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Background Coronavirus disease 2019 (COVID-19) is a life-threatening respiratory condition caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was initially detected in China in December 2019. Currently, in Germany >140 000 cases of COVID-19 are confirmed. Here we report a nosocomial outbreak of SARS-CoV-2 infections in the pediatric dialysis unit of the University Hospital Münster (UHM). Methods Single-step real-time reverse-transcription polymerase chain reaction (rRT-PCR) from nasopharyngeal swabs was used to diagnose the index patient and identify infected contacts. Epidemiological links were analyzed by patient interviews and medical record reviews. In addition, each contact was assessed for exposure to the index case and monitored for clinical symptoms. Cycle threshold (Ct) values of all positive test results were compared between symptomatic and asymptomatic cases. Results Forty-eight cases were involved in this nosocomial outbreak. Nine contact cases developed laboratory-confirmed COVID-19 infections. Two SARS-CoV-2–positive cases remained clinically asymptomatic. Eleven cases reported flulike symptoms without positive results. Ct values were significantly lower in cases presenting typical COVID-19 symptoms, suggesting high viral shedding (P = .007). Conclusions Person-to-person transmission was at the heart of a hospital outbreak of SARS-CoV-2 between healthcare workers (HCWs) and patients in the pediatric dialysis unit at UHM. Semiquantitative rRT-PCR results suggest that individuals with high viral load pose a risk to spread SARS-CoV-2 in the hospital setting. Our epidemiological observation highlights the need to develop strategies to trace and monitor SARS-CoV-2–infected HCWs to prevent COVID-19 outbreaks in the hospital setting.

Vancomycin is frequently used for the treatment of C. difficile infections (CDI). There are concerns that this might increase the risk of selecting vancomycin resistant enterococci (VRE). Here, we evaluated whether there is an increased risk of VRE acquisition following vancomycin for CDI specific treatment. Patients with CDI, metronidazole, or oral vancomycin treatment and without preexisting VRE were monitored for VRE acquisition. VRE isolates from patients with acquired and preexisting colonization were collected and subjected to whole genome sequencing. In total, 281 patients (median age 56 years, 54% of the male sex) presented with toxin positive C. difficile. Of them, 170 patients met the inclusion criteria, comprising 37 patients treated with metronidazole and 133 treated with oral vancomycin. In total, 14 patients meeting the inclusion criteria acquired VRE (vancomycin: n = 11, metronidazole: n = 3). Statistical analysis revealed no significant differences between both VRE acquisition rates. Genetic comparison of detected VRE isolates resulted in eight clusters of closely related genotypes comprising acquired and preexisting strains. Our results suggest that vancomycin and metronidazole likewise increase the risk of VRE acquisition. Genetic comparison indicates that VRE acquisition is a result of both antibiotic selection and pathogen transmission.

Vancomycin is frequently used for the treatment of C. difficile infections (CDI). There are concerns that this might increase the risk of selecting vancomycin resistant enterococci (VRE). Here, we evaluated whether there is an increased risk of VRE acquisition following vancomycin for CDI specific treatment. Patients with CDI, metronidazole, or oral vancomycin treatment and without preexisting VRE were monitored for VRE acquisition. VRE isolates from patients with acquired and preexisting colonization were collected and subjected to whole genome sequencing. In total, 281 patients (median age 56 years, 54% of the male sex) presented with toxin positive C. difficile. Of them, 170 patients met the inclusion criteria, comprising 37 patients treated with metronidazole and 133 treated with oral vancomycin. In total, 14 patients meeting the inclusion criteria acquired VRE (vancomycin: n = 11; metronidazole: n = 3). Statistical analysis revealed no significant differences between both VRE acquisition rates. Genetic comparison of detected VRE isolates resulted in eight clusters of closely related genotypes comprising acquired and preexisting strains. Our results suggest that vancomycin and metronidazole likewise increase the risk of VRE acquisition. Genetic comparison indicates that VRE acquisition is a result of both antibiotic selection and pathogen transmission.

