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Abstract Background Pancreatic stone protein (PSP) exhibits potential as a plasma biomarker for infection diagnosis and risk stratification in critically ill patients, but its significance in nosocomial infection and intensive care unit (ICU)-acquired bloodstream infection (BSI) remains unclear. This study explores the temporal responses of PSP in ICU-acquired BSI caused by different pathogens. Methods From a large cohort of ICU patients, we selected episodes of ICU-acquired BSI caused by Gram-negative rods (GNRs), enterococci, or Candida species. Events were matched on length of ICU stay at infection onset, Severe Organ Failure Assessment (SOFA) score, presence of immune deficiency, and use of renal replacement therapy. PSP concentrations were measured at infection onset (T0) and at 24, 48 and 72 h prior to infection onset as defined by the first occurrence of a positive blood culture. Absolute and trend differences in PSP levels between pathogen groups were analysed using one-way analysis of variance. Sensitivity analyses were performed in events with a new or worsening systematic inflammatory response based on C-reactive protein, white cell count and fever. Results We analysed 30 BSI cases per pathogen group. Median (IQR) BSI onset was on day 9 (6–12). Overall, PSP levels were high (381 (237–539) ng/ml), with 18% of values exceeding the assay’s measurement range. Across all 90 BSI cases, there was no clear trend over time (median change 34 (− 75–189) ng/ml from T-72 to T0). PSP concentrations at infection onset were 406 (229–497), 350 (223–608), and 480 (327–965) ng/ml, for GNR, enterococci, and Candida species, respectively (p = 0.32). At every time point, absolute PSP levels and trends did not differ significantly between pathogens. PSP values at T0 correlated with SOFA scores. Eighteen (20%) of 90 BSI events did not exhibit a systemic inflammatory response, primarily in Candida species. No clear change in PSP concentration before BSI onset or between-group differences were found in sensitivity analyses of 72 cases. Conclusions Against a background of overall (very) high plasma PSP levels in critically ill patients, we did not find clear temporal patterns or any pathogen-specific differences in PSP response in the days preceding onset of ICU-acquired BSI.

Abstract Background Impaired intestinal barrier function can enable passage of enteric microorganisms into the bloodstream and lead to nosocomial bloodstream infections during critical illness. We aimed to determine the relative importance of gut translocation as a source for ICU-acquired enterococcal bacteremia of unknown origin. Methods We conducted a nested case–control study in two mixed medical-surgical tertiary ICUs in the Netherlands among patients enrolled between 2011 and 2018. We selected 72 cases with ICU-acquired bacteremia due to enterococci (which are known gastrointestinal tract commensals) and 137 matched controls with bacteremia due to coagulase-negative staphylococci (CoNS) (which are of non-intestinal origin). We measured intestinal fatty acid-binding protein, trefoil factor-3, and citrulline 48 h before bacteremia onset. A composite measure for Gut Barrier Injury (GBI) was calculated as the sum of standardized z-scores for each biomarker plus a clinical gastrointestinal failure score. Results No single biomarker yielded statistically significant differences between cases and controls. Median composite GBI was higher in cases than in controls (0.58, IQR − 0.36–1.69 vs. 0.32, IQR − 0.53–1.57, p = 0.33) and higher composite measures of GBI correlated with higher disease severity and ICU mortality (p

SARS-CoV-2 infections elicit antibodies against the viral spike (S) and nucleocapsid (N) proteins; COVID-19 vaccines against the S-protein only. The BCG-Corona trial, initiated in March 2020 in SARS-CoV-2-naïve Dutch healthcare workers, captured several epidemic peaks and the introduction of COVID-19 vaccines during the one-year follow-up. We assessed determinants of systemic anti-S1 and anti-N immunoglobulin type G (IgG) responses using trial data. Participants were randomised to BCG or placebo vaccination, reported daily symptoms, SARS-CoV-2 test results, and COVID-19 vaccinations, and donated blood for SARS-CoV-2 serology at two time points. In the 970 participants, anti-S1 geometric mean antibody concentrations (GMCs) were much higher than anti-N GMCs. Anti-S1 GMCs significantly increased with increasing number of immune events (SARS-CoV-2 infection or COVID-19 vaccination): 104.7 international units (IU)/mL, 955.0 IU/mL, and 2290.9 IU/mL for one, two, and three immune events, respectively (p < 0.001). In adjusted multivariable linear regression models, anti-S1 and anti-N log10 concentrations were significantly associated with infection severity, and anti-S1 log10 concentration with COVID-19 vaccine type/dose. In univariable models, anti-N log10 concentration was also significantly associated with acute infection duration, and severity and duration of individual symptoms. Antibody concentrations were not associated with long COVID or long-term loss of smell/taste.

