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There has been a growing body of evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) shows enhanced transmissibility and increased viral loads compared to other variants. A recent study has even suggested that respiratory samples from people infected with the Delta variant can harbor up to 1000 times higher viral loads compared to samples with variants that are more closely related to the original Wuhan strain, although the sample size of this study (n = 125) was very limited. Here, we have compared the viral load in 16,185 samples that were obtained in periods during which non-VOC, the Alpha (B.1.1.7) or Delta variant (B.1.617.2) were dominant as evidenced by genomic surveillance. We found that the Delta variant contained about fourfold higher viral loads across all age groups compared to the non-VOC or Alpha variants, which is significantly lower than reported earlier. Interestingly, the increased viral load for the Delta variant seemed to be age-dependent, regardless of sex, as the viral load was about 14-fold higher for Delta compared to the non-VOC or Alpha variant in age group 0–20 years and fourfold higher in age group 21–40 years, while there was no difference in viral load between variants in age groups 41–60 and 61+ years, most likely as a consequence of a higher degree of vaccination in the older age groups.

Vancomycin-resistant enterococci (VRE) can rapidly spread through hospitals. Therefore, our hospital employs a screening program whereby rectal swabs are screened for the presence of vanA and vanB , and only PCR-positive broths are cultured on VRE selection agar. Early November 2016, a clinical vanA- / vanB -negative VRE isolate was detected in a vanA/vanB -screening-negative patient, giving the possibility that an undetected VRE might be spreading within our hospital. Whole-genome-sequencing of the isolate showed that resistance was vanD -mediated and core genome multilocus sequence typing showed it was a rare type: ST17/CT154. To determine the prevalence of vanA/B/C/D -carrying enterococci, we designed a real-time PCR for vanC1/2/3 and vanD and screened rectal swabs from 360 patients. vanD was found in 27.8% of the patients, yet culture demonstrated only E . faecium from vanA -positive broths and E . gallinarum from vanC1 -positive broths. No vanD -positive VRE were found, limiting the possibility of nosocomial spread of this VRE. Moreover, the high prevalence of non-VRE vanD in rectal swabs makes it unfeasible to include the vanD PCR in our VRE screening. However, having validated the vanC1/2/3 and vanD PCRs allows us to rapidly check future vanA/B -negative VRE for the presence of vanC and vanD genes.

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with l-arginine supplementation exhibited less consistent results; however, l-citrulline, the precursor of l-arginine, may be a promising alternative. In this study, we determined the effects of l-citrulline compared to l-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with l-citrulline or l-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. l-arginine and l-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that l-citrulline, and not l-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

Objectives Maintenance treatment with macrolide antibiotics has shown to be effective in reducing exacerbations in COPD patients. A major concern with prolonged treatment with antibiotics is the development of bacterial resistance. In this study we determined the effect of azithromycin on the development and acquisition of resistance to macrolides in the nasopharyngeal flora in COPD patients. Methods This study was part of the COLUMBUS trial, a randomised, double-blind, placebo-controlled trial to measure the effect of maintenance treatment with azithromycin in 92 COPD patients on the exacerbation rates during a 12-month period. In order to determine resistance to macrolides, we used a targeted metagenomic approach to measure the presence and relative abundance of specific macrolide resistance genes ermB , ermF and mefA in throat samples collected at different time-points during this 12-month period. Results There was no increased risk for acquisition of macrolide resistance genes in the azithromycin group compared to the placebo group in COPD patients. However, loss of the macrolide resistance gene ermB was increased overtime in the placebo treated group compared to the azithromycin group ( n  = 5 for the placebo group versus n  = 0 for the azithromycin group at 12 months; p  = 0.012). The change in relative abundance of the three macrolide-resistance genes showed that all but one ( ermF ) increased during treatment with azithromycin. Conclusions The acquisition rate of macrolide resistance genes in COPD patients treated with azithromycin maintenance therapy was limited, but the relative abundance of macrolide resistance genes increased significantly over time compared to placebo. This study was part of the COLUMBUS trial ( Clinicaltrials.gov , NCT00985244 ).

We investigated the longitudinal development of several antibiotic resistance genes (ARGs) of the infant gut resistome during the first months after birth. Fecal samples from 120 infants collected at the ages of 5, 13 and 31 weeks were analyzed and subjected to qPCR for the detection of several ARGs. The prevalence of ARGs significantly increased for and , while it decreased for . Birth mode and breastfeeding significantly affected prevalence. Correlations to bacterial taxa suggest that fluctuations in some ARGs are (partly) attributed to shifts in bacteroides colonization rates. Acquisition of ARGs in the gut microbiota occurs shortly after birth and resistome composition fluctuates over the course of several months, reflecting changes in microbial community structure.

Genotyping of Klebsiella pneumoniae is indispensable for management of nosocomial infections, monitoring of emerging strains--including extended-spectrum beta-lactamase (ESBL) producers-, and general epidemiology. Such objectives require a high-resolution genotyping method with a fixed scheme that allows (1) long-term retrospective and prospective assessment, (2) objective result readout and (3) library storage for database development and exchangeable results. We have developed a multiple-locus variable number tandem repeat analysis (MLVA) using a single-tube fluorescently primed multiplex PCR for 8 Variable Number Tandem Repeats (VNTRs) and automated fragment size analysis. The type allocation scheme was optimized using 224 K. pneumoniae clinical isolates, which yielded 101 MLVA types. The method was compared to the gold standard multilocus sequence typing (MLST) using a subset of these clinical isolates (n = 95) and found to be highly concordant, with at least as high a resolution but with considerably less hands-on time. Our results position this MLVA scheme as an appropriate, high-throughput and relatively low-cost tool for K. pneumoniae epidemiology.

Background: The patient environment is increasingly considered a major source of transmission of nosocomial bacteria to patients. In May 2019, a cluster of 3 patients with multiresistant Enterobacter cloacae was discovered in the hematology-oncology department of the Maastricht University Medical Center (built in 1991). The strains had an identical antibiogram: ESBL-positive, ciprofloxacin R, cotrimoxazole R, meropenem S, and colistin S. One neutropenic patient had a positive blood culture for this strain, resistant to the empiric treatment with piperacillin-tazobactam, but the patient recovered after switching the antibiotic regimen to meropenem. All strains were determined to be identical by amplified-fragment length polymorphism and whole-genome multi-locus sequencing typing (genotype A). New cases occurred, despite the introduction of contact isolation of positive and contact patients. Therefore, weekly point-prevalence screening was introduced, in which more newly colonized patients were identified in the subsequent weeks. Attention to hand hygiene was enforced, and the hypothesis of contamination from “wet” environmental locations was tested by performing cultures of sinks and shower drains. In June and July, 47 of 241 environmental cultures (19.5%) were positive for E. cloacae with an identical antibiogram, among which some were typed as genotype A. To diminish the environmental contamination, all siphons of sinks were replaced, and disinfection of sinks and shower drains was intensified using chlorine and soda on a daily basis. Replacement of shower drains was not possible. After this intervention, the incidence of newly colonized patients declined gradually. A change in the regimen of selective gut decontamination in hematology patients was considered as an alternative intervention, but with the decrease in new patient cases, this was not implemented. A final round of environmental cultures at the end of August revealed 8 positive cultures, of which 5 were positive for genotype A. In retrospect, this finding could be explained by the fact that the cleaning team did not follow the intensified instructions for disinfection. From week 29, genotype A E. cloacae was no longer cultured in weekly patient screenings. Based on this observation, it is important that in (re)building plans for hospitals, a master plan for the prevention of nosocomial transmission from environment to patients is incorporated. Funding: None Disclosures: None

Objectives: Maintenance treatment with macrolide antibiotics has shown to be effective in reducing exacerbations in COPD patients. A major concern with prolonged treatment with antibiotics is the development of bacterial resistance. In this study we determined the effect of azithromycin on the development and acquisition of resistance to macrolides in the nasopharyngeal flora in COPD patients. Methods: This study was part of the COLUMBUS trial, a randomised, double-blind, placebo-controlled trial to measure the effect of maintenance treatment with azithromycin in 92 COPD patients on the exacerbation rates during a 12-month period. In order to determine resistance to macrolides, we used a targeted metagenomic approach to measure the presence and relative abundance of specific macrolide resistance genes ermB, ermF and mefA in throat samples collected at different time-points during this 12-month period. Results: There was no increased risk for acquisition of macrolide resistance genes in the azithromycin group compared to the placebo group in COPD patients. However, loss of the macrolide resistance gene ermB was increased overtime in the placebo treated group compared to the azithromycin group (n=5 for the placebo group versus n=0 for the azithromycin group at 12 months; p=0.012). The change in relative abundance of the three macrolide-resistance genes showed that all but one (ermF) increased during treatment with azithromycin. Conclusions: The acquisition rate of macrolide resistance genes in COPD patients treated with azithromycin maintenance therapy was limited, but the relative abundance of macrolide resistance genes increased significantly over time compared to placebo. This study was part of the COLUMBUS trial (Clinicaltrials.gov, NCT00985244).