Franziska Schuler

Listeriosis is a severe foodborne infection and associated with high mortality. Treatment is based on ampicillin, amoxicillin or penicillin, often combined with gentamicin, but meropenem is also used occasionally. β-lactam resistant Listeria monocytogenes isolates are infrequently described but the mechanism of resistance is not known. A clinical L. monocytogenes isolate with reduced β-lactam susceptibility was collected from a German listeriosis patient. Resistance profiling, whole genome sequencing, comparative genomics and genetic experiments were used to identify the causative DNA polymorphism. A W428R substitution near the active site of penicillin binding protein B1 (PBPB1) was identified as the cause of reduced ampicillin, amoxicillin and meropenem susceptibility. Further clinical L. monocytogenes isolates with similar susceptibility profiles were found by searching the genome database of the German consultant laboratory for Listeria for pbpB1 W428R-positive isolates. Spontaneous suppressors of L. monocytogenes reference strain EGD-e with reduced β-lactam susceptibility were selected during ampicillin exposure and their genomes were sequenced. The same pbpB1 mutation emerged in strain EGD-e during cultivation in the presence of ampicillin. Further experiments showed that meropenem also promotes the development of resistant suppressor mutants with pbpB1 mutations. Our work demonstrates that β-lactam susceptibility of L. monocytogenes can be reduced through a specific substitution in PBPB1. This mutation is potentially selected for during β-lactam treatment.

Introduction. Rectovaginal colonization with Streptococcus agalactiae in pregnant women is a risk factor for invasive infections of the newborns. Various culture-based approaches are available (selective vs. non-selective media±enrichment). Hypothesis/Gap Statement. Comprehensive head-to-head comparisons of culture-based methods for S. agalactiae screening in pregnant women are largely missing. Aim. We compared the test accuracy of six culture-based approaches for the detection of S. agalactiae . Methodology. We performed a cross-sectional study on vaginal or rectovaginal swabs (in liquid Amies medium) from pregnant women. Samples were analysed in parallel in six study arms using (i) colistin nalidixic acid (CNA) agar, (ii) chromogenic S. agalactiae selective agar, (iii) CNA agar after enrichment in thioglycolate broth, (iv) chromogenic agar after enrichment in thioglycolate broth, (v) CNA agar after enrichment in Todd–Hewitt broth and (vi) chromogenic agar after enrichment in Todd–Hewitt broth. The test accuracy was calculated for each study arm using a composite reference standard. Results. In total, 244 pregnant women were included (median age: 32 years, median weeks of pregnancy: 28 3/7). Positivity was comparable between approaches with direct inoculation on solid media (study arms i and ii, 15.6–18.0%) and with primary enrichment (study arms iii–vi, 17.6–18.9%). Each study arm had a specificity of 100%, while the sensitivity varied between 81% (CNA agar alone, study arm i) and 98% (Todd–Hewitt broth for enrichment followed by subculture on chromogenic agar, study arm vi). Conclusion. Enrichment in Todd–Hewitt broth followed by subculture on chromogenic agar had the highest sensitivity (98%, specificity: 100%) for the detection of S. agalactiae in pregnant women.