Abstract Background Immune suppression has been implicated in the occurrence of pneumonia in critically ill patients. We tested the hypothesis that Intensive Care Unit (ICU)-acquired pneumonia is associated with broad host immune aberrations in the trajectory to pneumonia, encompassing inflammatory, endothelial and coagulation responses. We compared plasma protein biomarkers reflecting the systemic host response in critically ill patients who acquire a new pneumonia (cases) with those who do not (controls). Methods We performed a nested case–control study in patients undergoing mechanical ventilation at ICU admission with an expected stay of at least 48 h enrolled in 30 hospitals in 11 European countries. Nineteen host response biomarkers reflective of key pathophysiological domains were measured in plasma obtained on study inclusion and day 7, and—in cases—on the day of pneumonia diagnosis. Results Of 1997 patients, 316 developed pneumonia (15.8%) and 1681 did not (84.2%). Plasma protein biomarker analyses, performed in cases and a randomly selected subgroup of controls (1:2 ratio to cases, n = 632), demonstrated considerable variation across time points and patient groups. Yet, cases showed biomarker concentrations suggestive of enhanced inflammation and a more disturbed endothelial barrier function, both at study enrollment (median 2 days after ICU admission) and in the path to pneumonia diagnosis (median 5 days after ICU admission). Baseline host response biomarker aberrations were most profound in patients who developed pneumonia either shortly ( 10 days, n = 68) after ICU admission. Conclusions Critically ill patients who develop an ICU-acquired pneumonia, compared with those who do not, display alterations in plasma protein biomarker concentrations indicative of stronger proinflammatory, procoagulant and (injurious) endothelial cell responses. Trial registration: ClinicalTrials.gov Identifier: NCT02413242, posted April 9th, 2015. Graphical abstract

Abstract Background The Dutch Working Party on Antibiotic Policy (SWAB) in collaboration with relevant professional societies, has updated their evidence-based guidelines on empiric antibacterial therapy of sepsis in adults. Methods Our multidisciplinary guideline committee generated ten population, intervention, comparison, and outcome (PICO) questions relevant for adult patients with sepsis. For each question, a literature search was performed to obtain the best available evidence and assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system. The quality of evidence for clinically relevant outcomes was graded from high to very low. In structured consensus meetings, the committee formulated recommendations as strong or weak. When evidence could not be obtained, recommendations were provided based on expert opinion and experience (good practice statements). Results Fifty-five recommendations on the antibacterial therapy of sepsis were generated. Recommendations on empiric antibacterial therapy choices were differentiated for sepsis according to the source of infection, the potential causative pathogen and its resistance pattern. One important revision was the distinction between low, increased and high risk of infection with Enterobacterales resistant to third generation cephalosporins (3GRC-E) to guide the choice of empirical therapy. Other new topics included empirical antibacterial therapy in patients with a reported penicillin allergy and the role of pharmacokinetics and pharmacodynamics to guide dosing in sepsis. We also established recommendations on timing and duration of antibacterial treatment. Conclusions Our multidisciplinary committee formulated evidence-based recommendations for the empiric antibacterial therapy of adults with sepsis in The Netherlands.

ABSTRACT Bacillus Calmette-Guerin (BCG) vaccination has been hypothesized to reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severity, and/or duration via trained immunity induction. Health care workers (HCWs) in nine Dutch hospitals were randomized to BCG or placebo vaccination (1:1) in March and April 2020 and followed for 1 year. They reported daily symptoms, SARS-CoV-2 test results, and health care-seeking behavior via a smartphone application, and they donated blood for SARS-CoV-2 serology at two time points. A total of 1,511 HCWs were randomized and 1,309 analyzed (665 BCG and 644 placebo). Of the 298 infections detected during the trial, 74 were detected by serology only. The SARS-CoV-2 incidence rates were 0.25 and 0.26 per person-year in the BCG and placebo groups, respectively (incidence rate ratio, 0.95; 95% confidence interval, 0.76 to 1.21; P = 0.732). Only three participants required hospitalization for SARS-CoV-2. The proportions of participants with asymptomatic, mild, or moderate infections and the mean infection durations did not differ between randomization groups. In addition, unadjusted and adjusted logistic regression and Cox proportional hazards models showed no differences between BCG and placebo vaccination for any of these outcomes. The percentage of participants with seroconversion (7.8% versus 2.8%; P = 0.006) and mean SARS-CoV-2 anti-S1 antibody concentration (13.1 versus 4.3 IU/mL; P = 0.023) were higher in the BCG than placebo group at 3 months but not at 6 or 12 months postvaccination. BCG vaccination of HCWs did not reduce SARS-CoV-2 infections nor infection duration or severity (ranging from asymptomatic to moderate). In the first 3 months after vaccination, BCG vaccination may enhance SARS-CoV-2 antibody production during SARS-CoV-2 infection. IMPORTANCE While several BCG trials in adults were conducted during the 2019 coronavirus disease epidemic, our data set is the most comprehensive to date, because we included serologically confirmed infections in addition to self-reported positive SARS-CoV-2 test results. We also collected data on symptoms for every day during the 1-year follow-up period, which enabled us to characterize infections in detail. We found that BCG vaccination did not reduce SARS-CoV-2 infections nor infection duration or severity but may have enhanced SARS-CoV-2 antibody production during SARS-CoV-2 infection in the first 3 months after vaccination. These results are in agreement with other BCG trials that reported negative results (but did not use serological endpoints), except for two trials in Greece and India that reported positive results but had few endpoints and included endpoints that were not laboratory confirmed. The enhanced antibody production is in agreement with prior mechanistic studies but did not translate into protection from SARS-CoV-2 infection.