Background Currently, hospitals have been forced to divert substantial resources to cope with the ongoing coronavirus disease 2019 (COVID-19) pandemic. It is unclear if this situation will affect long-standing infection prevention practices and impact on healthcare associated infections. Here, we report a nosocomial cluster of vancomycin-resistant enterococci (VRE) that occurred on a COVID-19 dedicated intensive care unit (ICU) despite intensified contact precautions during the current pandemic. Whole genome sequence-based typing (WGS) was used to investigate genetic relatedness of VRE isolates collected from COVID-19 and non-COVID-19 patients during the outbreak and to compare them to environmental VRE samples. Methods Five VRE isolated from patients (three clinical and two screening samples) as well as 11 VRE and six vancomycin susceptible Enterococcus faecium (E. faecium) samples from environmental sites underwent WGS during the outbreak investigation. Isolate relatedness was determined using core genome multilocus sequence typing (cgMLST). Results WGS revealed two genotypic distinct VRE clusters with genetically closely related patient and environmental isolates. The cluster was terminated by enhanced infection control bundle strategies. Conclusions Our results illustrate the importance of continued adherence to infection prevention and control measures during the COVID-19 pandemic to prevent VRE transmission and healthcare associated infections.

During the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, healthcare systems worldwide have to prevent nosocomial SARS-CoV-2 transmission while maintaining duty of care. In our study, we characterize the transmission dynamic of SARS-CoV-2 in inpatients and healthcare workers (HCWs) at the University Hospital Münster (UHM) in northwest Germany. We identified 27 cases of healthcare-associated SARS-CoV-2 infections (4 inpatients and 23 HCWs) who had contact with patients and/or HCWs without the use of adequate PPE. The contacts of these index cases were followed up for SARS-CoV-2 infection after unprotected exposure and a quantitative measure of probability of becoming infected, the attack rate, was calculated. In addition, transmission was evaluated in the context of infection control measures established during the pandemic and we compared the epidemiological data of all index cases, including symptoms and Ct values of virology test results. The overall attack rate in the hospital setting was 1.3% (inpatients 0.9%, HCWs 1.6%). However, during an outbreak, the attack rate was 25.5% (inpatients 20.0%, HCWs 29.6%). For both scenarios, HCWs had a higher attack rate illustrating their role in healthcare-associated SARS-CoV-2 transmission. Taken together, our experiences demonstrate how infection control measures can minimize the transmission of SARS-CoV-2 in the healthcare setting.

Background Transplant recipients are at high risk for infections. However, donor-recipient transmission of multidrug-resistant organisms (MDROs) remains mostly unaddressed in the protocols of pre-transplant infection and colonization screening. Vancomycin-resistant enterococci (VRE) are MDROs that colonize the gastrointestinal tract and are associated with a significant burden of disease. Besides the high mortality of invasive VRE infections, chronic colonization leads to costly isolation measures in the hospital setting. Whereas most post-transplantation VRE infections are endogenous and thus preceded by colonization of the recipient, conclusive evidence of VRE transmission via allograft in the context of intestinal transplantation is lacking. Case presentation We describe a donor-derived VRE infection after intestinal transplantation including small bowel and right hemicolon. The recipient, a 54-year old male with history of mesenteric ischemia and small bowel perforation due to generalized atherosclerosis and chronic stenosis of the celiac trunk and the superior mesenteric artery, developed an intra-abdominal infection and bloodstream infection after transplantation. VRE isolates recovered from the patient as well as from the allograft prior to transplantation were analyzed via whole genome sequencing. Isolates showed to be genetically identical, thus confirming the transmission from donor to recipient. Conclusions This case underlines the relevance of donor-recipient VRE transmission and invasive infection in the context of intestinal transplantation, highlighting the need for preoperative MDRO screening that facilitates the prompt and effective treatment of possible infections as well as the timely establishment of contact precautions to prevent further spread.

On 7 January 2020, the World Health Organization (WHO) announced a novel coronavirus to be the cause of pneumonia cases whose cause was unclear in China. The infection came to be known as a coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After

Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target’s spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.

