Tobramycin and colistin represent 2 standard antimicrobial agents in the treatment of cystic fibrosis (CF) patients who are chronically colonized with Pseudomonas aeruginosa. In this study, we determined the rate of resistance to tobramycin and colistin in 1844 isolates of P. aeruginosa obtained from 22 CF patients under alternate therapy with inhaled tobramycin and colistin. Resistance to tobramycin was observed in 27.5% of isolates. In contrast, all isolates were susceptible to colistin. Molecular typing of selected isolates suggested that only 1 clone occurred over time in each patient. To conclude, resistance to tobramycin in P. aeruginosa isolates from CF patients under antimicrobial therapy may occur while colistin resistance remains uncommon.

Introduction: The Enterobacter cloacae complex (ECC) species are potential neonatal pathogens, and ECC strains are among the most commonly encountered Enterobacter spp. associated with nosocomial bloodstream infections. Outbreaks caused by ECC can lead to significant morbidity and mortality in susceptible neonates. At the molecular level, ECC exhibits genomic heterogeneity, with six closely related species and subspecies. Genetic variability poses a challenge in accurately identifying outbreaks by determining the clonality of ECC isolates. This difficulty is further compounded by the limitations of the commonly used molecular typing methods, such as pulsed field gel electrophoresis, which do not provide reliable accuracy in distinguishing between ECC strains and can lead to incorrect conclusions. Next-generation sequencing (NGS) offers superior resolution in determining strain relatedness. Therefore, we investigated the clinical pertinence of incorporating NGS into existing bundle measures to enhance patient management during an outbreak of ECC in a level-3 neonatal intensive care unit (NICU) in Germany. Methods: As the standard of care, all neonates on the NICU received weekly microbiological swabs (nasopharyngeal and rectal) and analysis of endotracheal secretion, where feasible. During the 2.5-month outbreak, colonisation with ECC was detected in n = 10 neonates. The phylogenetic relationship and potential antimicrobial resistance genes as well as mobile genetic elements were identified via bacterial whole-genome sequencing (WGS) using Illumina MiSeq followed by in silico data analysis. Results: Although all ECC isolates exhibited almost identical antimicrobial susceptibility patterns, the WGS data revealed the involvement of four different ECC clones. The isolates could be characterised as Enterobacter hormaechei subspecies steigerwaltii (n = 6, clonal), subsp. hoffmannii (n = 3, two clones) and subsp. oharae (n = 1). Despite the collection of environmental samples, no source of this diffuse outbreak could be identified. A new standardised operating procedure was implemented to enhance the management of neonates colonised with MRGN. This collaborative approach involved both parents and medical professionals and successfully prevented further transmission of ECC. Conclusions: Initially, it was believed that the NICU outbreak was caused by a single ECC clone due to the similarity in antibiotic resistance. However, our findings show that antibiotic susceptibility patterns can be misleading in investigating outbreaks of multi-drug-resistant ECC. In contrast, bacterial WGS accurately identified ECC at the clonal level, which significantly helped to delineate the nature of the observed outbreak.

Previously, we demonstrated that the majority of vancomycin-resistant Enterococcus faecium (VREfm) strains from in-patients of the University Hospital Erlangen, Germany, belonged to only three clonal lineages, namely ST117/CT71 vanB and two novel ST1299 vanA lineages classified as CT3109 and CT1903. The goal of the current study was (i) to investigate whether VREfm is also detectable in wastewater of the city of Erlangen, (ii) to identify their molecular features, and (iii) to clarify whether VREfm could arise from the community of the city of Erlangen or can be (directly) connected to nosocomial infections in the hospital setting. From April to May 2023, a total of 244 VREfm strains from raw wastewater of the city of Erlangen were analyzed by core genome multilocus sequence typing (cgMLST). Moreover, 20 of them were further investigated for single nucleotide polymorphisms (SNPs). The molecular characterization of the wastewater VREfm strains revealed a high prevalence (27.9%) of the recently identified clonal lineage ST1299/CT3109 vanA, which is mainly characterized by the presence of the tetracycline-resistance determinant tet(M ) and the virulence genes pilA and prpA . The SNPs analysis revealed the presence of two major clusters, namely cluster I (≤65 SNPs), which included well-known hospital-adapted vanB clonal lineages such as ST117/CT71 and ST80/CT1065 and cluster II (≤70 SNPs), which were mainly characterized by the lineage ST1299/CT3109 vanA . Based on the concomitant resistance to vancomycin and tetracycline, we propose that ST1299/CT3109 vanA primarily originated and spread outside of hospital settings. IMPORTANCE: This study provides a detailed genomic analysis of vancomycin-resistant Enterococcus faecium (VREfm) strains isolated from municipal wastewater with a particular focus on clonal lineages, antimicrobial resistance, and the presence of virulence genes. The high wastewater prevalence of the recently identified clonal lineage ST1299/CT3109 vanA , which has been previously detected in hospitals, suggests an enormous potential for future spread in Germany.