Abstract Background Immunomodulatory therapies that improve the outcome of sepsis are not available. We sought to determine whether treatment of critically ill patients with sepsis with low-dose erythromycin—a macrolide antibiotic with broad immunomodulatory effects—decreased mortality and ameliorated underlying disease pathophysiology. Methods We conducted a target trial emulation, comparing patients with sepsis admitted to two intensive care units (ICU) in the Netherlands for at least 72 h, who were either exposed or not exposed during this period to treatment with low-dose erythromycin (up to 600 mg per day, administered as a prokinetic agent) but no other macrolides. We used two common propensity score methods (matching and inverse probability of treatment weighting) to deal with confounding by indication and subsequently used Cox regression models to estimate the treatment effect on the primary outcome of mortality rate up to day 90. Secondary clinical outcomes included change in SOFA, duration of mechanical ventilation and the incidence of ICU-acquired infections. We used linear mixed models to assess differences in 15 host response biomarkers reflective of key pathophysiological processes from admission to day 4. Results In total, 235 patients started low-dose erythromycin treatment, 470 patients served as controls. Treatment started at a median of 38 [IQR 25–52] hours after ICU admission for a median of 5 [IQR 3–8] total doses in the first course. Matching and weighting resulted in populations well balanced for proposed confounders. We found no differences between patients treated with low-dose erythromycin and control subjects in mortality rate up to day 90: matching HR 0.89 (95% CI 0.64–1.24), weighting HR 0.95 (95% CI 0.66–1.36). There were no differences in secondary clinical outcomes. The change in host response biomarker levels from admission to day 4 was similar between erythromycin-treated and control subjects. Conclusion In this target trial emulation in critically ill patients with sepsis, we could not demonstrate an effect of treatment with low-dose erythromycin on mortality, secondary clinical outcomes or host response biomarkers.

Abstract Background Quantification of acquisition routes of antibiotic resistant bacteria (ARB) is pivotal for understanding transmission dynamics and designing cost-effective interventions. Different methods have been used to quantify the importance of transmission routes, such as relative risks, odds ratios (OR), genomic comparisons and basic reproduction numbers. We systematically reviewed reported estimates on acquisition routes’ contributions of ARB in humans, animals, water and the environment and assessed the methods used to quantify the importance of transmission routes. Methods PubMed and EMBASE were searched, resulting in 6054 articles published up until January 1st, 2019. Full text screening was performed on 525 articles and 277 are included. Results We extracted 718 estimates with S. aureus (n = 273), E. coli (n = 157) and Enterobacteriaceae (n = 99) being studied most frequently. Most estimates were derived from statistical methods (n = 560), mainly expressed as risks (n = 246) and ORs (n = 239), followed by genetic comparisons (n = 85), modelling (n = 62) and dosage of ARB ingested (n = 17). Transmission routes analysed most frequently were occupational exposure (n = 157), travelling (n = 110) and contacts with carriers (n = 83). Studies were mostly performed in the United States (n = 142), the Netherlands (n = 87) and Germany (n = 60). Comparison of methods was not possible as studies using different methods to estimate the same route were lacking. Due to study heterogeneity not all estimates by the same method could be pooled. Conclusion Despite an abundance of published data the relative importance of transmission routes of ARB has not been accurately quantified. Links between exposure and acquisition are often present, but the frequency of exposure is missing, which disables estimation of transmission routes’ importance. To create effective policies reducing ARB, estimates of transmission should be weighed by the frequency of exposure occurrence.

Abstract Background Surveillance is the cornerstone of surgical site infection prevention programs. The validity of the data collection and awareness of vulnerability to inter-rater variation is crucial for correct interpretation and use of surveillance data. The aim of this study was to investigate the reliability and validity of surgical site infection (SSI) surveillance after colorectal surgery in the Netherlands. Methods In this multicentre prospective observational study, seven Dutch hospitals performed SSI surveillance after colorectal surgeries performed in 2018 and/or 2019. When executing the surveillance, a local case assessment was performed to calculate the overall percentage agreement between raters within hospitals. Additionally, two case-vignette assessments were performed to estimate intra-rater and inter-rater reliability by calculating a weighted Cohen’s Kappa and Fleiss’ Kappa coefficient. To estimate the validity, answers of the two case-vignettes questionnaires were compared with the answers of an external medical panel. Results 1111 colorectal surgeries were included in this study with an overall SSI incidence of 8.8% (n = 98). From the local case assessment it was estimated that the overall percent agreement between raters within a hospital was good (mean 95%, range 90–100%). The Cohen’s Kappa estimated for the intra-rater reliability of case-vignette review varied from 0.73 to 1.00, indicating substantial to perfect agreement. The inter-rater reliability within hospitals showed more variation, with Kappa estimates ranging between 0.61 and 0.94. In total, 87.9% of the answers given by the raters were in accordance with the medical panel. Conclusions This study showed that raters were consistent in their SSI-ascertainment (good reliability), but improvements can be made regarding the accuracy (moderate validity). Accuracy of surveillance may be improved by providing regular training, adapting definitions to reduce subjectivity, and by supporting surveillance through automation.

