Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

BACKGROUND: Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. METHODS: A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. RESULTS: We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. CONCLUSIONS: We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

Design and use of photoactive ruthenium complexes to study electron transfer within cytochrome bc1 and from cytochrome bc1 to cytochrome c.

The cytochrome bc1 complex (ubiquinone:cytochrome c oxidoreductase) is the central integral membrane protein in the mitochondrial respiratory chain as well as the electron-transfer chains of many respiratory and photosynthetic prokaryotes. Based on X-ray crystallographic studies of cytochrome bc1, a mechanism has been proposed in which the extrinsic domain of the iron-sulfur protein first binds to cytochrome b where it accepts an electron from ubiquinol in the Qo site, and then rotates by 57° to a position close to cytochrome c1 where it transfers an electron to cytochrome c1. This review describes the development of a ruthenium photooxidation technique to measure key electron transfer steps in cytochrome bc1, including rapid electron transfer from the iron-sulfur protein to cytochrome c1. It was discovered that this reaction is rate-limited by the rotational dynamics of the iron-sulfur protein rather than true electron transfer. A conformational linkage between the occupant of the Qo ubiquinol binding site and the rotational dynamics of the iron-sulfur protein was discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method is also described for the measurement of electron transfer from cytochrome c1 to cytochrome c. This article is part of a Special Issue entitled: Respiratory Complex III and related bc complexes.

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL.

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc1 (cyt bc1) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c1. When cyt c1, the iron­sulfur center Fe2S2, and cyt bH were reduced before the reaction, photooxidation of cyt c1 led to electron transfer from Fe2S2 to cyt c1 with a rate constant of ka = 80,000 s-1, followed by bifurcated reduction of both Fe2S2 and cyt bL by QH2 in the Qo site with a rate constant of k2 = 3000 s-1. The resulting Q then traveled from the Qo site to the Qi site and oxidized one equivalent each of cyt bL and cyt bH with a rate constant of k3 = 340 s-1. The rate constant ka was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron­sulfur protein from the b state with reduced Fe2S2 close to cyt bL to one or more intermediate states, followed by rotation to the final c1 state with Fe2S2 close to cyt c1, and rapid electron transfer to cyt c1.

The acidic domain of cytochrome c1 in paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc1 complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c(552).

The cytochrome bc(1) complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c(1) subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c(552).

Photoinitiated electron transfer within the Paracoccus denitrificans cytochrome bc1 complex: mobility of the iron-sulfur protein is modulated by the occupant of the Q(o) site.

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c(1) redox centers plays a key role in the mechanism of the cyt bc(1) complex. Electron transfer within the cyt bc(1) complex of Paracoccus denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c(1) within 1 µs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc(1) complex at pH 8.0, electron transfer from reduced ISP to cyt c(1) was biphasic with rate constants of k(1f) = 6300 ± 3000 s(-1)and k(1s) = 640 ± 300 s(-1) and amplitudes of 10 ± 3% and 16 ± 4% of the total amount of cyt c(1) photooxidized. Upon addition of any of the P(m) type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc(1) in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the P(f) type inhibitors JG-144 and famoxadone decreased the rate constant k(1f) by 5-10-fold and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a 2-fold increase in the amplitude of the k(1f) phase. The effect of QH(2) on the kinetics of electron transfer from reduced ISP to cyt c(1) was thus similar to that of the P(m) inhibitors and very different from that of the P(f) inhibitors. The current results indicate that the species occupying the Q(o) site has a significant conformational influence on the dynamics of the ISP domain rotation.

A new approach to the furan degradation problem involving ozonolysis of the trans-enedione and its use in a cost-effective synthesis of eplerenone.

[reaction: see text] Whereas ozonization of furan 3a affords little or no carboxylic acid 5, ozonization of the corresponding trans-enedione 6 afforded carboxylic acid 5 in 82.4% yield (cryst., overall from furan, 100 g scale; after workup with dimethyl sulfide, followed by mildly basic hydrogen peroxide). This new approach to furan degradation is showcased in a cost-effective synthesis of eplerenone, an important new medicine for cardiovascular indications.

Early amidation approach to 3-[(4-amido)pyrrol-2-yl]-2-indolinones.

A new synthesis of 3-[(4-amido)pyrrol-2-yl]-2-indolinones has been developed, where the amide side chain was installed prior to pyrrole formation. This strategy precludes the need to use any coupling reagents to install the amide side chain. This process includes a zinc-free alternative to the Knorr pyrrole synthesis.