High incidence of Epstein-Barr virus, cytomegalovirus, and human-herpes virus-6 reactivations in critically ill patients with COVID-19.

BACKGROUND: Systemic reactivation of herpesviruses may occur in intensive care unit (ICU) patients and is associated with morbidity and mortality. Data on severe Coronavirus disease-19 (COVID-19) and concomitant reactivation of herpesviruses are lacking. METHODS: We selected patients admitted to ICU for confirmed COVID-19 who underwent systematic testing for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human-herpes virus-6 (HHV-6) DNAemia while in the ICU. We retrospectively analysed frequency, timing, duration and co-occurrence of viral DNAemia. RESULTS: Thirty-four patients were included. Viremia with EBV, CMV, and HHV-6 was detected in 28 (82%), 5 (15%), and 7 (22%) patients, respectively. EBV reactivation occurred early after ICU admission and was associated with longer ICU length-of-stay. CONCLUSIONS: While in the ICU, critically ill patients with COVID-19 are prone to develop reactivations due to various types of herpesviruses.

Impact of a targeted isolation strategy at intensive-care-unit-admission on intensive-care-unit-acquired infection related to multidrug-resistant bacteria: a prospective uncontrolled before-after study.

Isolation of patients with multidrug resistant (MDR) bacteria is recommended to reduce cross-transmission of these bacteria. However, isolation of critically ill patients has several negative side effects. Therefore, we hypothesized that a targeted isolation strategy, based on the presence of at least one risk factor for MDR bacteria, would be not inferior to a systematic isolation strategy at intensive-care unit (ICU) admission. This prospective before-after study was conducted in a mixed ICU, during two 12-month periods, separated by a 1-month 'wash-out' period. During the before period, isolation was systematically performed in all patients at admission. During the after period, isolation was only performed in patients with at least one risk factor for MDR bacteria at admission. During the two periods, routine screening for MDR bacteria was performed at ICU admission, and isolation prescription was modified after receipt of screening result. Primary outcome was the percentage of patients with ICU-acquired infection (ICUAI) related to MDR bacteria, measured from ICU admission until ICU discharge or day 28, whatever happens first. A total of 1221 patients were included. No significant difference was found in ICUAI related to MDR bacteria (85 of 585 (14.5%) vs. 84 of 636 (13.2%) patients, risk difference, -1.3%, 95% confidence interval [-5.2 to 2.6%]) between the two periods, confirming the non-inferiority hypothesis. Our results suggest that targeted isolation of patients at ICU admission is not inferior to systematic isolation, regarding the percentage of patients with ICUAI related to MDR bacteria. Further randomized controlled multicentre studies are needed to confirm our results.

Functioning of DcuC as the C4-dicarboxylate carrier during glucose fermentation by Escherichia coli.

The dcuC gene of Escherichia coli encodes an alternative C4-dicarboxylate carrier (DcuC) with low transport activity. The expression of dcuC was investigated. dcuC was expressed only under anaerobic conditions; nitrate and fumarate caused slight repression and stimulation of expression, respectively. Anaerobic induction depended mainly on the transcriptional regulator FNR. Fumarate stimulation was independent of the fumarate response regulator DcuR. The expression of dcuC was not significantly inhibited by glucose, assigning a role to DcuC during glucose fermentation. The inactivation of dcuC increased fumarate-succinate exchange and fumarate uptake by DcuA and DcuB, suggesting a preferential function of DcuC in succinate efflux during glucose fermentation. Upon overexpression in a dcuC promoter mutant (dcuC*), DcuC was able to compensate for DcuA and DcuB in fumarate-succinate exchange and fumarate uptake.

Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange.

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.

Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, and O2-responsive, Fe containing transcriptional regulator of Escherichia coli.

FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor. The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state. It was analyzed which residues become accessible under either condition. Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation. By N-terminal sequencing, the carboxymethylated cysteine residues were identified. The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent. It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function. Potential modes of Fe-binding by the cysteine residues are discussed. In addition, a different type of interaction of Fe(II) with FNR is described. The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues.

A third periplasmic transport system for L-arginine in Escherichia coli: molecular characterization of the artPIQMJ genes, arginine binding and transport.

A new binding-protein-dependent transport system of Escherichia coli specific for L-arginine was characterized by genetic and biochemical means. The system is encoded by five adjacent genes, artPIQMJ (art standing for arginine transport), which are organized in two transcriptional units (artPIQM and artJ). The artl and artJ gene products (Artl and ArtJ) are periplasmic binding proteins with sequence similarity to binding proteins for polar (basic) amino acids. The artQ, artM and artP products are similar to the transmembraneous proteins and the ATPase of binding-protein-dependent carriers. The mature Artl and J proteins were localized in the periplasm and lacked signal peptides of 19 amino acid residues. Artl and ArtJ were isolated from overproducing strains. ArtJ specifically binds L-arginine with high affinity and overproduction of ArtJ stimulated L-arginine uptake by the bacteria. The substrate for Artl is not known, and isolated Artl did not bind common amino acids, various basic uncommon amino acids or amines. It is concluded that the artPIQM artJ genes encode a third arginine-uptake system in addition to the known argT hisJQMP system of Salmonella typhimurium and E. coli and the arginine (-ornithine) carrier (aps) of E. coli.