Background and objectiveA recent study has suggested that circadian rhythm has an important impact on the immunological effects induced by Bacillus Calmette-Guérin (BCG) vaccination. The objective of this study was to evaluate whether the timing of BCG vaccination (morning or afternoon) affects its impact on severe acute respiratory syndrome–coronavirus-2 (SARS-CoV-2) infections and clinically relevant respiratory tract infections (RTIs).MethodsThis is a post-hoc analysis of the BCG-CORONA-ELDERLY (NCT04417335) multicenter, placebo-controlled trial, in which participants aged 60 years and older were randomly assigned to vaccination with BCG or placebo, and followed for 12 months. The primary endpoint was the cumulative incidence of SARS-CoV-2 infection. To assess the impact of circadian rhythm on the BCG effects, participants were divided into four groups: vaccinated with either BCG or placebo in the morning (between 9:00h and 11:30h) or in the afternoon (between 14:30h and 18:00h).ResultsThe subdistribution hazard ratio of SARS-CoV-2 infection in the first six months after vaccination was 2.394 (95% confidence interval [CI], 0.856-6.696) for the morning BCG group and 0.284 (95% CI, 0.055-1.480) for the afternoon BCG group. When comparing those two groups, the interaction hazard ratio was 8.966 (95% CI, 1.366-58.836). In the period from six months until 12 months after vaccination cumulative incidences of SARS-CoV-2 infection were comparable, as well as cumulative incidences of clinically relevant RTI in both periods.ConclusionAlthough there was a difference in effect between morning and afternoon BCG vaccination, the vaccine did not protect against SARS-COV-2 infections and clinically relevant RTI’s at either timepoint.

ABSTRACT Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales have been increasingly isolated from pigs, highlighting their potential for transmission to humans living and/or working within pig farms. As longitudinal data on the prevalence and the molecular characteristics of such isolates from the high-risk farming population remain scarce, we performed a long-term study on 39 Dutch pig farms. Fecal samples from pigs, farmers, family members, and employees were collected during four sampling occasions with a 6-month period. The presence of ESBL-producing Enterobacterales and their molecular characteristics (ESBL gene, plasmid, and sequence types) were determined by standard methods. Data on personal and farm characteristics were collected using questionnaires. ESBL-producing Escherichia coli was present in pigs at least once for 18 of 39 farms and in 17 of 146 farmers, family members, and/or employees. Among these 417 E. coli isolates, blaCTX-M-1 was the most frequently observed ESBL gene in pigs (n = 261) and humans (n = 25). Despite the great variety in plasmid (sub)types and E. coli sequence types (STs), we observed genetic similarity between human- and pig-derived isolates in (i) ESBL gene, plasmid (sub)type, and ST, suggesting potential clonal transmission in seven farms, and (ii) only ESBL gene and plasmid (sub)type, highlighting the possibility of horizontal transfer in four farms. Five pig farmers carried ESBL producers repeatedly, of whom two carried an identical combination of gene, plasmid (sub)type, and ST over time. Human ESBL carriage was associated with both presence of ESBL producers in pigs and average number of hours working on the pig farm per week, while prolonged human carriage was observed only incidentally. IMPORTANCE Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a public health hazard due to reduced therapeutic options for the treatment of infections. Although direct contact with pigs is considered a risk factor for human ESBL-producing E. coli carriage through occupational exposure, nationwide data regarding the occurrence of such isolates among pigs and humans living and/or working on farms remain scarce. Therefore, we determined (i) the longitudinal dynamics in prevalence and molecular characteristics of ESBL-producing E. coli in Dutch pig farmers and their pigs over time and (ii) the potential transmission events between these reservoirs based on genetic relatedness and epidemiological associations in longitudinal data. Our data suggesting the possibility of clonal and horizontal dissemination of ESBL-producing Escherichia coli between pigs and pig farmers can be used to inform targeted intervention strategies to decrease the within-farm human exposure to ESBL-producing E. coli.