Vancomycin-resistant enterococci (VRE) are relevant nosocomial pathogens with an increasing incidence in the last decades. Their transmission is optimal in the hospital setting, as it offers two potential, large reservoirs that are closely related: susceptible patients and their environment. Here we investigate the role of the hospital environment in the nosocomial transmission of VRE by establishing concrete links between contaminated surfaces and colonized/infected patients in outbreak and non-outbreak settings. Environmental and patient VRE isolates were collected between 2013 and 2019 and analyzed by whole-genome sequencing (WGS), subsequent multilocus sequence typing (MLST), and core genome (cg) MLST. Pairs of isolates differing in &lt, 3 alleles were rated as closely related, making a transmission likely. Fifty-three environmental VRE isolates were analyzed. MLST sequence types (ST) ST203 (50.0%), ST192 (21.3%), ST117 (17.3%), ST721 (8.8%), ST80 (2%), and ST1489 (0.7%) were detected, carrying the resistance determinants vanA (72.7%), vanB (24%), or both (3.3%). Of the 53 environmental isolates, 51 were found to form five clusters with genetically related patient isolates (n = 97 isolates). WGS confirms the role of the environment in the transmission dynamics of VRE in both the outbreak and non-outbreak settings, highlighting the importance of prevention and control of VRE spread.

Background Traveller's diarrhea (TD) is the leading cause of morbidity among travelers. Recently, molecular methods have been developed to detect the causative pathogens of TD. The aim of this study was to test a multiplex-PCR approach in patients with TD and asymptomatic controls. Methods In total, 91 travelers (61 TD cases, 30 asymptomatic controls) prospectively collected stool samples during travel and documented gastrointestinal symptoms. Samples were analyzed using the BioFire® FilmArray® Gastrointestinal Panel, which covers 13 enteric bacteria (e.g. diarrheagenic Escherichia coli), 4 protozoan parasites and 5 viruses. Results TD cases had more likely a positive test (46/61, 75%) compared to controls (17/30, 57%, p = 0.07). The most common enteric pathogens were enteroaggregative E. coli (36/91, 40%), followed by enterotoxigenic E. coli (31/91, 34%) and enteropathogenic E. coli (EPEC, 29/91, 32%). The only pathogens significantly associated with TD were EPEC (p = 0.01) and ETEC (p = 0.047). Protozoan parasites were not detected. Conclusion Our results challenge the widespread use of multiplex-approaches covering numerous enteric pathogens in the diagnostic workup of TD because only few (i.e. diarrheagenic E. coli) were related to symptoms of TD.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that require effective infection control measures, representing a challenge for healthcare systems. This study aimed at identifying risk factors associated with prolonged VRE carriage and determining the rate of clearance that allows the discontinuation of contact precautions. During a 2-year study, screening was performed in patients with a history of VRE or at risk of becoming colonized. After bacterial identification and antibiotic susceptibility testing, glycopeptide resistance was confirmed by PCR. Isolates were compared via whole genome sequence-based typing. Risk factors were recorded, and follow-up screening was performed upon readmission, defining patients as long-term carriers if still colonized &ge, 10 weeks after first detection. Of 1059 patients positive for VRE, carriage status was assessed upon readmission in 463 patients. VRE was cleared in 56.4% of the cases. Risk factors associated with long-term persistence were hospital stays (frequency, length), hemato-oncological disease, systemic treatment with steroids, and use of antibiotics. No specific genotypic clustering was observed in patients with VRE clearance or persistence. VRE clearance is possibly underestimated. The identification of risk factors favoring long-term carriage may contribute to a targeted implementation of infection control measures upon readmission of patients with history of VRE.

Coprinopsis cinerea is an environmental fungus which can cause disseminated infections in immunocompromised patients, often leading to death. Here we report the case of a paediatric patient with an invasive wound infection due to C. cinerea, which was successfully treated with surgical debridement and oral posaconazole. Keywords: Coprinopsis, hormographiella, immunosuppression, posaconazole, skin, wound infection

We report a case of a soft-tissue infection with Francisella philomiragia, a rare opportunistic pathogen in individuals with chronic granulomatous disease.

