Klebsiella pneumoniae is an opportunistic gram-negative bacterial pathogen responsible for a variety of infections in hospitals and in the community. Hypervirulent K. pneumoniae (hvKp) strains are characterized by their ability to cause liver abscesses and bloodstream infections. This study describes plasmid-based tetracycline-dependent regulation systems for induction or repression of target genes in hvKp. These plasmids encode a wild-type tetracycline repressor (TetR) or a reverse variant (revTetR), and carry tet-sensitive promoters, for anhydrotetracycline-dependent Tet-ON or Tet-OFF regulation, respectively. The dynamic range, determined by gfp under tet control, was up to 19.3-fold with wt-TetR and 5.5-fold with revTetR. As analyzed for up to 48 h, the reporter-gene responses in both systems were controllable in an inducer concentration-dependent manner. When the virulence-associated factor peg-344 was induced by the Tet-ON system, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella infection model. These tet vectors can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) strains are a serious threat in healthcare environments and beyond. Even in healthy individuals, infections often result in liver abscesses, and the bacteria can spread to multiple infection sites. We here describe two kinds of gene expression control in hvKp, dependent on anhydrotetracycline (ATc). The Tet-ON system uses wild-type tetracycline repressor (TetR), and addition of ATc leads to target gene induction. In the Tet-OFF mode, reverse TetR is co-repressed by ATc, resulting in target gene repression. We have quantified both systems by reporter gene assays and also subjected the hypervirulence marker gene peg-344 to Tet-ON control. When induced, two different hvKp strains exerted slightly increased pathogenicity in a Galleria mellonella (greater wax moth) infection model. These systems can be useful tools for characterizing potential virulence factors and other genes of interest in an important pathogen.

Linezolid serum concentrations in critically ill patients show high variability. In this retrospective study, we analysed the linezolid plasma through levels (Cmin) in patients on intensive care units (ICUs) under extracorporeal membrane oxygenation (ECMO) support. The aim was to evaluate if patients’ clinical or demographic characteristics influence drug concentrations and if these are within the therapeutic range. In total, 156 linezolid trough plasma concentrations from 52 ICU ECMO patients were analysed. Linezolid trough levels were correlated with the following clinical and demographic characteristics: Age, sex, body mass index (BMI), dosage per day, creatinine clearance (CrCl), and requirement for renal replacement therapy (RRT). Drug concentrations were quantified by liquid chromatography with tandem mass spectrometry. The European Committee on Antimicrobial Susceptibility Testing susceptibility breakpoints of 4 mg/L of linezolid for staphylococci and enterococci and of 2 mg/L for streptococci and other Gram-positive rods were used as minimum target concentration. Patients were treated with standard linezolid dosage (2 × 600 mg iv per day; n = 88 Cmin, 56.4

Brain abscesses can lead to severe clinical outcomes, including death. Most studies on brain abscesses focus either on patient cohorts including postsurgical/posttraumatic abscesses. This study aimed to assess the clinical presentation and postoperative outcomes in a homogeneous group of patients with primary, pyogenic brain abscesses who underwent surgical treatment. We retrospectively analyzed consecutive patients with pyogenic brain abscesses treated surgically at our center from 2008 to 2023. The primary endpoint was a modified Rankin Scale (mRS) score of ≥ 3 at discharge. Secondary endpoint was preoperative clinical status (mRS ≥ 3). We statistically correlated clinical, radiographic, and microbiological parameters with these endpoints. A total of 60 patients (36.7

As a potential side effect of the severe acute respiratory syndrome coronavirus type 2 pandemic, invasive group A Streptococcus (iGAS) infections in Europe have increased dramatically in both children and adults in the end of 2022. This epidemiological and molecular study describes the distributions of streptococcal genes encoding the M antigen (emm types) and superantigens in patients with invasive and non-invasive GAS infections. From December 2022 to December 2023, a total of 163 GAS isolates were collected from sterile and non-sterile sites of patients at five hospitals in Germany including two tertiary care centers. Genes encoding M protein and superantigens were determined following the guidelines of CDC Streptococcus laboratory. Patients' characteristics were reviewed retrospectively. Correlations of clinical factors, emm types, and superantigens with rates of invasive infections were analyzed. Of the 163 included GAS cases, 112 (69%) were considered as invasive. In total, 33 different emm types were observed, of which emm1.0 (n = 49; 30%), emm89.0 (n = 15; 9%), and emm12.0 (n = 14; 9%) were most prevalent. In total, 70% of emm1.0 isolates belonged to M1UK lineage. No difference in invasive infections was observed for the M1UK lineage compared with other emm1.0 isolates. However, the emm1.0 type, presence of speA1-3, speG, or speJ, as well as adulthood were significantly associated with invasive infections. In contrast, emm12.0 isolates were significantly less associated with invasive infections. Multivariable analysis confirmed a significant influence of speJ and adulthood on iGAS infections. This study underlines the importance of continuous monitoring of genomic trends and identification of emerging GAS variants. This may aid in delineating pathogenicity factors of Streptococcus pyogenes that propel invasive infections.

