Asymptomatic Healthcare Worker PCR Screening during SARS-CoV-2 Omicron Surge, Germany, 2022.

During 2022, a total of 9,515 asymptomatic healthcare workers of a large hospital in Germany underwent SARS-CoV-2 PCR screening twice weekly. Of 398,784 saliva samples, 3,555 (0.89%) were PCR positive (median cycle threshold value 30). Early identification of infected healthcare workers can help reduce SARS-CoV-2 transmission in the hospital environment.

Microbiologic diagnostics and pathogen spectrum in infective endocarditis of surgically treated patients: a five-year, retrospective, monocentric study.

PURPOSE: The spectrum of causative organisms in infective endocarditis (IE) has changed significantly in the last decades. Reliable pathogen detection is crucial for appropriate antimicrobial therapy for IE. The aim of the study was to evaluate the diagnostic value of microbiological methods for detecting the causative microorganism of IE and to analyze the spectrum of pathogens. METHODS: A total of 224 cases (211 unique patients, some with multiple surgeries) were included into this retrospective study. Patients were diagnosed with IE according to the modified Duke criteria from January 2016 to July 2021 and underwent heart valve surgery in a tertiary hospital. Pathogen detection was performed by blood culture, microbiological culture and 16S rDNA PCR of explanted heart valve material. RESULTS: A causative pathogen of IE was detected in 95.5% (n = 214) of cases. Blood cultures were positive in 83.3%, while a pathogen in the examined heart valve samples was identified in 32.6% by culture and in 88.2% by 16S rDNA PCR. A microorganism was identified by 16S rDNA PCR in 61.1% of blood culture negative cases but only in 19.4% by heart valve culture. The most common pathogens were Staphylococcus aureus (27%), viridans group streptococci (20%), enterococci (19%) and coagulase-negative staphylococci (CoNS 8%). Cutibacterium acnes (7%) was detected in prosthetic valve IE cases only. CONCLUSION: Blood culture as a comparatively non-invasive and straightforward technique remains an important and reliable method for initial detection of the causative organism in IE. Diagnostic stewardship programs should broadly emphasize proper collection of blood cultures, particularly sampling prior to any antibiotic treatment. Additionally, molecular testing using 16S rDNA tissue PCR can be used with culture techniques to increase the diagnostic yield, especially in the case of a negative blood culture.

Clinical and demographic factors affecting trough levels of isavuconazole in critically ill patients with or without COVID-19.

BACKGROUND: The broad-spectrum antifungal isavuconazole is administered to treat invasive aspergillosis and mucormycosis. OBJECTIVES: Isavuconazole plasma concentrations in critically ill ICU patients with or without COVID-19 and invasive fungal infection were determined, and factors for sub-therapeutic drug levels (<1 µg/mL) were evaluated. PATIENTS AND METHODS: Isavuconazole plasma levels were measured as part of therapeutic drug monitoring (TDM) in ICUs of a tertiary hospital. Concentrations determined 20-28 h after previous dosing were defined as trough (Cmin ) levels. A total of 160 Cmin levels from 62 patients with invasive fungal infections were analysed, 30 of which suffering from COVID-19. Patient characteristics included into univariable and multivariable analyses were gender, age, COVID-19 status, body mass index (BMI), sepsis-related organ failure (SOFA) score, renal replacement therapy (RRT) and extracorporeal membrane oxygenation (ECMO) requirement. RESULTS: The mean Cmin of isavuconazole in all patients was 1.64 µg/mL (interquartile range 0.83-2.24 µg/mL, total range 0.24-5.67 µg/mL). In total, 34.4% of the Cmin values (corresponding to 46.8% of patients) were below a threshold concentration of 1 µg/mL. Drug concentrations between patients with or without COVID-19 did not differ (p = .43). In contrast, levels were significantly lower in patients with female sex (p = .0007), age ≤ 65 years (p = .002), BMI > 25 (p = .006), SOFA score > 12 (p = .026), RRT (p = .017) and ECMO requirement (p = .001). CONCLUSIONS: Isavuconazole plasma levels can be negatively affected by patients' risk factors, supportive renal replacement and ECMO therapy. Future prospective studies analysing the relevance of isavuconazole drug levels in ICU patient outcome are urgently needed.

