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Summary Introduction The large number of asymptomatic SARS-CoV-2 infections necessitates general screening of employees. We evaluate the performance of a SARS-CoV-2 screening program in asymptomatic healthcare-workers (HCW), utilizing self-sampled gargling-solution and sample pooling for RT-qPCR. Methods We conducted a cross-sectional retrospective study to collect real-life data on the performance of a screening-workflow based on automated-pooling and high-throughput qPCR testing over a 3-month-period at the University Hospital Hamburg. Results Matrix validation reveals that lower limit of detection for SARS-CoV-2 RNA in gargling-solution was 180 copies/mL (5-sample-pool). A total of 55,122 self-collected gargle samples (= 7513 HCWs) was analyzed. The median time to result was 8.5 hours (IQR 7.2–10.8). Of 11,192 pools analyzed, 11,041 (98.7%) were negative, 69 (0.6%) were positive and 82 (0.7%) were invalid. Individual testing of pool participants revealed 57 SARS-CoV-2 previously unrecognized infections. All 57 HCWs were either pre-symptomatic or asymptomatic (prevalence 0.76%,CI95%0.58–0.98%). Accuracy based on HCWs with gargle-solution and NP-swab available within 3-day-interval (N = 521) was 99.5% (CI95%98.3–99.9%), sensitivity 88.9% (CI95%65.3–98.6%) while specificity 99.8% (CI95%98.9–99.9). Conclusion This workflow was highly effective in identifying SARS-CoV-2 positive HCWs, thereby lowering the potential of inter-HCW and HCW-patient transmissions. Automated-sample-pooling helped to conserve qPCR reagents and represents a promising alternative strategy to antigen testing in mass-screening programs.

Objective We aim to describe the epidemiological, clinical and microbiological characteristics of the linezolid- and vancomycin- resistant Enterococcus faecium (LVRE) in a tertiary care hospital in Germany. Methods We conducted a retrospective analysis of 196 LVRE cases observed from 1st January 2012 to 31th December 2018. Patients’ medical charts were reviewed and available LVRE (n = 102) were subjected to whole-genome-sequencing. Antibiotic consumption was measured in defined daily dose (DDD)/100 bed-days (BD). Results The prevalence of LVRE isolates among VRE was 6.3 % in 2018. Most patients had an onco-hematological disease (134/196, 68.4 %). From 2012–2018 an increase of +356.7 % of linezolid defined daily dose/100 bed-days was observed. In 71.4 % (90/126, 70 missing values) of the patients, linezolid was prescribed in the previous 6 months. The median exposure to linezolid was 15 days (Interquartile, IQR 9–23). 42/196 (21.4 %) patients had an LVRE-related infection with an overall 30-day mortality rate of 33 %. In 121/196 (61.7 %) patients, linezolid-susceptible VREfm were isolated before LVRE, suggesting secondary acquisition of linezolid resistance. Genetic analysis revealed that most isolates belonged to ST117 (64/102 available isolates, 62.7 %). The G2576T 23S rDNA mutation was identified as the most common resistance mechanism (96/102, 94.1 %). poxtA was identified in two isolates, while cfr, and optrA were not detected. Conclusions Incidence of LVRE related to 23S rDNA mutations is rising and probably associated with antibiotic consumption. Restrictions in the use of linezolid may be needed in order to retain therapeutic options in VRE.

To evaluate the effectiveness of automated room decontamination devices, a common aerosolized hydrogen peroxide (aHP) as well as a recent gaseous ozone-based device, which produces the disinfectant reagent without the need of consumables, were tested under real-life conditions. Twenty-two contaminated surfaces were positioned in different areas in a patient room with adjacent bathroom and anteroom. Following the decontamination process bacteria were recovered and reduction factors were calculated after performing quantitative culture. Following the manufactures instructions, the ozone-based device displayed a bactericidal effect (log10 > 5), whereas the aHP system failed for a high bacterial burden and achieves only a complete elimination of a realistic bioburden (log10 2). After increasing the exposure time to 30 min, the aHP device also reached a bactericidal effect. Nevertheless, our results indicate, that further research and development is necessary, to get knowledge about toxicity, efficacy and safety by using in complex hospital conditions and achieve meaningful integration in cleaning procedures, to reach positive effects on disinfection performance.