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.

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.

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 mu g/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 mu g/mL (interquartile range 0.83-2.24 mu g/mL, total range 0.24-5.67 mu g/mL). In total, 34.4% of the Cmin values (corresponding to 46.8% of patients) were below a threshold concentration of 1 mu g/mL. Drug concentrations between patients with or without COVID-19 did notdiffer (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.

Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.

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.

As an important element in the regional containment of the COVID-19 pandemic a PCR testing laboratory with a cooperative character was founded in spring 2021 to screen for SARS-CoV-2 in the Nuremberg region, Germany. The aim was to detect asymptomatic infections in day care facilities for children, schools, and companies. The laboratory used an established RT-PCR protocol and analyzed approximately 18,500 pools of up to 25 pooled samples each from gargles or swabs ("lollipops") from up to 135 facilities between July 2021 and June 2022. Usually, the participating facilities were informed about positive pools within a few hours. Retention samples from positive pools were usually analyzed on the same day, and the results were reported to the facilities as well as the German Electronic Reporting and Information System (DEMIS). In the laboratory results, both the local incidences and the transition from the Delta- to the Omicron surge in early 2022 were well reflected. It is plausible that about 4,800 secondary infections could be prevented from the approximately 1,570 positive individual samples detected in conjunction with appropriate isolation measures. Such a PCR laboratory, which is characterized by short response times and high flexibility, can thus provide valuable services for regional surveillance of infection incidence.

In early 2022, the Coronavirus disease 2019 (COVID-19) remains a global challenge. COVID-19 is caused by an increasing number of variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we report an outbreak of SARS-CoV-2 breakthrough infections related to a student festive event with 100 mostly vaccinated guests, which took place in Northern Bavaria, Germany, in October 2021. The data were obtained by retrospective guest interviews. In total, 95 students participated in the study, with 94 being fully vaccinated and 24 reporting infection by the delta variant. Correlation analyses among 15 examined variables revealed that time spent at the event, conversation with the supposed index person, and a homologous viral vector vaccination regime were significant risk factors for infection. Non-significant observations related to higher rates of infection included time since last vaccination, shared use of drinking vessels, and number of individual person-to-person contacts at the event. Our data suggest that a high rate of breakthrough infections with the delta variant occurs if no preventive measures are practiced. To limit infection risk, high-quality testing of participants should be considered a mandatory measure at gatherings, irrespective of the participants' vaccination status.

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.

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 (C-max), those 20-28 h after last dosing as trough levels (C-min). Results Overall, 223 isavuconazole levels of 41 patients were analysed, divided into 141 peak levels and 82 trough levels. The overall median C-max was 2.36 mu g/ml (mean 2.43 mu g/ml, range 0.41-7.79 mu g/ml) and the overall median C-min was 1.74 mu g/ml (mean 1.77 mu g/ml, range 0.24-4.96 mu g/ml). In total, 31.7% of the C-min values of the total cohort were below the plasma target concentrations of 1 mu g/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.

AbstractWe report an outbreak with SARS-CoV-2 breakthrough infections related to a festive event in Northern Bavaria, Germany in October 2021, with 24 of 95 participants infected. Correlation analyses among 15 interrogated variables revealed that duration at the event and conversation with the supposed index person were significant risk factors.Article Summary LineThe risk of infection with SARS-CoV-2 in a vaccinated cohort associated with a private festive event was significantly increased upon conversation with the putative index person and positively correlated to the duration of stay at the event.

