv) are homotetramers composed of four voltage sensors and one pore domain. Because of high-level structural flexibility, the first mammalian Kv structure, Kv1.2 at 2.9 Å, has about 37% molecular mass of the transmembrane portion not resolved. In this study, by applying a novel normal-mode-based X-ray crystallographic refinement method to the original diffraction data and structural model, we established the structure of full-length Kv1.2 in its native form. This structure offers mechanistic insights into voltage sensing. Particularly, it shows a hydrophobic layer of about 10 Å at the midpoint of the membrane bilayer, which is likely the molecular basis for the observed “focused electric field” of Kv1.2 between the internal and external solutions. This work also demonstrated the potential of the refinement method in bringing up large chunks of missing densities, thus beneficial to structural refinement of many difficult systems.     

A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis

Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H⁻, and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates.     

Endemically acquired foodborne outbreak of enterotoxin-producing Escherichia coli serotype O169:H41

Purpose

Enterotoxigenic Escherichia coli (ETEC) is traditionally recognized as a common cause of traveler’s diarrhea, but is becoming a more frequent cause of foodborne disease outbreaks in the United States. It is important for public health practitioners and clinicians to be aware of ETEC as a domestic cause of gastroenteritis. We investigated a foodborne disease outbreak to understand the epidemiology of ETEC in this setting.

Methods

We conducted a cohort study of 63 employees of Company A. A case was defined as an employee who experienced diarrhea or vomiting or fever and cramps after eating a catered meal at Company A from August 14th-15th. A standardized questionnaire was administered to cases and controls.

Results

Of 63 employees, 36 met the case definition (Attack Rate = 57.1%). Diarrhea (94%) and cramps (74%) were common, whereas vomiting was not (3%). Mean duration of illness was 2.7 days. Coleslaw at the August 15th lunch was significantly associated with illness (Odds ratio = 4.4, 95% CI = 1.1-17). Stool specimens were positive for heat-stable enterotoxin-producing E. coli O169:H41. Contamination likely occurred at the point of service.

Conclusions

This outbreak illustrates the changing epidemiology of enterotoxigenic E. coli and the importance for healthcare practitioners to consider ETEC as a potential cause of domestically acquired gastroenteritis.     

Surveillance of ESBL producing multidrug resistant Escherichia coli in a teaching hospital in India

Objective

To record nosocomial and community-acquired accounts of antibiotic resistance in Escherichia coli (E. coli) strains, isolated from clinical samples of a teaching hospital by surveillance, over a period of 39 months (November 2009-January 2013).

Methods

Clinical samples from nosocomial sources, i.e., wards and cabins, intensive care unit (ICU) and neonatal intensive care unit (NICU), and community (outpatient department, OPD) sources of the hospital, were used for isolating strains of E. coli, which were subjected for testing for production of ‘extended spectrum beta-lactamase’-(ESBL) enzyme as well as determining antibiotic sensitivity pattern with 23 antibiotics.

Results

Of the total 1642 (100%) isolates, 810 (49.33%) strains were from OPD and 832 (50.66%) were from hospital settings. Occurrence of infectious E. coli strains increased in a mathematical progression in community sources, but in nosocomial infections, such values remained almost constant in each quarter. A total of 395 (24.05%) ESBL strains were isolated from the total 810 isolates of community; of the total of 464 (28.25%) isolates of wards and cabins, 199 (12.11%) were ESBL strains; and among the total of 368 (22.41%) isolates of ICU and NICU, ESBLs were 170 (10.35%); the total nosocomial ESBL isolates, 369 (22.47%) were from the nosocomial total of 832 (50.66%) isolates. Statistically, it was confirmed that ESBL strains were equally distributed in community or hospital units. Antibiogram of 23 antibiotics revealed progressive increases of drug-resistance against each antibiotic with the maximum resistance values were recorded against gentamicin: 92% and 79%, oxacillin: 94% and 69%, ceftriaxone: 85% and 58%, and norfloxacin 97% and 69% resistance, in nosocomial and community isolates, respectively.

Conclusions

This study revealed the daunting state of occurrence of multidrug resistant E. coli and its infection dynamics in both community and hospital settings.     

Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete ~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.     

Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA–oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA–oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.     

Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K+ channel subunit

During nervous system development, synapses undergo morphological change as a function of electrical activity. In Drosophila, enhanced activity results in the expansion of larval neuromuscular junctions. We have examined whether these structural changes involve the pre- or postsynaptic partner by selectively enhancing electrical excitability with a Shaker dominant-negative (SDN) potassium channel subunit. We find that the SDN enhances neurotransmitter release when expressed in motoneurons, postsynaptic potential broadening when expressed in muscles and neurons, and selectively suppresses fast-inactivating, Shaker-mediated IA currents in muscles. SDN expression also phenocopies the canonical behavioral phenotypes of the Sh mutation. At the neuromuscular junction, we find that activity-dependent changes in arbor size occur only when SDN is expressed presynaptically. This finding indicates that elevated postsynaptic membrane excitability is by itself insufficient to enhance presynaptic arbor growth. Such changes must minimally involve increased neuronal excitability.     