OBJECTIVES During allogeneic hematopoietic stem cell transplantation (allo-SCT), infections significantly contribute to morbidity and mortality. A monocentric prospective analysis was performed to assess epidemiology, risk factors, and outcomes of infections during the peri-transplant period. METHODS Data were recorded prospectively using a predefined questionnaire. RESULTS In 2015, 163 consecutive patients, 37.4% female, median age 59 (range 18-79) years received 166 allo-SCT. Median duration of leukopenia

Background Avibactam is a novel broad-range β-lactamase inhibitor active against Ambler class A (including ESBL and KPC) and some Ambler class C and D (e.g. OXA-48) enzymes. We here report on the emergence of ceftazidime/avibactam resistance in clinical, multiresistant, OXA-48 and CTX-M-14-producing Klebsiella pneumoniae isolate DT12 during ceftazidime/avibactam treatment. Methods and results Comparative whole-genome sequence analysis identified two SNPs in the CTX-M-14-encoding gene leading to two amino acid changes (P170S and T264I). Compared with WT CTX-M-14, expression of the CTX-M-14Δ170Δ264 isoform in Escherichia coli led to a >64- and 16-fold increase in ceftazidime and ceftazidime/avibactam MICs, respectively, functionally linking the observed SNPs and elevated MICs. The mutated CTX-M-14 isoform exhibited augmented ceftazidime hydrolytic activity, which was a reasonable cause for impaired susceptibility to avibactam inhibition. The P170S exchange in CTX-M-14 was found in association with elevated ceftazidime/avibactam MICs for independent K. pneumoniae isolates, but was not sufficient for full resistance. Apparently, additional CTX-M-independent mechanisms contribute to ceftazidime/avibactam resistance in K. pneumoniae DT12. Conclusions This study on the molecular basis of ceftazidime/avibactam resistance in clinical K. pneumoniae emerging in vivo underscores the need for continuous monitoring of ceftazidime/avibactam susceptibility during therapy. Despite sustained inhibition of OXA-48, rapid development of CTX-M-14 isoforms exhibiting augmented ceftazidime hydrolytic activity may limit the usefulness of ceftazidime/avibactam monotherapies in infections caused by isolates carrying blaCTX-M-14 and blaOXA-48.

Antibiotic use in animal husbandry has raised concerns on the spread of resistant bacteria. Currently animal products are traded globally with unprecedented ease, which has been challenging the control of antimicrobial resistance. This study aims to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from imported and locally produced poultry products sold in Ghana. Local and imported chicken meat was collected from 94 stores and markets throughout Kumasi (Ghana) and cultured on selective ESBL screening agar. Phenotypic ESBL-producing E. coli and K. pneumoniae isolates were confirmed by combined disc test and further characterized by antibiotic susceptibility testing, amplification of the blaCTX-M, blaTEM and blaSHV genes as well as multilocus sequence typing (MLST) and linked to the country of origin. Out of 200 meat samples, 71 (36%) samples revealed 81 ESBL-producing isolates (46 E. coli and 35 K. pneumoniae), with 44% (30/68) of local poultry and 31% (41/132) of imported products being contaminated. Most ESBL-producing isolates harboured the blaCTX-M-15 gene (61/81, 75%) and the dominant Sequence Types (ST) were ST2570 (7/35, 20%) among K. pneumoniae and ST10 (5/46, 11%) among E. coli. High numbers of ESBL-producing bacteria, particularly on local but also imported poultry meat, represent a potential source for human colonization and infection as well as spread within the community. Surveillance along the poultry production-food-consumer chain would be a valuable tool to identify sources of emerging multidrug resistant pathogens in Ghana.

Background Central line-associated bloodstream infections (CLABSI) are a major source of sepsis in modern intensive care medicine. Some years ago bundle interventions have been introduced to reduce CLABSI. The use of checklists may be an additional tool to improve the effect of these bundles even in highly specialized institutions. In this study we investigate if the introduction of a checklist reduces the frequency of CLABSI. Methods During the study period from October 2011 to September 2012, we investigated the effect of implementing a checklist for the placement of central venous lines (CVL). Patients were allocated either to the checklist group or to the control group, roughly in a 1:2 ratio. The frequency of CLABSI was compared between the two groups. Results During the study period 4416 CVL were inserted; 1518 in the checklist group and 2898 in the control group. The use of the checklist during CVL placement resulted in a lower CLABSI frequency. The incidence in the checklist group was 3.8 per 1000 catheter days as compared to 5.9 per 1000 catheter days in the control group (IRR = 0.57; p = 0.001). The use of the checklist also reduced the frequency of catheter colonisation significantly, 36.3 per 1000 catheter days in the checklist group vs 21.2 per 1000 catheter days in the control group, respectively (IRR = 0.58; p