Isavuconazole therapeutic drug monitoring in critically ill ICU patients: A monocentric retrospective analysis.

BACKGROUND: The broad-spectrum triazole isavuconazole is used for the treatment of invasive aspergillosis and mucormycosis. Data regarding human plasma concentrations in clinical routine of the drug are rare. OBJECTIVES: Plasma concentrations of isavuconazole were determined in critically ill ICU patients while considering different patients' characteristics. METHODS: Retrospective analysis of isavuconazole plasma concentrations were obtained as part of routine therapeutic drug monitoring (TDM) of ICU patients with invasive aspergillosis or other fungal infections treated with isavuconazole. Plasma levels 0-4 h after last dosing were defined as peak levels (Cmax ), those 20-28 h after last dosing as trough levels (Cmin ). RESULTS: Overall, 223 isavuconazole levels of 41 patients were analysed, divided into 141 peak levels and 82 trough levels. The overall median Cmax was 2.36 µg/ml (mean 2.43 µg/ml, range 0.41-7.79 µg/ml) and the overall median Cmin was 1.74 µg/ml (mean 1.77 µg/ml, range 0.24-4.96 µg/ml). In total, 31.7% of the Cmin values of the total cohort were below the plasma target concentrations of 1 µg/ml, defined as EUCAST antifungal clinical breakpoint for Aspergillus fumigatus. Both peak and trough plasma levels of isavuconazole were significantly lower among patients with a body mass index (BMI) ≥25. In addition, a significant correlation was observed between isavuconazole trough levels and sepsis-related organ failure assessment (SOFA) score. CONCLUSIONS: This study shows that isavuconazole plasma concentrations vary in critical ill ICU patients. Significantly lower isavuconazole levels were associated with elevated BMI and higher SOFA score indicating a need of isavuconazole TDM in this specific patient population.

TetR-dependent gene regulation in intracellular Listeria monocytogenes demonstrates the spatiotemporal surface distribution of ActA.

To enable specific and tightly controlled gene expression both in vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic promoter Pt17 . Anhydrotetracycline (ATc) as inducer permitted concentration-dependent, fine-tuned expression of genes under control of the tetO operator and a suitable promoter. The actin-polymerizing ActA protein represents a major virulence factor of L. monocytogenes, required for actin-based motility and cell-to-cell spread in infected host cells. To be able to observe its spatial and temporal distribution on intracellular L. monocytogenes cells, conditional mutants featuring actA placed under TetR control were used to infect PtK2 epithelial cells. Following induction at different time intervals, the subsequent recruitment of actin by L. monocytogenes could be monitored. We found that cells displayed functional ActA after approximately 15 min, while formation of polarized actin tail was complete after 90-120 min. At this point, intracellular motility of the induced mutants was indistinguishable from wild-type bacteria. Interestingly, de novo ActA synthesis in intracellular Listeria also demonstrated the temporal, asymmetric redistribution of the membrane-anchored proteins from the lateral walls toward the cell poles.

Bet 1: Can induced hypertension improve outcome following acute traumatic spinal cord injury?

A shortcut review was carried out to establish whether augmentation of blood pressure to a high mean arterial pressure (MAP) target in the early phase of traumatic spinal cord injury (SCI) could lead to improvements in morbidity or mortality. 23 directly relevant papers were found using the reported search strategy. Of these, two systematic reviews collated the best evidence to answer the clinical question. The author, date and country of publication; patient group studied; study type; relevant outcomes; results and study weaknesses of the best papers are tabulated. It is concluded that data from observational cohort studies support high MAP targets and avoidance of hypotension in the early stages of traumatic SCI, but there are insufficient trial data to support routine use as best practice. Given the intervention carries risk, induced hypertension requires careful consideration on a case-by-case basis.

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon.

