Hospital-onset COVID-19 infection surveillance systems: a systematic review.

Hospital-onset COVID-19 infections (HOCIs) are associated with excess morbidity and mortality in patients and healthcare workers. The aim of this review was to explore and describe the current literature in HOCI surveillance. Medline, EMBASE, the Cochrane Database of Systematic Reviews, the Cochrane Register of Controlled Trials, and MedRxiv were searched up to 30 November 2020 using broad search criteria. Articles of HOCI surveillance systems were included. Data describing HOCI definitions, HOCI incidence, types of HOCI identification surveillance systems, and level of system implementation were extracted. A total of 292 citations were identified. Nine studies on HOCI surveillance were included. Six studies reported on the proportion of HOCI among hospitalized COVID-19 patients, which ranged from 0 to 15.2%. Six studies provided HOCI case definitions. Standardized national definitions provided by the UK and US governments were identified. Four studies included healthcare workers in the surveillance. One study articulated a multimodal strategy of infection prevention and control practices including HOCI surveillance. All identified HOCI surveillance systems were implemented at institutional level, with eight studies focusing on all hospital inpatients and one study focusing on patients in the emergency department. Multiple types of surveillance were identified. Four studies reported automated surveillance, of which one included real-time analysis, and one included genomic data. Overall, the study quality was limited by the observational nature with short follow-up periods. In conclusion, HOCI case definitions and surveillance methods were developed pragmatically. Whilst standardized case definitions and surveillance systems are ideal for integration with existing routine surveillance activities and adoption in different settings, we acknowledged the difficulties in establishing such standards in the short-term.

Genome sequencing and characterization of an extensively drug-resistant sequence type 111 serotype O12 hospital outbreak strain of Pseudomonas aeruginosa.

A series of extensively drug-resistant isolates of Pseudomonas aeruginosa from two outbreaks in UK hospitals were characterized by whole genome sequencing (WGS). Although these isolates were resistant to antibiotics other than colistin, we confirmed that they are still sensitive to disinfectants. The sequencing confirmed that isolates in the larger outbreak were serotype O12, and also revealed that they belonged to sequence type ST111, which is a major epidemic strain of P. aeruginosa throughout Europe. As this is the first reported sequence of an ST111 strain, the genome was examined in depth, focusing particularly on antibiotic resistance and potential virulence genes, and on the reported regions of genome plasticity. High degrees of sequence similarity were discovered between outbreak isolates collected from recently infected patients, isolates from sinks, an isolate from the sewer, and a historical isolate, suggesting that the ST111 strain has been endemic in the hospital for many years. The ability to translate easily from outbreak investigation to detailed genome biology by use of the same data demonstrates the flexibility of WGS application in a clinical setting.

Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

Data reduction in headspace analysis of blood and urine samples for robust bacterial identification.

This paper demonstrates the application of chemical headspace analysis to the problem of classifying the presence of bacteria in biomedical samples by using computational tools. Blood and urine samples of disparate forms were analysed using a Cyrano Sciences C320 electronic nose together with an Agilent 4440 Chemosensor. The high dimensional data sets resulting from these devices present computational problems for parameter estimation of discriminant models. A variety of data reduction and pattern recognition techniques were employed in an attempt to optimise the classification process. A 100% successful classification rate for the blood data from the Agilent 4440 was achieved by combining a Sammon mapping with a radial basis function neural network. In comparison a successful classification rate of 80% was achieved for the urine data from the C320 which were analysed using a novel nonlinear time series model.

19F NMR study on the regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate by wild-type and Y385F p-hydroxybenzoate hydroxylase: evidence for a consecutive oxygenolytic dehalogenation mechanism.

The regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate (F4-POHB) by p-hydroxybenzoate hydroxylase (PHBH) and its active site mutant Y385F was investigated by 19F NMR. Evidence is provided that the hydroxylation of F4-POHB is not restricted to the C3 center of the aromatic ring but rather involves sequential oxygenation and dehalogenation steps. The catalytic efficiency of PHBH and Y385F with F4-POHB was optimal near pH 6.5. Below pH 7.0, substantial substrate inhibition occurred. Dianionic F4-POHB was a competent effector, highly stimulating upon binding the rate of flavin reduction by NADPH. Hydroxylation of F4-POHB involved the formation of quinone intermediates as primary products of oxygenolytic defluorination. Ascorbate competed favorably with NADPH for the nonenzymatic reduction of these reactive intermediates and prevented the accumulation of nonspecific oxidation products. 19F NMR showed that the initial aromatic product 2,5,6-trifluoro-3,4-dihydroxybenzoate (F3-DOHB) was further converted to 5,6-difluoro-2,3,4-trihydroxybenzoate (5,6-F2-TOHB). This reaction was most efficient with Y385F. F3-DOHB was not bound in a unique regiospecific orientation as also 2,6-difluoro-3,4, 5-trihydroxybenzoate (2,6-F2-TOHB) was formed. The oxygenolytic dehalogenation of F3-DOHB by PHBH and Y385F is consistent with the electrophilic aromatic substitution mechanism proposed for this class of flavoenzymes. Nucleophilic attack of the carbon centers of F3-DOHB onto the distal oxygen of the electrophilic flavin C(4a)-hydroperoxide occurs when the carbon center has a relatively high HOMO density and is relatively close to the distal oxygen of the flavin C(4a)-hydroperoxide.

Flavin motion in p-hydroxybenzoate hydroxylase. Substrate and effector specificity of the Tyr22-->Ala mutant.

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemistry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring.

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.

The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.

Selective cysteine-->serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.

Role of Tyr201 and Tyr385 in substrate activation by p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The crystal structure of the enzyme-substrate complex of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr-->Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201-->Phe and Tyr385-->Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p-hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzyme Tyr201-->Phe has a high Km for NADPH and produces only 5% of 3,4-dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the Tyr201-->Phe mutant does not stabilize the phenolate form of 4-hydroxybenzoate. With enzyme Tyr385-->Phe, flavin reduction is rate-limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385-->Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimulates the stabilization of the 4 alpha-hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3,4-dihydroxybenzoate. The affinity of enzyme Tyr201-->Phe for the dianionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment. In contrast to data reported for p-hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxycinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201-->Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385-->Phe, the phenolic pKa of bound 4-hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385-->Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.