Abstract Background Emergence of more transmissible SARS-CoV-2 variants requires more efficient control measures to limit nosocomial transmission and maintain healthcare capacities during pandemic waves. Yet the relative importance of different strategies is unknown. Methods We developed an agent-based model and compared the impact of personal protective equipment (PPE), screening of healthcare workers (HCWs), contact tracing of symptomatic HCWs and restricting HCWs from working in multiple units (HCW cohorting) on nosocomial SARS-CoV-2 transmission. The model was fit on hospital data from the first wave in the Netherlands (February until August 2020) and assumed that HCWs used 90% effective PPE in COVID-19 wards and self-isolated at home for 7 days immediately upon symptom onset. Intervention effects on the effective reproduction number (R E ), HCW absenteeism and the proportion of infected individuals among tested individuals (positivity rate) were estimated for a more transmissible variant. Results Introduction of a variant with 56% higher transmissibility increased — all other variables kept constant — R E from 0.4 to 0.65 (+ 63%) and nosocomial transmissions by 303%, mainly because of more transmissions caused by pre-symptomatic patients and HCWs. Compared to baseline, PPE use in all hospital wards (assuming 90% effectiveness) reduced R E by 85% and absenteeism by 57%. Screening HCWs every 3 days with perfect test sensitivity reduced R E by 67%, yielding a maximum test positivity rate of 5%. Screening HCWs every 3 or 7 days assuming time-varying test sensitivities reduced R E by 9% and 3%, respectively. Contact tracing reduced R E by at least 32% and achieved higher test positivity rates than screening interventions. HCW cohorting reduced R E by 5%. Sensitivity analyses show that our findings do not change significantly for 70% PPE effectiveness. For low PPE effectiveness of 50%, PPE use in all wards is less effective than screening every 3 days with perfect sensitivity but still more effective than all other interventions. Conclusions In response to the emergence of more transmissible SARS-CoV-2 variants, PPE use in all hospital wards might still be most effective in preventing nosocomial transmission. Regular screening and contact tracing of HCWs are also effective interventions but critically depend on the sensitivity of the diagnostic test used.

Clostridioides difficile infection (CDI) is the most common cause of antibiotic-associated diarrhoea (AAD); however, markers predictive of CDI or AAD development are as yet lacking. Here, to identify markers predictive of CDI, the authors profile the intestinal microbiota of 945 hospitalised patients from 34 hospitals in 6 different European countries and show distinct microbiota enriched in Enterococcus and depleted of Ruminococcus, Blautia, Prevotella and Bifidobacterium compared to non-CDI patients.

Here, the authors report the incidence of Clostridioides difficile infection (CDI) and assess potential clinical characteristics and biomarkers to predict CDI in 1,007 patients of 50 years and above receiving newly initiated antibiotic treatment.

The role of school-based contacts in the epidemiology of SARS-CoV-2 is incompletely understood. Here, the authors use an age-structured transmission model fitted to age-specific seroprevalence and hospital admission data to assess the effects of school-based measures during the COVID-19 pandemic in the Netherlands.

Background Repeated rotation of empiric antibiotic treatment strategies is hypothesized to reduce antibiotic resistance. Clinical rotation studies failed to change unit-wide prevalence of antibiotic resistant bacteria (ARB) carriage, including an international cluster-randomized crossover study. Unit-wide effects may differ from individual effects due to “ecological fallacy”. This post-hoc analysis of a cluster-randomized crossover study assesses differences between cycling and mixing rotation strategies in acquisition of carriage with Gram-negative ARB in individual patients. Methods This was a controlled cluster-randomized crossover study in 7 ICUs in 5 European countries. Clinical cultures taken as routine care were used for endpoint assessment. Patients with a first negative culture and at least one culture collected in total were included. Community acquisitions (2 days of admission or less) were excluded. Primary outcome was ICU-acquisition of Enterobacterales species with reduced susceptibility to: third- or fourth generation cephalosporins or piperacillin-tazobactam, and Acinetobacter species and Pseudomonas aeruginosa with reduced susceptibility for piperacillin-tazobactam or carbapenems. Cycling (altering first-line empiric therapy for Gram-negative bacteria, every other 6-weeks), to mixing (changing antibiotic type every empiric antibiotic course). Rotated antibiotics were third- or fourth generation cephalosporins, piperacillin-tazobactam and carbapenems. Results For this analysis 1,613 admissions were eligible (855 and 758 during cycling and mixing, respectively), with 16,437 microbiological cultures obtained. Incidences of acquisition with ARB during ICU-stay were 7.3% (n = 62) and 5.1% (n = 39) during cycling and mixing, respectively (p-value 0.13), after a mean of 17.7 (median 15) and 20.8 (median 13) days. Adjusted odds ratio for acquisition of ARB carriage during mixing was 0.62 (95% CI 0.38 to 1.00). Acquired carriage with ARB were Enterobacterales species (n = 61), Pseudomonas aeruginosa (n = 38) and Acinetobacter species (n = 20), with no statistically significant differences between interventions. Conclusions There was no statistically significant difference in individual patients’ risk of acquiring carriage with Gram-negative ARB during cycling and mixing. These findings substantiate the absence of difference between cycling and mixing on the epidemiology of Gram-negative ARB in ICU. Trial registration This trial is registered with ClinicalTrials.gov, registered 10 January 2011, NCT01293071.