Since 2019, we have observed a remarkable rise of vancomycin resistance among invasive strains of Enterococcus (E.) faecium at the University Hospital Erlangen (UKER), a tertiary-care hospital in Germany. Here, we analyzed the genetic diversity of E. faecium isolated from blood cultures of in-patients by core genome multilocus sequence typing to see whether the occurrence of vancomycin resistance was associated with the spread of new clonal lineages. Between January and December 2022, 37 consecutive non-duplicate isolates were included in this study. Resistance to vancomycin was detected in 15 isolates (40.5%). The affected patients had a median age of 65 years, and 26 of them (70.3%) were males. Patients with bloodstream infection caused by vancomycin-resistant E. faecium (VREfm) or vancomycin-susceptible E. faecium (VSEfm) showed a similar in-hospital mortality rate (33.3% vs 27.3%, P = 0.692), indicating that VREfm were not more virulent than VSEfm. In contrast, the genetic diversity was higher in VSEfm (15 clonal lineages) than in VREfm isolates (seven clonal lineages). The majority of VREfm isolates (73.2%) belonged to only three clonal lineages: ST117/CT71 vanB ( n = 4), which is the most commonly detected vancomycin-resistant lineage in Germany, and two novel ST1299 vanA lineages classified as CT3109 ( n = 4) and CT1903 ( n = 3). We conclude that the high rate of vancomycin resistance among invasive E. faecium isolates at UKER could be associated with the emergence of novel ST1299 vanA lineages, which is likely to be relevant also for other hospitals and countries. Future studies will focus on the prevalence of ST1299 vanA in the general population. IMPORTANCE: The proportion of VREfm among all Enterococcus faecium isolated from blood cultures in German hospitals has increased in the period 2015–2020 from 11.9% to 22.3% with a country-wide spread of the clonal lineage ST117/CT71 vanB . In this study, we provided useful information about the genetic diversity of invasive strains of E. faecium . Moreover, our findings confirm the nosocomial spread of novel ST1299 vanA lineages, which recently had a rapid expansion in Austria and the south-eastern part of Germany.

Invasive Candida infection (ICI) is the most common fungal infection in critically ill patients. This study analyzed the performance of various biomarkers in sera from the CandiSep trial, a randomized, multicenter trial including 342 sepsis patients at high risk for ICI across 18 German intensive care units. ICI and candidemia were diagnosed in 48 (14.0%) and 14 (4.1%) patients, respectively. Sera collected on 2 consecutive days at sepsis onset were analyzed for β-(1→3)-D-glucan (BDG; Fungitell), mannan (Platelia- Candida -Ag-Plus [Platelia-Mn] and Serion-ELISA-antigen- Candida [Serion-Mn]), and anti- Candida antibodies (Platelia- Candida -Ab-Plus, Serion-ELISA- Candida albicans -IgA/IgM/IgG, and Virclia- Candida albicans -germ-tube-antibody-IgG-Monotest). Only antigen levels (BDG, Platelia-Mn, Serion-Mn), but not anti-Candida antibody levels, were significantly elevated in ICI patients. Antigen levels were unaffected by Candida colonization, while antibody levels were significantly increased. Sensitivity and specificity at the manufacturer’s cutoffs were unsatisfactory. Performance improved by adjusting cutoffs: BDG required a 3-fold increase, while all others required lowering. At 80% specificity, sensitivities (95% confidence intervals) for the diagnosis of ICI and candidemia were as follows: BDG (cutoff >280 pg/mL), 46% (31.4–60.8) and 64% (35.1–87.2); Platelia-Mn (cutoff >50 pg/mL), 38% (24.0–52.6) and 64% (35.1–87.2); Serion-Mn (cutoff >0.7 U/mL), 38% (24.0–52.6) and 50% (23.0–77.0), and below 28% and 43% for all antibody assays. Area under the receiver operating characteristic curve comparisons showed no significant differences between antigen assays. Combining biomarkers, either simultaneously or sequentially, offered no diagnostic advantage over single-biomarker use. In conclusion, anti- Candida antibody assays are likely influenced by colonization and have limited diagnostic utility. Antigen assays offer similar diagnostic value but require cutoff optimization. Combining biomarkers did not enhance diagnostic yield in our cohort. IMPORTANCE: Our main findings based on our specific ICU cohort were as follows: (i) only Candida antigen levels, not anti- Candida antibody levels, were significantly elevated in ICI and candidemia. (ii) In patients without ICI, Candida colonization was not associated with altered antigen levels, but with elevated antibody levels. (iii) Sensitivity and specificity at the manufacturer’s cutoffs were unsatisfactory, and cutoffs should be significantly adjusted. Potential optimal cutoff values are proposed by us. (iv) The evaluated antigen assays demonstrated overall comparable diagnostic performance in this study. (v) Anti- Candida antibody assays did not provide a meaningful diagnostic contribution. (vi) The combination of biomarkers offered no diagnostic advantage over the use of individual biomarkers, neither simultaneously nor sequentially. Our results suggest that the use of a single Candida antigen test with a cohort-adapted cutoff value may be sufficient. Furthermore, avoiding biomarker combinations could potentially reduce healthcare costs. The clinical implications of these findings should be interpreted with caution, given the limited data with associated large confidence intervals for candidemia and the cohort-specific nature of the study. Our results should therefore be confirmed in larger studies and additionally with other patient groups.