Objectives Travellers may be colonized with antimicrobial-resistant bacteria on return, but little is known about colonization during travel. Our objectives were to assess the acquisition and colonization dynamics during the stay abroad for a broad range of antimicrobial-resistant bacteria and resistance phenotypes and to identify risk factors for faecal carriage of antimicrobial-resistant bacteria. Methods German and Dutch participants (n = 132) of this prospective cohort study (2016–2018) completed a questionnaire on risk factors and provided daily stool samples before, during, and after travel. Samples were screened for extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenem-resistant (CarbR-GN), and non-intrinsically colistin-resistant Gram-negative rods (ColR-GN), vancomycin-resistant Enterococcus faecium/faecalis (VRE), and Clostridioides difficile. Results Colonization rates reached a plateau within a week after departure fluctuating around 48.5% (63/130) and 58.4% (45/77, ESBL-E), 10.4% (11/106) and 23.4% (18/77, ColR-GN), or 3.0% (4/132) and 6.8% (8/118, CarbR-GN). Colonization rates after the travel were 46.2% (61/132, ESBL-E), 9.0% (12/132, ColR-GN), and 3.4% (5/132, CarbR-GN). Travellers carried mcr-1- (15/132; 11.4%) or blaNDM-positive (4/132; 3.0%) Enterobacterales. A vegetarian diet was associated with a lower risk for the acquisition of ESBL-E (OR = 0.4, p 0.04) and ColR-GN (OR = 0.1, p 0.01) during travel in a multivariable model. Similarly, travellers visiting friends and relatives had a lower risk for the acquisition of ESBL-E (OR = 0.3, p 0.009) and CarbR-GN (OR = 0.3, p 0.01). VRE and C. difficile were not detected. Conclusion The number of travellers with a temporary colonization during the journey exceeded the number of travellers still colonized after return.

Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed. Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT. Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Background Most phenotypic methods routinely employed for the detection of ESBL and AmpC producing Enterobacteriaceae require 18 hours of incubation. Aiming to offer this clinically relevant information in a shorter time, we developed a MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) as a one-step screening and confirmation panel in accordance with the EUCAST guidelines. Methods DOT-MGA was performed on 12 clinical Enterobacteriaceae strains displaying resistance against third-generation cephalosporins plus four control strains recommended by EUCAST for detection of ESBL production. Microdroplets (6 µL) containing bacterial suspension and antibiotics (cefpodoxime, cefotaxime, ceftazidime, cefepime with or without clavulanic acid, and/or cloxacillin) in cation-adjusted Mueller–Hinton broth (CA-MHB) were spotted directly onto MBT Biotargets 96 (Bruker Daltonics, Germany). Targets were incubated for 4 hours at 36°C in plastic transport boxes in order to avoid evaporation. Subsequently, culture medium was removed and MALDI-TOF MS of the cells adhered to the target’s surface was performed. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration at which the MALDI Biotyper software (Bruker) provided no species identification. ESBL/AmpC production was defined as an 8-fold or greater decrease of the cephalosporin MIC when combined with clavulanic acid and/or cloxacillin, respectively. A computer-based algorithm was established to interpret the results, which were compared with those of broth microdilution (reference method). Resistance mechanisms of the clinical isolates were assessed by PCR. Results The method correctly identified the ESBL and AmpC production of both control and clinical strains (positive agreement, 100%; negative agreement, 100%), allowing the differentiation of each mechanism even in isolates displaying combined resistance. The results agreed with the characterization of the strains by PCR. Conclusion We present a rapid method for phenotypic detection of ESBL and AmpC that yields reliable results in a significantly shorter time, thus representing a potentially valuable tool in the routine detection of multidrug-resistant pathogens. Disclosures E. A. Idelevich, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium. K. Sparbier, Bruker Daltonik: Employee, Salary. M. Kostrzewa, Bruker Daltonik: Employee, Salary. K. Becker, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium.