Abstract Background Advanced age is associated with increased mortality in acute respiratory distress syndrome (ARDS) patients. Preclinical studies suggest that the host response to an injurious challenge is age-dependent. In ARDS patients, we investigated whether the association between age and mortality is mediated through age-related differences in the host response. Methods This was a prospective longitudinal observational cohort study, performed in the ICUs of two university-affiliated hospitals. The systemic host response was characterized in three predefined age-groups, based on the age-tertiles of the studied population: young (18 to 54 years, N = 209), middle-aged (55 to 67 years, N = 213), and elderly (67 years and older, N = 196). Biomarkers of inflammation, endothelial activation, and coagulation were determined in plasma obtained at the onset of ARDS. The primary outcome was 90-day mortality. A mediation analysis was performed to examine whether age-related differences in biomarker levels serve as potential causal pathways mediating the association between age and mortality. Results Ninety-day mortality rates were 30% (63/209) in young, 37% (78/213) in middle-aged, and 43% (84/196) in elderly patients. Middle-aged and elderly patients had a higher risk of death compared to young patients (adjusted odds ratio, 1.5 [95% confidence interval 1.0 to 2.3] and 2.1 [1.4 to 3.4], respectively). Relative to young patients, the elderly had significantly lower systemic levels of biomarkers of inflammation and endothelial activation. Tissue plasminogen activator, a marker of coagulation, was the only biomarker that showed partial mediation (proportion of mediation, 10 [1 to 28] %). Conclusion Little evidence was found that the association between age and mortality in ARDS patients is mediated through age-dependent differences in host response pathways. Only tissue plasminogen activator was identified as a possible mediator of interest. Trial registration This trial was registered at ClinicalTrials.gov (identifier NCT01905033, date of registration July 23, 2013).

Abstract Background Early recognition of sepsis is challenging, and diagnostic criteria have changed repeatedly. We assessed the robustness of sepsis-3 criteria in intensive care unit (ICU) patients. Methods We studied the apparent incidence and associated mortality of sepsis-3 among patients who were prospectively enrolled in the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) cohort in the Netherlands, and explored the effects of minor variations in the precise definition and timing of diagnostic criteria for organ failure. Results Among 1081 patients with suspected infection upon ICU admission, 648 (60%) were considered to have sepsis according to prospective adjudication in the MARS study, whereas 976 (90%) met sepsis-3 criteria, yielding only 64% agreement at the individual patient level. Among 501 subjects developing ICU-acquired infection, these rates were 270 (54%) and 260 (52%), respectively (yielding 58% agreement). Hospital mortality was 234 (36%) vs 277 (28%) for those meeting MARS-sepsis or sepsis-3 criteria upon presentation (p < 0.001), and 121 (45%) vs 103 (40%) for those having sepsis onset in the ICU (p < 0.001). Minor variations in timing and interpretation of organ failure criteria had a considerable effect on the apparent prevalence of sepsis-3, which ranged from 68 to 96% among those with infection at admission, and from 22 to 99% among ICU-acquired cases. Conclusion The sepsis-3 definition lacks robustness as well as discriminatory ability, since nearly all patients presenting to ICU with suspected infection fulfill its criteria. These should therefore be specified in greater detail, and applied more consistently, during future sepsis studies. Trial registration The MARS study is registered at ClinicalTrials.gov (identifier NCT 01905033).

Abstract Background Guidelines recommend macrolides and fluoroquinolones in patients hospitalized with community-acquired pneumonia (CAP), but their use has been associated with cardiac events. We quantified associations between macrolide and fluoroquinolone use and cardiac events in patients hospitalized with CAP in non-ICU wards. Methods This was a post-hoc analysis of a cluster-randomized trial as a cohort study; including patients with a working diagnosis of CAP admitted to non-ICU wards without a cardiac event on admission. We calculated cause-specific hazard ratio’s (HR’s) for effects of time-dependent macrolide and fluoroquinolone exposure as compared to beta-lactam monotherapy on cardiac events, defined as new or worsening heart failure, arrhythmia, or myocardial ischemia during hospitalization. Results Cardiac events occurred in 146 (6.9%) of 2107 patients, including heart failure (n = 101, 4.8%), arrhythmia (n = 53, 2.5%), and myocardial ischemia (n = 14, 0.7%). These occurred in 11 of 207 (5.3%), 18 of 250 (7.2%), and 31 of 277 (11.2%) patients exposed to azithromycin, clarithromycin, and erythromycin for at least one day, and in 9 of 234 (3.8%), 5 of 194 (2.6%), and 23 of 566 (4.1%) exposed to ciprofloxacin, levofloxacin, and moxifloxacin, respectively. HR’s for erythromycin, compared to beta-lactam monotherapy, on any cardiac event and heart failure were 1.60 (95% CI 1.09;2.36) and 1.89 (95% CI 1.22;2.91), respectively. HR’s for levofloxacin and moxifloxacin, compared to beta-lactam monotherapy, on any cardiac event were 0.40 (95% CI 0.18;0.87)and 0.56 (95% CI 0.36;0.87), respectively. Findings remained consistent after adjustment for confounders and/or in a sensitivity analysis of radiologically confirmed CAP (n = 1604, 76.1%). Conclusions Among patients with CAP hospitalized to non-ICU wards, erythromycin use was associated with a 68% increased risk of hospital-acquired cardiac events, mainly heart failure. Levofloxacin and moxifloxacin were associated with a lower risk of heart failure. Although our study does not fully exclude confounding bias, findings remained largely unchanged in crude, adjusted, and sensitivity analyses. These findings may caution the use of erythromycin as empirical therapy in these patients. Trial registration The original trial was retrospectively registered under ClinicalTrials.gov Identifier NCT01660204 on August 8th, 2012.