Understanding the mechanisms of how bacteria become tolerant toward antibiotics during clinical therapy is a very important object. In a previous study, we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (Pi) and polyphosphate (polyP). DAP tolerance in the pitA6 mutant differs from classical resistance mechanisms since there is no increase in the MIC. In this follow-up study, we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that 234 genes were at least 2.0-fold differentially expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism, and replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt operon, which is induced by the accumulation of intracellular Pi The GraXRS system, known as an activator of the dlt operon (d-alanylation of teichoic acids) and of the mprF gene (multiple peptide resistance factor), is not involved in DAP tolerance of the pitA6 mutant. In conclusion, DAP tolerance of the pitA6 mutant is due to an upregulation of the dlt operon, triggered directly or indirectly by the accumulation of Pi.

A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus. Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (Pi) transporter gene pitA. Drug tolerance caused by this allele, termed pitA6, was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of Pi and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.

Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator of tricarboxylic acid cycle activity in Staphylococcus aureus.

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

Metabolic and transcriptional activities of Staphylococcus aureus challenged with high-doses of daptomycin.

Treatment of stationary growth phase Staphylococcus aureus SA113 with 100-fold of the MIC of the lipopeptide antibiotic daptomycin leaves alive a small fraction of drug tolerant albeit genetically susceptible bacteria. This study shows that cells of this subpopulation exhibit active metabolism even hours after the onset of the drug challenge. Isotopologue profiling using fully (13)C-labeled glucose revealed de novo biosynthesis of the amino acids Ala, Asp, Glu, Ser, Gly and His. The isotopologue composition in Asp and Glu suggested an increased activity of the TCA cycle under daptomycin treatment compared to unaffected stationary growth phase cells. Microarray analysis showed differential expression of specific genes 10 min and 3 h after addition of the drug. Besides factors involved in drug response, a number of metabolic genes appear to shape the signature of daptomycin-tolerant S. aureus cells. These observations will be useful toward the development of new strategies against persisters and related forms of bacterial cells with downshifted physiology.

Characterization of multi-drug tolerant persister cells in Streptococcus suis.

BACKGROUND: Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial 'bet-hedging' strategy and are of particular importance in pathogenic bacteria. RESULTS: We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. CONCLUSIONS: Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis.

Feasibility of a virtual reality course on adult tracheostomy safety skills.

The National Tracheostomy Safety Project has run high-quality, face-to-face skills courses since 2009. The aim of this project was to produce a virtual reality version of the established course and evaluate its impact on participant learning, and participant and faculty satisfaction. Healthcare staff and students were recruited and randomised to attend one of (1) a face-to-face traditional course (control); (2) a virtual reality course at a conference centre with on-site technical support; (3) a fully remote virtual reality course; the virtual reality groups were combined for the analysis of learning outcomes and satisfaction. The primary outcome was the difference in pre/post-course knowledge scores on a 30-item questionnaire; secondary outcomes included knowledge retention, usability, comfort/side effects and participant performance in a simulated tracheostomy emergency. Thirty-seven participants and 15 faculty participated in this study. There was no significant difference between mean pre/post-course scores from the face-to-face (from 21.1 to 23.1; +2) and combined virtual reality (from 17.1 to 21.1; +4) groups, with both showing improvement (p = 0.21). The mean System Usability Scale score for virtual reality was 76.8 (SD 12.6), which is above average; the median Simulator Sickness Questionnaire score was 7.5 (IQR 3.7-22.4), indicating minimal symptoms. All participants resolved the primary clinical problem in the simulated emergency, but the virtual reality (VR) group was slower overall (mean difference 61.8 s, p = 0.003). This technical feasibility study demonstrated that there was no difference in participant knowledge immediately after and 4 weeks following face-to-face and virtual reality courses. Virtual reality offers an immersive experience that can be delivered remotely and offers potential benefits of reducing travel and venue costs for attendees, therefore increasing the flexibility of training opportunities.

Characterization of a mazEF toxin-antitoxin homologue from Staphylococcus equorum.