Dissemination of antimicrobial resistance (AMR) is a growing global public health burden. The aim of this study was to characterize AMR plasmid transmissions within a tertiary care hospital and identify relevant AMR plasmid transmission pathways. During an 18-month observation period, 540 clinical gram-negative multidrug-resistant bacterial (MDRB) isolates were collected during routine hospital surveillance and subjected to Pacific Biosciences long-read whole genome sequencing. Potential clonal transmissions were determined based on core genome multilocus sequence typing (cgMLST), and plasmid transmissions were detected using a novel real-time applicable tool for plasmid transmission detection. Potential transmissions were validated using epidemiological data. Among the 471 eligible MDRB isolates, we detected 1,539 plasmids; 84.41% of these were circularized. We identified 38 potential clonal transmissions in 24 clusters based on cgMLST and 121 potential plasmid transmissions in 24 clusters containing genetically related AMR plasmids. Among the latter clusters, 10 contained different multilocus sequence types (involving 2–38 isolates, median: 3 isolates), and nine contained multiple species (2–18 isolates, median: 4). Epidemiological data confirmed 19 clonal transmissions (in seven clusters) and an additional 12 plasmid transmissions (within eight plasmid clusters). Among these, we identified seven cases of intra-host and five patient-to-patient plasmid transmissions. We demonstrate that intra-host and patient-to-patient transmissions of AMR plasmids can be identified by combining long-read sequencing with real-time applicable tools during routine molecular surveillance. In addition, our study highlights that more than a decade of bacterial genomic surveillance missed at least one-third of all AMR transmission events due to plasmids. IMPORTANCE: Antimicrobial resistance (AMR) poses a significant threat to human health. Most AMR determinants are encoded extra-chromosomally on plasmids. Although current infection control strategies primarily focus on clonal transmission of multidrug-resistant bacteria, until today, AMR plasmid transmission routes are neither understood nor analyzed in the hospital setting. In our study, we simultaneously determined both clonal, that is, based on chromosomes, and AMR plasmid transmissions during routine molecular surveillance by combining long-read sequencing with a novel real-time applicable software tool and validated all potential transmission events with epidemiological data. Our analysis determined not only the yet unknown plasmid transmissions within healthcare facilities or within the community but also resulted, in addition to the clonal transmissions, in at least a third more transmissions due to AMR plasmids.

Objectives: Listeriosis is a severe foodborne infection and associated with high mortality. Treatment is based on ampicillin, amoxicillin or penicillin, often combined with gentamicin, but meropenem is also used occasionally. β-lactam resistant Listeria monocytogenes isolates are infrequently described but the mechanism of resistance is not known. Methods: A clinical L. monocytogenes isolate with suspected β-lactam resistance was collected from a German listeriosis patient. Resistance profiling, whole genome sequencing, comparative genomics and genetic experiments were used to identify the causative DNA polymorphism. Spontaneous ampicillin resistant suppressors of L. monocytogenes reference strain EGD-e were selected and their genomes sequenced. Results: A W428R substitution near the active site of penicillin binding protein B1 (PBP B1) was identified as the cause of ampicillin, amoxicillin and meropenem resistance. Further clinical L. monocytogenes isolates with similar resistance profiles were found by searching the genome database of the German consultant laboratory for Listeria for pbpB1 W428R -positive isolates. Furthermore, the same mutation was selected for in strain EGD-e during cultivation in the presence of ampicillin. Conclusions: L. monocytogenes can develop β-lactam resistance by a specific substitution in PBP B1, likely selected for during β-lactam treatment. Antibiotic susceptibility testing should be considered an important part of adequate listeriosis therapy.

A commercially available loop-mediated isothermal amplification assay (LAMP) for the detection of Pneumocystis jirovecii (P. jirovecii) has been evaluated for the diagnosis of Pneumocystis pneumonia (PcP) in critically ill patients. Altogether, 109 lower respiratory tract specimens from 95 patients with a positive P. jirovecii test in routine diagnostics were collected from five distinct university hospitals in Germany. All samples were tested with a qPCR and eazyplex^® LAMP assay. qPCR was set as the gold standard and was evaluated beforehand with samples from 100 patients categorized to have proven, probable, and possible PcP according to the EORTC/MSGERC guidelines. The sensitivity, specificity, and positive and negative predictive value (PPV and NPV) of the LAMP were assessed. Sensitivity was 68%, specificity was 86%, and PPV and NPV were 99% and 16%, respectively. All patients with proven PcP were positive in the LAMP. There was a weak correlation between the time to positivity and the fungal load (squared Pearson correlation coefficient (r²) = 0.5653). A positive result in the LAMP indicates a PcP. Because of the low sensitivity, negative results do not rule out an infection and should be clarified with further molecular methods. The LAMP should be used in patients in whom a PcP is expected, not for screening only.

Purpose: Staphylococcus aureus bacteremia (SAB) is associated with a 90-day mortality of 28–34%. Many SAB-patients (7.8–39%) have a secondary S. aureus bacteriuria (SABU) mainly without symptoms of a urinary tract infection. Due to high morbidity and mortality, there is an interest in rapid detection of S. aureus bacteremia. Here, we compared a rapid nucleic acid amplification test (NAAT) with conventional culture to detect S. aureus in urine and to identify cases with increased risk for SAB. Methods: In a cross-sectional study, we assessed urine samples (mid-stream, clean catch and catheter urine) of patients with SAB and bacteremia other than SAB (non-SAB). Urine samples were collected ± 3 days to the collection of the positive blood culture and were cultured on a set of selective and non-selective agar plates. NAAT was performed using a commercial test (Xpert^® SA Nasal Complete G3, Cepheid) from a sterile swab soaked in urine. Results: We included samples from 100 patients (68% male, median age: 67.4 years) with SAB and 20 patients (75% male, median age: 65.84 years) with non-SAB. The sensitivity of detecting SAB from urine samples was 47% (specificity: 90%) for NAAT, when applying a Ct-value of ≤ 37.4 for positive results. Urine culture had a sensitivity of 25% and a specificity of 95%. Molecular and culture methods showed a moderate agreement (80%, Cohens kappa: 0.55). Conclusion: NAAT from urine has a higher sensitivity than culture in patients with SAB and could potentially identify cases with increased risk for SAB. Future studies should investigate whether this characteristic could translate into a clinical benefit through rapid detection of SAB.

Staphylococcus epidermidis (S. epidermidis) is part of the human skin flora but can also cause nosocomial infections, such as device-associated infections, especially in vulnerable patient groups. Here, we investigated clinical isolates of linezolid-resistant S. epidermidis (LRSE) collected from blood cultures at the University Hospital Münster (UHM) during the period 2020–2022. All detected isolates were subjected to whole genome sequencing (WGS) and the relatedness of the isolates was determined using core genome multilocus sequence typing (cgMLST). The 15 LRSE isolates detected were classified as multilocus sequence type (ST) 2 carrying the staphylococcal cassette chromosome mec (SCCmec) type III. All isolates showed high-level resistance for linezolid by gradient tests. However, no isolate carried the cfr gene that is often associated with linezolid resistance. Analysis of cgMLST data sets revealed a cluster of six closely related LRSE isolates, suggesting a transmission event on a hematological/oncological ward at our hospital. Among the included patients, the majority of patients affected by LRSE infections had underlying hematological malignancies. This confirms previous observations that this patient group is particularly vulnerable to LRSE infection. Our data emphasize that the surveillance of LRSE in the hospital setting is a necessary step to prevent the spread of multidrug-resistant S. epidermidis among vulnerable patient groups, such as patients with hematological malignancies, immunosuppression or patients in intensive care units.

Background: Capnocytophaga canimorsus, a Gram-negative rod, belongs to the Flavobacteriaceae family and colonizes the oropharynx of dogs and cats. Infections with C. canimorsus are rare and can induce a systemic infection with a severe course of the disease. So far, only five case reports of C. canimorsus infections associated with Waterhouse–Friderichsen Syndrome (WFS) have been reported with only two of the patients having a history of splenectomy. Case presentation: Here, we report a fatal case of WFS due to C. canimorsus bacteremia and mycetal superinfection in a 61-year-old female asplenic patient. Despite extensive therapy including mechanical ventilation, antibiotic coverage with meropenem, systemic corticosteroids medication, vasopressor therapy, continuous renal replacement therapy, therapeutic plasma exchange, multiple transfusions of blood products and implantation of a veno-arterial extracorporeal membrane oxygenation the patient died 10 days after a dog bite. The autopsy showed bilateral hemorrhagic necrosis of the adrenal cortex and septic embolism to heart, kidneys, and liver. Diagnosis of C. canimorsus was prolonged due to the fastidious growth of the bacteria. Conclusions: The occurrence of a severe sepsis after dog bite should always urge the attending physician to consider C. canimorsus as the disease-causing pathogen. A therapeutic regimen covering C. canimorsus such as aminopenicillins or carbapenems should be chosen. However, despite maximum therapy, the prognosis of C. canimorsus-induced septic shock remains very poor. Asplenic or otherwise immunocompromised patients are at higher risk for a severe course of disease and should avoid exposure to dogs and cats and consider antibiotic prophylaxis after animal bite.