Background The accurate identification of carbapenem resistance mechanisms is decisive for the appropriate selection of antibiotic regimens. Numerous methods can detect carbapenemase-producing carbapenem-resistant bacteria (CPCR). However, non-CPCR (NCPCR) are routinely assumed to display porin loss as a diagnosis of exclusion. No further confirmatory tests are performed since the gold standard (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS–PAGE) is laborious and time consuming. We propose a test for rapid and easy detection of porin loss by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical meropenem-resistant Enterobacterales strains (10 CPCR, 10 NCPCR) and control strains recommended by EUCAST (5 carbapenemase-producing, one with porin loss, one-negative control) were analyzed. Membrane proteins were extracted by successive centrifugation of bacterial suspensions (McFarland 0.5) and addition of ethanol, formic acid and acetonitrile. MALDI-TOF MS of the protein extracts was performed on a 96-spot target (Bruker Daltonics, Germany). Peaks between 35 and 40 kDa were analyzed for the presence of porins and compared with the bands observed in the SDS–PAGE of the protein extracts. Results Within the molecular weight range of 35–40 kDa, the MALDI-TOF MS-based method revealed peaks in all CPCR isolates corresponding to those observed in the carbapenemase-producing control strains. In contrast, the control strain with porin loss as well as all CNCR isolates showed a lower quantity of peaks in this range. All peaks observed correlated with the bands observed in the SDS–PAGE of the protein extracts at the corresponding molecular weight (Figure 1). Conclusion Yielding results that reliably correspond to the current gold standard, we propose a method for accelerated detection of porin loss as an alternative to the diagnosis of exclusion usually made in routine settings. With a processing time of approximately 20 minutes, the method can be easily implemented in the clinical setting. Applying this MALDI-TOF MS-based approach, valuable information will be provided about a resistance mechanism that otherwise remains unexplained. Disclosures All authors: No reported disclosures.

Background The differential identification of carbapenemases relies mostly on molecular techniques. Current phenotypic methods require 18 hours of incubation. We propose a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) aiming to offer an easy and rapid phenotypic identification of AmpC, KPC, MBL and OXA production. Methods Seven well-characterized Enterobacteriaceae strains recommended by EUCAST for carbapenemase detection were analyzed. Synergy between meropenem and carbapenemase inhibitors (phenylboronic acid, aminophenylboronic acid, cloxacillin, dipicolinic acid, ethylenediaminetetraacetic acid, and avibactam) and temocillin resistance were determined using a testing panel developed on a 96-spot MALDI-TOF MS target (MBT Biotarget 96, Bruker Daltonics, Germany). Microdroplets (6 µl) containing bacterial suspension and antibiotic or antibiotic/inhibitor in cation-adjusted Mueller–Hinton broth were spotted on the target and incubated for 4 hours at 36°C in a humidity chamber to avoid evaporation. The medium was subsequently removed and MALDI-TOF MS of the cells adhered to the target’s surface was performed. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration at which the MALDI Biotyper software yielded no organism identification. Synergy was defined by an eightfold or greater reduction of the meropenem MIC in the presence of an inhibitor. The absence of synergy between meropenem and inhibitors as well as high-level temocillin resistance was considered suggestive of OXA production. Results were processed and interpreted with a computer-based algorithm. Results After 4 hours, the method was able to correctly detect the foreknown resistance mechanisms of all tested strains (KPC, MBL, OXA, and AmpC), yielding results that agreed with those obtained by performing broth microdilution with 18 hours of incubation. Conclusion The DOT-MGA approach allowed easy identification and differentiation of carbapenemase production, delivering reliable results one day earlier than the usual phenotypic methods, thus displaying great potential for the clinical setting. Disclosures E. A. Idelevich, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium. K. Sparbier, Bruker Daltonik: Employee, Salary. M. Kostrzewa, Bruker Daltonik: Employee, Salary. K. Becker, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium.