CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

Low Risk of Severe Acute Respiratory Syndrome Coronavirus 2 Transmission by Fomites: A Clinical Observational Study in Highly Infectious Coronavirus Disease 2019 Patients.

BACKGROUND: The contribution of droplet-contaminated surfaces for virus transmission has been discussed controversially in the context of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. More importantly, the risk of fomite-based transmission has not been systematically addressed. Therefore, the aim of this study was to evaluate whether confirmed hospitalized coronavirus disease 2019 (COVID-19) patients can contaminate stainless steel carriers by coughing or intensive moistening with saliva and to assess the risk of SARS-CoV-2 transmission upon detection of viral loads and infectious virus in cell culture. METHODS: We initiated a single-center observational study including 15 COVID-19 patients with a high baseline viral load (cycle threshold value ≤25). We documented clinical and laboratory parameters and used patient samples to perform virus culture, quantitative polymerase chain reaction, and virus sequencing. RESULTS: Nasopharyngeal and oropharyngeal swabs of all patients were positive for viral ribonucleic acid on the day of the study. Infectious SARS-CoV-2 could be isolated from 6 patient swabs (46.2%). After coughing, no infectious virus could be recovered, however, intensive moistening with saliva resulted in successful viral recovery from steel carriers of 5 patients (38.5%). CONCLUSIONS: Transmission of infectious SARS-CoV-2 via fomites is possible upon extensive moistening, but it is unlikely to occur in real-life scenarios and from droplet-contaminated fomites.

Virucidal Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome Coronavirus 2.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic creates a significant threat to global health. Recent studies suggested the significance of throat and salivary glands as major sites of virus replication and transmission during early coronavirus disease 2019, thus advocating application of oral antiseptics. However, the antiviral efficacy of oral rinsing solutions against SARS-CoV-2 has not been examined. Here, we evaluated the virucidal activity of different available oral rinses against SARS-CoV-2 under conditions mimicking nasopharyngeal secretions. Several formulations with significant SARS-CoV-2 inactivating properties in vitro support the idea that oral rinsing might reduce the viral load of saliva and could thus lower the transmission of SARS-CoV-2.

Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 by WHO-Recommended Hand Rub Formulations and Alcohols.

Infection control instructions call for use of alcohol-based hand rub solutions to inactivate severe acute respiratory syndrome coronavirus 2. We determined the virucidal activity of World Health Organization-recommended hand rub formulations, at full strength and multiple dilutions, and of the active ingredients. All disinfectants demonstrated efficient virus inactivation.

Comparative Analysis of the Euroimmun CXCL13 Enzyme-Linked Immunosorbent Assay and the ReaScan Lateral Flow Immunoassay for Diagnosis of Lyme Neuroborreliosis.

Diagnosis of Lyme neuroborreliosis (LNB) is challenging, as long as Borrelia-specific intrathecal antibodies are not yet detectable. The chemokine CXCL13 is elevated in the cerebrospinal fluid (CSF) of LNB patients. Here, we compared the performances of the Euroimmun CXCL13 enzyme-linked immunosorbent assay (CXCL13 ELISA) and the ReaScan CXCL13 lateral flow immunoassay (CXCL13 LFA), a rapid point-of-care test, to support the diagnosis of LNB. In a dual-center case-control study, CSF samples from 90 patients (34 with definite LNB, 10 with possible LNB, and 46 with other central nervous system [CNS] diseases [non-LNB group]) were analyzed with the CXCL13 ELISA and the CXCL13 LFA. Classification of patients followed the European Federation of Neurological Societies (EFNS) guidelines on LNB. The CXCL13 ELISA detected elevated CXCL13 levels in all patients with definite LNB (median, 1,409 pg/ml) compared to the non-LNB controls (median, 20.7 pg/ml; P < 0.0001), with a sensitivity of 100% and a specificity of 84.8% (cutoff value, 78.6 pg/ml; area under the receiver operating characteristic [ROC] curve, 0.93). Similarly, the CXCL13 LFA yielded elevated CXCL13 levels in 31 patients with definite LNB (median arbitrary value, 223.5) compared to the non-LNB control patients (median arbitrary value, 0; P < 0.0001) and had a sensitivity and specificity of 91.2% and 93.5%, respectively (cutoff arbitrary value, 22.5; area under the ROC curve, 0.94). The correlation between the CXCL13 levels obtained by ELISA and LFA was strong (Spearman correlation coefficient r = 0.89; P < 0.0001). The CXCL13 ELISA and the CXCL13 LFA are comparable diagnostic tools for the detection of CXCL13 in the CSF of patients with definite LNB. The advantage of the CXCL13 LFA is the shorter time to result.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings.IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.

SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene.

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.

Risk and clinical impact of bacterial resistance/susceptibility to silver-based wound dressings: a systematic review.

OBJECTIVE: To perform a systematic review of the literature on bacterial resistance, tolerance and susceptibility of silver within the context of wound therapy using silver-based dressings. METHODS: A literature search was carried out using PubMed, Embase and Cochrane Library databases, the focus was whether results from microbiological experimental in vitro tests with reference strains and clinical wound isolates are reflected in clinical practice with regards to their 'resistance' profiles, comparable with those observed for antibiotics. The search results were allocated to six categories: resistance and resistance mechanism, in vitro tests with standard strains and wound isolates, prevalence and incidence, impact on clinical practice and impact on antibiotic therapy as well as reviews, expert opinions and consensus. RESULTS: Based on all findings of the literature, it cannot be confirmed that a related clinical resistance to silver-ions in silver-based dressings has clinical impact, although endogenous and exogenous genetic resistance patterns have been described and intensively investigated. A translation of these genetic resistance-expression structures to phenotypic appearances, similar to those known for antibiotics, has not been demonstrated for silver in the literature. CONCLUSION: It can be concluded that there is no definitive evidence available and further studies should be conducted.

Comparative LC-MS/MS and HPLC-UV Analyses of Meropenem and Piperacillin in Critically Ill Patients.

BACKGROUND: Therapeutic drug monitoring (TDM) of beta-lactam antibiotics has become a valuable tool to guide dosing in critically ill patients. The main goal of the study was to compare two routinely used techniques for beta-lactam TDM in intensive care unit (ICU) patient samples, namely isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and high-performance liquid chromatography combined with ultra-violet detection (HPLC-UV). METHODS: A set of 80 sera/plasma samples from ICU patients receiving therapeutic meropenem or piperacillin dosage was investigated. Sample duplicates and quality assessment samples were assayed in parallel with an in-house LC-MS/MS and a commercially available IVD HPLC-UV kit. A pharmacokinetic and pharmacodynamic (PK/PD) target with ≥ 22.5 mg/L for piperacillin and ≥ 8.0 mg/L for meropenem was used for medical assessment of trough sample (n = 40) antibiotic concentrations. RESULTS: There was no difference between serum and Li-heparin plasmas. Concentration deviations were found for 4% of meropenem and 17% of piperacillin samples. Eliminating the influence of the systemic bias of approximately 10% for piperacillin, measurement discrepancies ≥ 25% between LC-MS/MS and HPLC-UV analyses were only observed for ≈ 4 - 6% of all samples. In the same way, identical PK/PD target attainment rates of 50 - 60% could be obtained. CONCLUSIONS: After correction of the analytical bias for piperacillin measurements, both methods showed comparable results, also with respect to clinical decision limits. HPLC-UV analysis is an adequate TDM methodology for testing of beta-lactam antibiotics in centers where no special knowledge in LC-MS/MS based TDM is present. However, potential matrix effects, interferences, and calibration issues for both methods must be taken into account.

Multiplex PCR of bronchoalveolar lavage fluid in children enhances the rate of pathogen detection.