O2-sensing and O2-dependent gene regulation in facultatively anaerobic bacteria.

Availability of O2 is one of the most important regulatory signals in facultatively anaerobic bacteria. Various two- or one-component sensor/regulator systems control the expression of aerobic and anaerobic metabolism in response to O2. Most of the sensor proteins contain heme or Fe as cofactors that interact with O2 either by binding or by a redox reaction. The ArcA/ArcB regulator of aerobic metabolism in Escherichia coli may use a different sensory mechanism. In two-component regulators, the sensor is located in the cytoplasmic membrane, whereas one-component regulators are located in the cytoplasm. Under most conditions, O2 can readily reach the cytoplasm and could provide the signal in the cytoplasm. The transcriptional regulator FNR of E. Coli controls the expression of many genes required for anaerobic metabolism in response to O2. Functional homologs of FNR are present in facultatively anaerobic Proteobacteria and presumably also in gram-positive bacteria. The target genes of FNR are mostly under multiple regulation by FNR and other regulators that respond to O2, nitrate, or glucose. FNR represents a 'one-component' sensor/regulator and contains Fe for signal perception. In response to O2 availability, FNR is converted reversibly from the aerobic (inactive) state to the anaerobic (active) state. Experiments suggest that the Fe cofactor is bound by four essential cysteine residues. The O2-triggered transformation between active and inactive FNR presumably is due to a redox reaction at the Fe cofactor, but other modes of interaction cannot be excluded. O2 seems to affect the site-specific DNA binding of FNR at target genes or the formation of an active transcriptional complex with RNA polymerase.

Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct).

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Oxygen regulated gene expression in facultatively anaerobic bacteria.

In facultatively anaerobic bacteria such as Escherichia coli, oxygen and other electron acceptors fundamentally influence catabolic and anabolic pathways. E. coli is able to grow aerobically by respiration and in the absence of O2 by anaerobic respiration with nitrate, nitrite, fumarate, dimethylsulfoxide and trimethylamine N-oxide as acceptors or by fermentation. The expression of the various catabolic pathways occurs according to a hierarchy with 3 or 4 levels. Aerobic respiration at the highest level is followed by nitrate respiration (level 2), anaerobic respiration with the other acceptors (level 3) and fermentation. In other bacteria, different regulatory cascades with other underlying principles can be observed. Regulation of anabolism in response to O2 availability is important, too. It is caused by different requirements of cofactors or coenzymes in aerobic and anaerobic metabolism and by the requirement for different O2-independent biosynthetic routes under anoxia. The regulation mainly occurs at the transcriptional level. In E. coli, 4 global regulatory systems are known to be essential for the aerobic/anaerobic switch and the described hierarchy. A two-component sensor/regulator system comprising ArcB (sensor) and ArcA (transcriptional regulator) is responsible for regulation of aerobic metabolism. The FNR protein is a transcriptional sensor-regulator protein which regulates anaerobic respiratory genes in response to O2 availability. The gene activator FhlA regulates fermentative formate and hydrogen metabolism with formate as the inductor. ArcA/B and FNR directly respond to O2, FhlA indirectly by decreased levels of formate in the presence of O2. Regulation of nitrate/nitrite catabolism is effected by two 2-component sensor/regulator systems NarX(Q)/NarL(P) in response to nitrate/nitrite. Co-operation of the different regulatory systems at the target promoters which are in part under dual (or manifold) transcriptional control causes the expression according to the hierarchy. The sensing of the environmental signals by the sensor proteins or domains is not well understood so far. FNR, which acts presumably as a cytoplasmic 'one component' sensor-regulator, is suggested to sense directly cytoplasmic O2-levels corresponding to the environmental O2-levels.

Characterization of the FNR protein of Escherichia coli, an iron-binding transcriptional regulator.

FNR is a transcriptional regulator mediating the activation or repression of a variety of Escherichia coli genes in response to anoxia. The FNR protein resembles CRP (the cyclic-AMP receptor protein) except for the presence of a cysteine-rich N-terminal segment which may form part of an iron-binding redoxsensing domain. The FNR protein was purified by a new procedure. It was monomeric (Mr = 30,000) and contained as much as 1.1 mol of iron per monomer when purified in the presence of added iron. This iron was associated with cysteine residues, because there was an inverse relation between iron content and titratable sulphydryl groups. Other physical and chemical properties are reported including evidence for a potential disulphide group or analogous modification. The interaction between FNR protein and target DNA appeared weak and non-specific in gel-retardation assays, but specific binding to the proposed DNA-binding site was shown for the first time in footprinting studies. A role for iron in FNR-mediated gene expression was confirmed by using cultures in which FNR was inactivated by growth in the presence of the specific chelator, ferrozine, but protected by ferrous iron.