Advances in Host Depletion and Pathogen Enrichment Methods for Rapid Sequencing-Based Diagnosis of Bloodstream Infection.

Bloodstream infection is a major cause of morbidity and death worldwide. Timely and appropriate treatment can reduce mortality among critically ill patients. Current diagnostic methods are too slow to inform precise antibiotic choice, leading to the prescription of empirical antibiotics, which may fail to cover the resistance profile of the pathogen, risking poor patient outcomes. Additionally, overuse of broad-spectrum antibiotics may lead to more resistant organisms, putting further pressure on the dwindling pipeline of antibiotics, and risk transmission of these resistant organisms in the health care environment. Therefore, rapid diagnostics are urgently required to better inform antibiotic choice early in the course of treatment. Sequencing offers great promise in reducing time to microbiological diagnosis; however, the amount of host DNA compared with the pathogen in patient samples presents a significant obstacle. Various host-depletion and bacterial-enrichment strategies have been used in samples, such as saliva, urine, or tissue. However, these methods have yet to be collectively integrated and/or extensively explored for rapid bloodstream infection diagnosis. Although most of these workflows possess individual strengths, their lack of analytical/clinical sensitivity and/or comprehensiveness demands additional improvements or synergistic application. This review provides a distinctive classification system for various methods based on their working principles to guide future research, and discusses their strengths and limitations and explores potential avenues for improvement to assist the reader in workflow selection.

Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study.

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster under iron limitation.

IMPORTANCE: Campylobacter jejuni is a bacterium that is prevalent in the ceca of farmed poultry such as chickens. Consumption of ill-prepared poultry is thus the most common route by which C. jejuni infects the human gut to cause a typically self-limiting but severe gastrointestinal illness that can be fatal to very young, old, or immunocompromised people. The lack of a vaccine and an increasing resistance to current antibiotics highlight a need to better understand the mechanisms that make C. jejuni a successful human pathogen. This study focused on the functional components of one such mechanism-a molecular system that helps C. jejuni thrive despite the restriction on growth-available iron by the human body, which typically defends against pathogens. In providing a deeper understanding of how this system functions, this study contributes toward the goal of reducing the enormous global socioeconomic burden caused by C. jejuni.

Acidic pH modulates Burkholderia cenocepacia antimicrobial susceptibility in the cystic fibrosis nutritional environment.

IMPORTANCE: Burkholderia cenocepacia causes severe infections in cystic fibrosis (CF) patients. CF patients are prone to reoccurring infections due to the accumulation of mucus in their lungs, where bacteria can adhere and grow. Some of the antibiotics that inhibit B. cenocepacia in the laboratory are not effective for CF patients. A major contributor to poor clinical outcomes is that antibiotic testing in laboratories occurs under conditions that are different from those of sputum. CF sputum may be acidic and have increased concentrations of iron and zinc. Here, we used a medium that mimics CF sputum and found that acidic pH decreased the activity of many of the antibiotics used against B. cenocepacia. In addition, we assessed susceptibility to more than 500 antibiotics and found four active compounds against B. cenocepacia. Our findings give a better understanding of the lack of a relationship between susceptibility testing and the clinical outcome when treating B. cenocepacia infections.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster in iron scavenging.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be antecedent to highly debilitating long-term sequelae. Expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution by each of the downstream genes ( fetABCDEF ) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA , fetB, fetC , and fetD , suggesting a role in iron acquisition for each encoded protein. FetA expression was modulated by iron-availability but not dependent on the presence of FetB, FetC, FetD, FetE or FetF. Functions of the putative thioredoxins FetE and FetF were redundant in iron scavenging, requiring a double deletion (Δ fetEF ) to exhibit a growth defect. C. jejuni FetE was expressed and the structure solved to 1.50 Å, revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than E. coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo . This study advances our understanding of the contributions by the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge towards mitigating C. jejuni -related morbidity and mortality.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Aldehyde-Based Inhibitors of the Peptidoglycan O-Acetylesterase Ape.