Toxin-antitoxin (TA) systems encoded in prokaryotic genomes fall into five types, typically composed of two distinct small molecules, an endotoxic protein and a cis-encoded antitoxin of ribonucleic or proteinaceous nature. In silico analysis revealed seven putative type I and three putative type II TA systems in the genome of the nonpathogenic species strain Staphylococcus equorum SE3. Among these, a MazEF system orthologue termed MazEF(seq) was further characterized. 5' rapid amplification of cDNA ends (RACE) revealed the expression and the transcriptional start site of mazE(seq), indicating an immediately upstream promoter. Heterologous expression of the putative toxin-encoding mazF(seq) gene imposed growth cessation but not cell death on Escherichia coli. In vivo and in vitro, MazF(seq) was shown to cleave at UACAU motifs, which are remarkably abundant in a number of putative metabolic and regulatory S. equorum gene transcripts. Specific interaction between MazF(seq) and the putative cognate antitoxin MazE(seq) was demonstrated by bacterial two-hybrid analyses. These data strongly suggest that MazEF(seq) represents the first characterized TA system in a nonpathogenic Staphylococcus species and indicate that MazEF modules in staphylococci may also control processes beyond pathogenicity.

The mevalonate auxotrophic mutant of Staphylococcus aureus can adapt to mevalonate depletion.

In this study, we attempted to adopt the auxotrophic mevalonate synthase mutant (ΔmvaS mutant) of Staphylococcus aureus to study whether a nongrowing but viable cell population is tolerant to bactericidal antibiotics. The mevalonate-depleted nongrowing ΔmvaS mutant was found tolerant to antibiotics. Surprisingly, after prolonged cultivation, we obtained stable ΔmvaS variants that were able to grow without mevalonate, which suggested unknown mechanisms for compensating undecaprenyl pyrophosphate production without mevalonate in S. aureus.

An update on the molecular genetics toolbox for staphylococci.

Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developments of various genetic tools and molecular methods for staphylococcal research, which include reporter systems and vectors for controllable gene expression, gene inactivation, gene essentiality testing, chromosomal integration and transposon delivery. It is furthermore illustrated how mutant strain construction by homologous or site-specific recombination benefits from sophisticated counterselection methods. The underlying genetic components have been shown to operate in wild-type staphylococci or modified chassis strains. Finally, possible future developments in the field of applied Staphylococcus genetics are highlighted.

Two paralogous yefM-yoeB loci from Staphylococcus equorum encode functional toxin-antitoxin systems.

Toxin-antitoxin (TA) systems are small genetic elements of prokaryotes associated with persister cell formation, phage defence, stress regulation and programmed cell arrest. In this study, we characterized two paralogues of the ribosome-dependent RNase YefM-YoeB TA system from the Gram-positive organism Staphylococcus equorum SE3. 5' Rapid amplification of cDNA ends confirmed transcriptional activity in the exponential growth phase and revealed an extended 5' untranslated region upstream of the yefM-seq1 gene. Inducible expression of the putative yoeB-seq1/2 toxins led to growth defects of Escherichia coli, which were counteracted by simultaneous induction of the cognate yefM-seq1/2 antitoxin candidates in a strictly pairwise manner. Bacterial two-hybrid assays revealed interaction between YoeB-seq1 and YefM-seq1 but not YoeB-seq1 and YefM-seq2, also indicating two independent systems. In vivo primer extensions demonstrated specific RNA cleavage adjacent to the start codons by YoeB-seq proteins, and YoeB-seq2 activity could be neutralized by the corresponding antitoxin YefM-seq2. Together, these results indicate that the two yefM-yoeB-seq1/2 paralogues from S. equorum encode functional TA systems.

tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Status quo of tet regulation in bacteria.

The tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other natural or synthetic origins have been used for tetracycline-dependent gene regulation (tet regulation) in at least 40 bacterial genera. Tet regulation serves several purposes such as conditional complementation, depletion of essential genes, modulation of artificial genetic networks, protein overexpression or the control of gene expression within cell culture or animal infection models. Adaptations of the promoters employed have increased tet regulation efficiency and have made this system accessible to taxonomically distant bacteria. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.