Collateral effects of antibiotics on mammalian gut microbiomes

Antibiotics are an essential component of the modern lifestyle. They improve our lives by treating disease, preventing disease, and in the case of agricultural animals by improving feed efficiency. However, antibiotic usage is not without collateral effects. The development and spread of antibiotic resistance is the most notorious concern associated with antibiotic use. New technologies have enabled the study of how the microbiota responds to the antibiotic disturbance, including how the community recovers after the antibiotic is removed. One common theme in studies of antibiotic effects is a rapid increase in Escherichia coli followed by a gradual decline. Increases in E. coli are also associated with systemic host stresses, and may be an indicator of ecosystem disturbances of the intestinal microbiota. Moreover, recent studies have shown additional effects mediated by antibiotics on the gut microbiota, such as the stimulation of gene transfer among gut bacteria and the reduction of immune responses in peripheral organs. Querying the microbiota after antibiotic treatment has led to intriguing hypotheses regarding predicting or mitigating unfavorable treatment outcomes. Here we explore the varied effects of antibiotics on human and animal microbiotas.     

Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO₂, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H⁺-ATPase, and plasma membrane inward-rectifying K⁺ channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO₂ uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H⁺-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42–63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36–41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K⁺ channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H⁺-ATPase in guard cells is useful for the promotion of plant growth.In the present era of global climate changes and the threat of food insufficiency, finding ways to improve the uptake of CO₂ by terrestrial plants is an increasingly important problem. Stomata, key organs in the uptake of CO₂, are microscopic pores surrounded by two specialized cells in the epidermis (named guard cells) and are mainly found on the leaf surface in terrestrial plants. Because the leaf surface is nearly impermeable to air and water, stomata provide the major pathway for the diffusion of CO₂, O₂, and water vapor between the ambient atmosphere and the interior of the leaf. This facilitation of gas exchange by stomatal opening is one of the most essential processes in plant photosynthesis and transpiration (1, 2). A recent study indicated that stomatal transpiration limited photosynthesis in rice (3). Therefore, increased stomatal opening/transpiration is expected to promote photosynthesis and thereby increase plant growth. Condon et al. (4) examined diverse wheat genotypes and showed that increased stomatal conductance, especially abaxial stomatal conductance, may have a positive effect on crop biomass. However, to our knowledge, no previous studies have determined stomatal effects on plant growth by manipulating stomatal aperture via gene regulation in guard cells, perhaps because of the difficulty in balancing the counteracting effects of taking up CO₂ while losing water vapor through the stomata (5).Light is one of the principal factors that stimulates stomatal opening, and various mechanisms underlie stomatal opening in response to different light wavelengths (6–8). Red light is thought to induce stomatal opening via photosynthesis in the mesophyll and guard cell chloroplasts, as well as the reduction of the intercellular CO₂ concentration (Ci) (5, 9, 10). However, the detailed mechanisms of stomatal responses to red light are under debate (11, 12). In contrast, blue light acts as a signal and exerts the most pronounced effect on stomatal opening. The blue light receptors phototropins (phot1 and phot2) activate plasma membrane H⁺-ATPase through the phosphorylation of the penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated threonine (13–15). Blue light-activated H⁺-ATPase induces hyperpolarization of the plasma membrane, which allows K⁺ uptake through inward-rectifying K⁺ (K⁺_in) channels (16–21). Accumulation of K⁺ induces the swelling of guard cells and stomatal opening. Thus, these three components (phototropins, plasma membrane H⁺-ATPase, and K⁺_in channels) have important roles in blue light-induced stomatal opening. In addition to these components, FLOWERING LOCUS T (FT) is suggested to be a positive regulator for stomatal opening via its effect on the activation status of the plasma membrane H⁺-ATPase (22).In this study, we produced transgenic plants expressing key components active in stomatal opening under the control of the strong guard cell promoter GC1 to promote stomatal opening in Arabidopsis thaliana (23). We showed that transgenic Arabidopsis plants overexpressing H⁺-ATPase in guard cells exhibited enhanced light-induced stomatal opening, photosynthesis, and plant growth, and that stomatal aperture is a limiting factor in photosynthesis and plant growth.     

Impermanence of bacterial clones

Bacteria reproduce asexually and pass on a single genome copied from the parent, a reproductive mode that assures the clonal descent of progeny; however, a truly clonal bacterial species is extremely rare. The signal of clonality can be interrupted by gene uptake and exchange, initiating homologous recombination that results in the unique sequence of one clone being incorporated into another. Because recombination occurs sporadically and on local scales, these events are often difficult to recognize, even when considering large samples of completely sequenced genomes. Moreover, several processes can produce the appearance of clonality in populations that undergo frequent recombination. The rates and consequences of recombination have been studied in Escherichia coli for over 40 y, and, during this time, there have been several shifting views of its clonal status, population structure, and rates of gene exchange. We reexamine the studies and retrace the evolution of the methods that have assessed the extent of DNA flux, largely focusing on its impact on the E. coli genome.     

High frequency of hotspot mutations in core genes of Escherichia coli due to short-term positive selection

Core genes comprising the ubiquitous backbone of bacterial genomes are not subject to frequent horizontal transfer and generally are not thought to contribute to the adaptive evolution of bacterial pathogens. We determined, however, that at least one-third and possibly more than one-half of the core genes in Escherichia coli genomes are targeted by repeated replacement substitutions in the same amino acid positions—hotspot mutations. Occurrence of hotspot mutations is driven by positive selection, as their rate is significantly higher than expected by random chance alone, and neither intragenic recombination nor increased mutability can explain the observed patterns. Also, commensal E. coli strains have a significantly lower frequency of mutated genes and mutations per genome than pathogenic strains. E. coli strains causing extra-intestinal infections accumulate hotspot mutations at the highest rate, whereas the highest total number of mutated genes has been found among Shigella isolates, suggesting the pathoadaptive nature of such mutations. The vast majority of hotspot mutations are of recent evolutionary origin, implying short-term positive selection, where adaptive mutations emerge repeatedly but are not sustained in natural circulation for long. Such pattern of dynamics is consistent with source-sink model of virulence evolution.