The O-acetylation of the muramic acid residues in peptidoglycan (PG) is a modification that protects the bacteria from lysis due to the action of lysozyme. In Gram-negative bacteria, deacetylation is required to allow lytic transglycosylases to promote PG cleavage during cell growth and division. This deacetylation is catalyzed by O-acetylpeptidoglycan esterase (Ape) which is a serine esterase and employs covalent catalysis via a serine-linked acyl enzyme intermediate. Loss of Ape activity affects the size and shape of bacteria and dramatically reduces virulence. In this work, we report the first rationally designed aldehyde-based inhibitors of Ape from Campylobacter jejuni. The most potent of these acts as a competitive inhibitor with a Ki value of 13 µM. We suspect that the inhibitors are forming adducts with the active site serine that closely mimic the tetrahedral intermediate of the normal catalytic cycle. Support for this notion is found in the observation that reduction of the aldehyde to an alcohol effectively abolishes the inhibition.

A multicentre evaluation and expert recommendations of use of the newly developed BioFire Joint Infection polymerase chain reaction panel.

Septic arthritis is a serious condition with significant morbidity and mortality, routinely diagnosed using culture. The FDA has recently approved the rapid molecular BioFire® Joint Infection Panel (BJIP) for synovial fluid. We aimed to evaluate the BJIP compared to culture and its potential use in patient management. A multicentre retrospective evaluation of BJIP was conducted in the UK and Ireland. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated between the BJIP and routine culture. A multidisciplinary team (MDT) discussion addressing the optimal or potential case use of the assay practice was facilitated. Three hundred ninety-nine surplus synovial fluid samples (~ 70% from native joints) from eight centres were processed using BJIP in addition to routine culture. An increased yield of positive results was detected using BJIP compared to routine culture (98 vs 83), giving an overall PPA of 91.6% and overall NPA of 93% for the BJIP compared to culture results. The BJIP detected resistant markers and additional organisms that could influence antibiotic choices including Neisseria gonorrhoeae and Kingella kingae. The MDT agreed that the assay could be used, in addition to standard methods, in adult and children patients with specialist advice use based on local needs. Rapid results from BJIP were assessed as having potential clinical impact on patient management. Organisms not included in the panel may be clinically significant and may limit the value of this test for PJI.

Sequence Divergence in the Arginase Domain of Ornithine Decarboxylase/Arginase in Fusobacteriacea Leads to Loss of Function in Oral Associated Species.

A number of species within the Fusobacteriaceae family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/ß arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60-100 M-1 s-1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but kcat/KM of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

SARS-CoV-2 Aptasensors Based on Electrochemical Impedance Spectroscopy and Low-Cost Gold Electrode Substrates.

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter.

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6.

Lignostilbene-α,ß-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from ß-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

An electrochemical SARS-CoV-2 biosensor inspired by glucose test strip manufacturing processes.

Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Why the Maternal Medication List Matters: Neonatal Toxicity From Combined Serotonergic Exposures.

Serotonergic medications are used for the prevention and treatment of depression during pregnancy. Selective serotonin reuptake inhibitors and serotonin-norepinephrine reuptake inhibitors (SNRIs) can cause poor neonatal adaptation, which has been attributed to withdrawal versus toxicity. Bupropion, a norepinephrine-dopamine reuptake inhibitor, is often used as an adjunctive agent to selective serotonin reuptake inhibitors or SNRIs for refractory depression. Quetiapine, an atypical antipsychotic, may also be used in more complex cases. When combined with serotonergic drugs, bupropion and quetiapine are associated with increased risk of serotonin syndrome in adults. We describe a neonate exposed to venlafaxine (an SNRI), bupropion, and quetiapine in utero who presented nearly immediately after birth with encephalopathy and abnormal movements. The severity and rapidity of symptoms may be attributable to potentiation of venlafaxine's serotonergic effects by bupropion and quetiapine. Neonatal providers should be aware of maternal medications and prepare for possible adverse effects, particularly from common psychotropic exposures.