Abstract Background Cytomegalovirus (CMV) reactivation in previously immunocompetent critically ill patients is associated with increased mortality, which has been hypothesized to result from virus-induced immunomodulation. Therefore, we studied the effects of CMV reactivation on the temporal course of host response biomarkers in patients with sepsis. Methods In this matched cohort study, each sepsis patient developing CMV reactivation between day 3 and 17 (CMV+) was compared with one CMV seropositive patient without reactivation (CMVs+) and one CMV seronegative patient (CMVs−). CMV serostatus and plasma loads were determined by enzyme-linked immunoassays and real-time polymerase chain reaction, respectively. Systemic interleukin-6 (IL-6), IL-8, IL-18, interferon-gamma–induced protein-10 (IP-10), neutrophilic elastase, IL-1 receptor antagonist (RA), and IL-10 were measured at five time points by multiplex immunoassay. The effects of CMV reactivation on sequential concentrations of these biomarkers were assessed in multivariable mixed models. Results Among 64 CMV+ patients, 45 could be matched to CMVs+ or CMVs− controls or both. The two baseline characteristics and host response biomarker levels at viremia onset were similar between groups. CMV+ patients had increased IP-10 on day 7 after viremia onset (symmetric percentage difference +44% versus −15% when compared with CMVs+ and +37% versus +4% when compared with CMVs−) and decreased IL-1RA (−41% versus 0% and −49% versus +10%, respectively). However, multivariable analyses did not show an independent association between CMV reactivation and time trends of IL-6, IP-10, IL-10, or IL-1RA. Conclusion CMV reactivation was not independently associated with changes in the temporal trends of host response biomarkers in comparison with non-reactivating patients. Therefore, these markers should not be used as surrogate clinical endpoints for interventional studies evaluating anti-CMV therapy.

Abstract Background Febrile Urinary Tract Infection (FUTI) is frequently treated initially with intravenous antibiotics, followed by oral antibiotics guided by clinical response and bacterial susceptibility patterns. Due to increasing infection rates with multiresistant Enterobacteriaceae, antibiotic options for stepdown treatment decline and patients more frequently require continued intravenous antibiotic treatment for FUTI. Fosfomycin is an antibiotic with high bactericidal activity against Escherichia coli and current resistance rates are low in most countries. Oral Fosfomycin-Trometamol 3000 mg (FT) reaches appropriate antibiotic concentrations in urine and blood and is considered safe. As such, it is a potential alternative for stepdown treatment. Methods The FORECAST study (Fosfomycin Randomized controlled trial for E.coli urinary tract infections as Alternative Stepdown Treatment) is a randomized, double-blind, double-dummy, non-inferiority trial in which 240 patients will be randomly allocated to a stepdown treatment with FT or ciprofloxacin (standard of care) for FUTI, caused by Escherichia coli with in vitro susceptibility to both antibiotics. The study population consists of consenting female patients (≥18 years) with community acquired E. coli FUTI. After intravenous antibiotic treatment during at least 48 (but less than 120) hours, and if eligibility criteria for iv-oral switch are met, patients receive either FT (3 g every 24 h) or ciprofloxacin (500 mg every 12 h) for a total antibiotic duration of 10 days. The primary endpoint is clinical cure (resolution of symptoms) 6–10 days post-treatment. Secondary endpoints are microbiological cure 6–10 days post-treatment, clinical cure, mortality, ICU admittance, relapse, reinfection, readmission, additional antibiotic use for UTI, early study discontinuation, adverse events, days of hospitalization and days of absenteeism within 30–35 days post-treatment. The sample size is based on achieving non-inferiority on the primary endpoint, applying a non-inferiority margin of 10%, a two-sided p-value of

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient’s physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU.