The capability of Pseudomonas aeruginosa and Staphylococcus aureus to form biofilm on varying CI component materials differs in the presence and absence of bioactive glass (BAG). The application of BAG induces significant changes in biofilm morphology which can be visualized via scanning electron microscopy (SEM). Bacterial biofilm formation on medical devices, such as cochlear implants (CI), can lead to chronic infections. Interestingly, BAG of type S53P4 seems to be a promising tool for use in the reduction of biofilm development. Primarily, four bacterial species known to cause implant-related infections, P.aeruginosa (ATCC9027), S. aureus (ATCC6538), Staphylococcus epidermidis (ATCC12228) and Streptococcus pyogenes (ATCC19615) were analyzed regarding their capacity to form biofilm on CI components manufactured from three kinds of material: silicone, platinum and titanium. Subsequently, P. aeruginosa and S. aureus biofilms were visualized using scanning electron microscopy, comparing BAG-treated biofilm with non-treated biofilm. The four bacterial species presented biofilm-forming capabilities in a species and surface dependent manner. Metal CI components allowed for the greatest proliferation of biofilm. S. aureus and P. aeruginosa showed the highest rate of biofilm formation on polystyrene surfaces. For both species, SEM revealed altered biofilm morphology after treatment of S53P4 BAG. This study indicates that bacterial biofilm formation and structure on CI components is dependent on the surface composition, altering between metal and silicone surfaces. After application of BAG, changes in biofilm morphology on CI components were observed. These data highlight the impact of BAG on bacterial biofilm morphology.

Invasion and persistence of bacteria within host cells requires that they adapt to life in an intracellular environment. This adaptation induces bacterial stress through events such as phagocytosis and enhanced nutrient-restriction. During stress, bacteria synthesize a family of proteins known as heat shock proteins (HSPs) to facilitate adaptation and survival. Previously, we determined the Staphylococcus aureus HSP ClpC temporally alters bacterial metabolism and persistence. This led us to hypothesize that ClpC might alter intracellular survival. Inactivation of clpC in S. aureus strain DSM20231 significantly enhanced long-term intracellular survival in human epithelial (HaCaT) and endothelial (EA.hy926) cell lines, without markedly affecting adhesion or invasion. This phenotype was similar across a genetically diverse collection of S. aureus isolates, and was influenced by the toxin/antitoxin encoding locus mazEF. Importantly, MazEF alters mRNA synthesis and/or stability of S. aureus virulence determinants, indicating ClpC may act through the mRNA modulatory activity of MazEF. Transcriptional analyses of total RNAs isolated from intracellular DSM20231 and isogenic clpC mutant cells identified alterations in transcription of α-toxin (hla), protein A (spa), and RNAIII, consistent with the hypothesis that ClpC negatively affects the intracellular survival of S. aureus in non-professional phagocytic cells, via modulation of MazEF and Agr.

Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro) tetracycline-inducible promoter P-xyl/tet from staphylococcal plasmid pRAB11 to control gene expression in Streptococcus pneumoniae. The system was evaluated by expressing genes of the two-component regulatory system ciaRH of S. pneumoniae. With full induction of P-xyl/tet, wild-type levels of the response regulator CiaR were obtained, while theuninducedbasal expression was low. Hyperactive variants of the kinase gene ciaH normally causing pronounced genetic instability could be handled without any problems upon cloning into pTEX2. Therefore, the expression system is well suited to express physiological levels of proteins in S. pneumoniae and also to aid regulatory studies.

Biofilms play a crucial role in the pathogenicity of Staphylococcus epidermidis, while little is known about whether the essential YycFG two-component signal transduction system (TCS) is involved in biofilm formation. We used antisense RNA (asRNA) to silence the yycFG TCS in order to study its regulatory functions in S. epidermidis. Strain 1457 expressing asRNA yycF exhibited a significant delay (~4-5 h) in entry to log phase, which was partially complemented by overexpressing ssaA. The expression of asRNA yycF and asRNA yycG resulted in a 68 and 50% decrease in biofilm formation at 6 h, respectively, while they had no significant inhibitory effect on 12 h biofilm formation. The expression of asRNA yycF led to a ~5-fold increase in polysaccharide intercellular adhesion (PIA) production, but it did not affect the expression of accumulation-associated protein (Aap) or the release of extracellular DNA. Consistently, quantitative real-time PCR showed that silencing yycF resulted in an increased transcription of biofilm-related genes, including icaA, arlR, sarA, sarX, and sbp. An in silico search of the YycF regulon for the conserved YycF recognition pattern and a modified motif in S. epidermidis, along with additional gel shift and DNase I footprinting assays, showed that arlR, sarA, sarX, and icaA are directly regulated by YycF. Our data suggests that YycFG modulates S. epidermidis biofilm formation in an ica-dependent manner.

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.