Biophysical characterization of the ETV6 PNT domain polymerization interfaces.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Severe Bronchoconstriction Caused by Administration of Rocuronium in a 3-Month-Old Infant: Case Report.

We present the case of a 3-month-old infant with severe, persistent bronchoconstriction following administration of rocuronium. This observation raises awareness of a rare but potentially life-threatening reaction to neuromuscular blocking agents.

A copper site is required for iron transport by the periplasmic proteins P19 and FetP.

Campylobacter jejuni is a leading cause of food-borne gastrointestinal disease in humans and uropathogenic Escherichia coli is a leading cause of urinary tract infections. Both human pathogens harbour a homologous iron uptake system (termed cjFetM-P19 in C. jejuni and ecFetM-FetP in E. coli). Although these systems are important for growth under iron limitation, the mechanisms by which these systems function during iron transport remain undefined. The copper ions bound to P19 and FetP, the homologous periplasmic proteins, are coordinated in an uncommon penta-dentate manner involving a Met-Glu-His3 motif and exhibit positional plasticity. Here we demonstrate the function of the Met and Glu residues in modulating copper binding and controlling copper positioning through site-directed variants, binding assays, and crystal structures. Growth of C. jejuni strains with these p19 variants is impaired under iron limited conditions as compared to the wild-type strain. Additionally, an acidic residue-rich secondary site is required for binding iron and function in vivo. Finally, western blot analyses demonstrate direct and specific interactions between periplasmic P19 and FetP with the large periplasmic domain of their respective inner membrane transporters cjFetM and ecFetM.

Evaluating the use of a 22-pathogen TaqMan array card for rapid diagnosis of respiratory pathogens in intensive care.

Introduction. Pneumonia is highly prevalent in intensive care units (ICUs), with high associated mortality. Empirical treatment prioritizes breadth of coverage while awaiting laboratory diagnosis, often at the expense of antimicrobial stewardship. Microarrays use multiple parallel polymerase chain reactions to enable a rapid syndromic approach to laboratory diagnosis.Aim. To evaluate the clinical and laboratory implications of introducing a bespoke 22-pathogen TaqMan Array Card (TAC) for rapid pathogen detection in deep respiratory samples from adult ICUs.Methodology. TAC results from all ICU patients prospectively tested over a 9-month period at Cambridge's Clinical Microbiology and Public Health Laboratory were compared to those of corresponding conventional microbiological assays (culture-, PCR- or serology-based) in terms of result agreement and time-to-result availability. Clinical impact was assessed by retrospective review of medical records.Results. Seventy-one patients were included [45 (63 %) male, median age 59). Overall result agreement was 94 %, with TAC detecting more pathogens than conventional methods. TAC detected Streptococcus pneumoniae more readily than culture (7 vs 0 cases; P=0.02). TAC did not detect Aspergillus spp. in eight culture- or galactomannan-positive cases. The median turnaround time (1 day) was significantly shorter than that of bacterial/fungal culture, Pneumocystis jirovecii PCR and galactomannan testing (each 3 days; P<0.001), atypical bacteria serology (13 days; P<0.001) and Mycobacterium tuberculosis culture (46 days; P<0.001). Earlier result availability prompted discontinuation of unnecessary antimicrobials in 15/71 (21 %) cases, but had no bearing on patient isolation/deisolation.Conclusion. TAC provided greater overall yield of pathogen detection and faster turnaround times, permitting earlier discontinuation of unnecessary antimicrobials.

ArnD is a deformylase involved in polymyxin resistance.

The modification of lipid A with cationic 4-amino-4-deoxy-l-arabinose residues serves to confer resistance against cationic peptide antibiotics in Gram-negative bacteria. In this work, the enzyme ArnD is shown to act as a metal-dependent deformylase in the biosynthesis of this carbohydrate.