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

Abstract Background Colorectal surgery is frequently complicated by surgical site infections (SSIs). The most important consequences of SSIs are prolonged hospitalization, an increased risk of surgical reintervention and an increase in mortality. Perioperative intravenously administered antibiotic prophylaxis is the standard of care to reduce the risk of SSIs. In the last few decades, preoperative orally administered antibiotics have been suggested as additional prophylaxis to further reduce the risk of infection, but are currently not part of routine practice in most hospitals. The objective of this study is to evaluate the efficacy of a preoperative orally administered antibiotic prophylaxis (Pre-OP) in addition to intravenously administered perioperative antibiotic prophylaxis to reduce the incidence of deep SSIs and/or mortality after elective colorectal surgery. Methods/design The PreCaution trial is designed as a multicenter, double-blind, randomized, placebo-controlled clinical trial that will be carried out in The Netherlands. Adult patients who are scheduled for elective colorectal surgery are eligible to participate. In total, 966 patients will be randomized to receive the study medication. This will either be Pre-OP, a solution that consists of tobramycin and colistin sulphate, or a placebo solution. The study medication will be administered four times daily during the 3 days prior to surgery. Perioperative intravenously administered antibiotic prophylaxis will be administered to all patients in accordance with national infection control guidelines. The primary endpoint of the study is the cumulative incidence of deep SSIs and/or mortality within 30 days after surgery. Secondary endpoints include both infectious and non-infectious complications of colorectal surgery, and will be evaluated 30 days and/or 6 months after surgery. Discussion To date, conclusive evidence on the added value of preoperative orally administered antibiotic prophylaxis in colorectal surgery is lacking. The PreCaution trial should determine the effects of orally administered antibiotics in preventing infectious complications in elective colorectal surgery. Trial registration Netherlands Trial Register, ID: NTR6113 . Registered on 11 October 2016; EudraCT 2015-005736-17.

Abstract Background The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq). Results We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745. Conclusions Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

Abstract Background The epidemiology of ICU pneumonia caused by Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) is not fully described, but is urgently needed to support the development of effective interventions. The objective of this study is to estimate the incidence of S. aureus and P. aeruginosa ICU pneumonia and to assess its association with patient-related and contextual risk factors. Methods ASPIRE-ICU is a prospective, observational, multi-center cohort study nested within routine surveillance among ICU patients in Europe describing the occurrence of S. aureus and P. aeruginosa ICU pneumonia. Two thousand (2000) study cohort subjects will be enrolled (50% S. aureus colonized) in which specimens and data will be collected. Study cohort subjects will be enrolled from a larger surveillance population, in which basic surveillance data is captured. The primary outcomes are the incidence of S. aureus ICU acquired pneumonia and the incidence of P. aeruginosa ICU acquired pneumonia through ICU stay. The analysis will include advanced survival techniques (competing risks and multistate models) for each event separately as well as for the sub-distribution of ICU pneumonia to determine independent association of outcomes with risk factors.. A risk prediction model will be developed to quantify the risk for acquiring S. aureus or P. aeruginosa ICU pneumonia during ICU stay by using a composite score of independent risk factors. Discussion The diagnosis of pathogen-specific ICU pneumonia is difficult, however, the criteria used in this study are objective and comparable to those in the literature. Trial registration This study is registered on clinicaltrials.gov under identifier NCT02413242 .

Abstract Background The gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed “selective decontamination of the digestive tract” (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (“the resistome”) of ICU patients during SDD and of healthy subjects. Results From ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6′)-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. Conclusions The gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.

Abstract Objective To determine the incidence of P. aeruginosa (PA) ICU pneumonia and its independent association with PA colonization at ICU admission. Methods This was a post-hoc analysis of a prospectively collected cohort study. Adult ICU patients with a length of stay of ≥48 h were included and assessed for microbiologically confirmed PA ICU pneumonia. Multivariate survival analysis was performed, including the covariates age, gender, PA colonization at ICU admission, ICU admission specialty and mechanical ventilation at ICU admission, while taking into account the effect of competing risks. Results We included 5093 patients, 2447 (48%) were tested for colonization; of those 226 (9.2%) were PA colonized at ICU admission. The incidence of PA ICU pneumonia was 1.34% (n = 68). PA colonization was an independent risk factor (subdistribution hazard ratio [SHR] 8.8; 95% confidence interval [CI] 4.9–15.7), as was mechanical ventilation (SHR 5.3, 95% CI 2.7–10.6). Conclusion In this study the incidence of P. aeruginosa ICU pneumonia was 1.34%. Hazard ratios for PA colonized patients compared to non-colonized to develop PA ICU pneumonia were 8.8. The high risk associated with P. aeruginosa colonization for subsequent infection may offer a target for future interventions.

ABSTRACT The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca2+ levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks. IMPORTANCE Infections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the antibiotics used in hospitals. During antibiotic treatment, the normal bacteria are replaced by resistant enterococci within the gut, from which they can spread and cause infections. We studied antibiotic-mediated intestinal proliferation of multidrug-resistant Enterococcus faecium and the effects on intestinal architecture. We demonstrated that antibiotics allow proliferation of E. faecium in the gut, alter the mucus-associated gut bacterial layer, and reduce the colon wall, mucus thickness, and amount of Muc-2 protein. E. faecium is agglutinated in the intestine in a matrix consisting of host molecules. We hypothesize that this matrix maintains a segregation of E. faecium from the epithelium. Understanding the processes that occur in the gut during antibiotic treatment may provide clues for future mucosal vaccination strategies to control E. faecium or other multidrug-resistant opportunistic pathogens, thereby preventing infections, hospital transmission, and outbreaks.

ABSTRACT Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfm disruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfm is associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfm mutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfm mutant. These findings indicate that AtlAEfm is important in biofilm and collagen binding in E. faecium, making AtlAEfm a promising target for treatment of E. faecium infections.