BACKGROUND: Culturing of bronchoalveolar lavage (BAL) fluid is a commonly used method for pathogen detection in pneumonia. However, the sensitivity is low, especially in patients pre-treated with anti-infective agents. The early detection of a pathogen is crucial for the outcome of respiratory tract infections. For bloodstream infections, a multiplex polymerase chain reaction (PCR) assay (SeptiFast®, SF) is available for improved pathogen detection from blood. OBJECTIVE: The aim of the present study was to determine whether the SF assay is applicable to the BAL of children with pulmonary infections and whether the frequency of pathogen detection is enhanced by the use of this multiplex PCR method. METHODS: We investigated 70 BAL samples of 70 children simultaneously by culture and multiplex PCR. The frequency of pathogen detection was compared. RESULTS: Pathogens were detected more frequently by SF than by culture (83% vs. 31%; p < 0.001). This advantage was shown for immunocompetent patients (p = 0.001) as well as for immunocompromised patients (p = 0.003). The majority (38/44; 86%) of the Gram positive cocci were only detected by SF. Fungal organisms were detected in 7/70 patients (10%) by SF and in 2/70 (3%) by culture (p = 0.125). CONCLUSION: Compared to conventional culture, the use of the SF assay on the BAL of children with pneumonia increases pathogen detection rates and therefore adds important information to guide anti-infective therapy.

FunResDB-A web resource for genotypic susceptibility testing of Aspergillus fumigatus.

Therapy of invasive aspergillosis is becoming more difficult due to the emergence of azole resistance in Aspergillus fumigatus. A majority of resistant strains carries mutations in the CYP51A gene. Due to a lack of sensitivity of culture-based methods, molecular detection of A. fumigatus has become an important diagnostic tool. We set up the database FunResDB (www.nrz-myk.de/funresdb) to gather all available information about CYP51A-dependent azole resistance from published literature. In summary, the screening resulted in 79 CYP51A variants, which are linked to 59 nonsynonymous mutations. A tailor-made online sequence analysis tool allows for genotypic susceptibility testing of A. fumigatus.

Donor Leucocytosis Predicts Bacterial and Fungal Contamination of the Preservation Solution in Visceral Organ Transplantation.

INTRODUCTION: Contamination of the preservation solution may contribute to septic complications that can occur after transplantation and cause higher morbidity and mortality among recipients. The aim of this study was to determine potential donor-related predictors of positive microbiological findings in the preservation solution. DESIGN: We retrospectively studied 16 donor parameters on data from our center for microbiological findings in the preservation solution used in solid-organ recovery. From January 2008 through December 2011, 976 solid organs were transplanted, and in 167, the solution was positive for contaminants. RESULTS: The most frequently detected contaminant was coagulase-negative staphylococci. Only the donor leucocyte count (cutoff at 9.1 × 109/L) predicted positive microbiological findings in the preservation solution ( P = .0024). Multivariable regression analysis found that donor age, donor sex, intensive care unit stay, total number of organs recovered, and leucocyte count differentiated various categories of potentially pathogenic bacteria. CONCLUSION: Donor leucocyte count higher than 9.1 × 109/L predicts contamination of preservation solution.

Comparison of Phenotypic Tests and an Immunochromatographic Assay and Development of a New Algorithm for Detection of OXA-48-like Carbapenemases.

OXA-48 is the most prevalent carbapenemase in Enterobacteriaceae in Europe and the Middle East, but it is frequently missed because many isolates display low MICs for carbapenems. Furthermore, in contrast to metallo-ß-lactamases or Klebsiella pneumoniae carbapenemases (KPC), no specific inhibitor is available for the phenotypic detection of OXA-48. Molecular detection of blaOXA-48 is the "gold standard" but is not available in many laboratories. A few phenotypic assays have been described but have not been independently evaluated. The aim of this study was the systematic comparison of phenotypic tests and an immunochromatographic assay (ICT) for the detection of OXA-48/OXA-48-like carbapenemases and the development of an algorithm for reliable phenotypic detection of OXA-48. Four phenotypic tests (temocillin disk test, faropenem disk test, OXA-48 disk test, and high-inoculum [HI] OXA-48 disk test) and a new ICT (OXA-48 K-SeT) were compared by using a set of 166 Enterobacteriaceae isolates, including isolates producing OXA-48/OXA-48-like carbapenemases (n = 84) or Ambler class A and B carbapenemases (n = 41) and carbapenemase-negative isolates (n = 41). The sensitivity and specificity for the different assays were 100% and 43.9% for temocillin, 57.1% and 98.8% for faropenem, 53.6% and 100% for the OXA-48 disk test, 98.8% and 97.6% for the HI OXA-48 disk test, and 100% and 100% for the ICT, respectively. The ICT displayed the highest sensitivity and specificity and was the most rapid assay, but it is more costly than phenotypic assays. Based on these results, a new algorithm incorporating temocillin, faropenem, and ICT which allows cost-effective detection of OXA-48 with 100% sensitivity and specificity was developed.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

Moxifloxacin monotherapy versus combination therapy in patients with severe community-acquired pneumonia evoked ARDS.

BACKGROUND: We tested the hypothesis that moxifloxacin monotherapy is equally effective and safe as a betalactam antibiotic based combination therapy in patients with acute respiratory distress syndrome (ARDS) evoked by severe community acquired pneumonia (CAP). METHODS: In a retrospective chart review study of 229 patients with adult respiratory distress syndrome (ARDS) admitted to our intensive care unit between 2001 and 2011, 169 well-characterized patients were identified to suffer from severe CAP. Patients were treated with moxifloxacin alone, moxifloxacin in combination with ß-lactam antibiotics, or with another antibiotic regimen based on ß-lactam antibiotics, at the discretion of the admitting attending physician. The primary endpoint was 30-day survival. To assess potential drug-induced liver injury, we also analyzed biomarkers of liver cell integrity. RESULTS: 30-day survival (69% overall) did not differ (p = 0.89) between moxifloxacin monotherapy (n = 42), moxifloxacin combination therapy (n = 44), and other antibiotic treatments (n = 83). We found significantly greater maximum activity of aspartate transaminase (p = 0.048), alanine aminotransferase (p = 0.003), and direct bilirubin concentration (p = 0.01) in the moxifloxacin treated groups over the first 10-20 days. However, these in-between group differences faded over time, and no differences remained during the last 10 days of observation. CONCLUSIONS: In CAP evoked ARDS, moxifloxacin monotherapy and moxifloxacin combination therapy was not different to a betalactam based antibiotic regimen with respect to 30-day mortality, and temporarily increased markers of liver cell integrity had no apparent clinical impact. Thus, in contrast to the current S3 guidelines, moxifloxacin may also be safe and effective even in patients with severe CAP evoked ARDS while providing coverage of an extended spectrum of severe CAP evoking bacteria. However, further prospective studies are needed for definite recommendations.

Multicentre investigation of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in German hospitals.

Aim of this study was to determine the incidence and molecular epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Germany. E. coli and K. pneumoniae isolates from clinical samples which were non-susceptible to carbapenems were collected in laboratories serving 20 hospitals throughout Germany from November 2013 to April 2014. The isolates were tested for the presence of carbapenemases by PCR and phenotypic methods and typed by multilocus sequence typing. Risk factors including a previous hospitalization abroad were analysed. Carbapenemases were detected in 24 isolates from 22 patients out of 464,514 admissions. Carbapenemases included OXA-48 (n=14), KPC-2 (n=8) and NDM-1 (n=2). Except for two K. pneumoniae isolates with ST101, all OXA-48 producing strains belonged to different clones. In contrast, half of KPC-2 producing K. pneumoniae were of ST258 and both NDM-1 producing strains were of ST11. Compared to carbapenem-susceptible controls, patients with carbapenemase-producing strains differed by a significantly higher proportion of males, a higher proportion of isolates from wound samples and a more frequent previous stay abroad in univariate analysis. This multicentre study demonstrated an incidence of carbapenemase-producing E. coli and K. pneumoniae from clinical samples in Germany of 0.047 cases per 1000 admissions. OXA-48 was more frequent than KPC-2 and NDM-1 and showed a multiclonal background.

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

BACKGROUND: Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. METHODS: Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. RESULTS: Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. CONCLUSIONS: The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.