Given constantly high or even rising incidences of both colonization and infection with vancomycin-resistant enterococci (VRE), timely and accurate identification of carriers in high-risk patient populations is of evident clinical importance. In this study, a two-tier approach consisting of PCR-based screening and cultural confirmation of positive results is compared to the conventional approach solely based on culture on selective media. The 2-tier strategy was highly consistent with the conventional approach, and was found to possess high sensitivity and specificity (93.1% and 100%, respectively). The introduction of the PCR-based combined VRE screening approach significantly (P0.0001) reduced median overall time to result by 44.3hours. The effect was found to be most pronounced in VRE negative samples. Positive vanA PCR was highly consistent with culture (PPV: 92.0%, 95% CI: 72.5-98.6%, NPV: 99.6%, 95% CI: 98.9-99.6%), thus allowing for preliminary reporting of VRE detection. In contrast, a vanB positive PCR does not allow for preliminary reporting (PPV: 58.5%, 95% CI: 44.2-71.6%, NPV: 99.8%, 95% CI: 99.2-100%). The introduction of a molecular assay for rapid detection of VRE from rectal swabs combined with cultural confirmation proved to be reliable and time saving, especially in a setting of low VRE prevalence and predominance of vanA positive strains.

Mutations in ramR, a negative regulator of ramA which stimulates transcription of acrA/-B encoding the multidrug efflux pump AcrAB-TolC, were recently shown to result in reduced susceptibility to tigecycline in Klebsiella pneumoniae. We analysed six non-duplicate K. pneumoniae isolates with elevated MICs to tigecycline. All isolates showed transcriptional up-regulation of ramA and acrB as demonstrated by Northern blot and quantitative real-time PCR. Sequencing of the ramR gene revealed deletions in five of the isolates and a premature stop codon in one isolate. Transformation of the wild-type ramR gene but not of any of the detected mutant ramR genes into a ramR-mutant K. pneumoniae strain restored tigecycline susceptibility and repressed ramA and acrB transcription to wild type levels. Thus, our study confirms the role of inactivating mutations in the ramR gene in tigecycline resistance.

Background Acinetobacter baumannii is an emerging multiresistant Gram-negative rod, which has caused multiple hospital outbreaks. A. baumannii can display a high ability to survive on inanimate surfaces. Therefore, cleaning and disinfection is an important part in the prevention of A. baumannii transmission. No-touch room decontamination is performed with increasing frequency to reach more standardization in hospital cleaning. In this study, we investigated the efficacy of an automated room decontamination using ozone (Sterisafe™ pro) against A. baumannii under complex room conditions. Methods A Carbapenemase-producing A. baumannii outbreak strain was analyzed with respect to its ability to survive on dry surfaces. The Sterisafe™ pro instrument was used in a patient room with an attached bathroom. The A. baumannii strain was dried on three different carriers (ceramic tiles, stainless steel, solid core furniture board) and placed at eight different positions in the rooms. A standard disinfection cycle (80 ppm ozone; 90 % RH; 60 minutes) was conducted in three independent experiments. Results The A. baumannii strain displayed a long-term survival on surfaces under dry conditions sufficient for further disinfection experiments. Interestingly, the mean reduction rates of A. baumannii dried on three surfaces displayed significant differences. Reduction rates greater than 5 log were reached on all stainless steel and ceramic carriers even under the complex room conditions using the standard disinfection cycle of the Sterisafe™ pro instrument. In contrast, on furniture board individual carriers displayed reduction rates of even less than 4 log. The mean reduction rate was still 5 log for A. baumannii on furniture board. Conclusion A. baumannii dried on different surfaces display a differential susceptibility against automated ozone disinfection. However, the SterisafeTM pro instrument displayed a sufficient reduction of A. baumannii for all tested surfaces even under complex room conditions. The individual behavior of A. baumannii on different materials indicate the necessity for the validation of automated room decontamination systems under varying conditions. Disclosures All authors: No reported disclosures.

Objectives To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. Methods The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . Results P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. Conclusions The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

Background High prevalence of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae threatens treatment options for invasive bloodstream infections in sub-Saharan Africa. Objectives To explore the frequency and genotype distribution of ESBL producing Enterobacteriaceae causing bloodstream infections in a primary health care setting in rural Ghana. Methods Blood cultures from all patients with fever ≥38 °C within 24 h after admission (community-acquired) and from all neonates with suspected neonatal sepsis (hospital-acquired) were obtained. ESBL-producing isolates were characterized by combined disc test and by amplifying the blaCTX-M, blaTEM and blaSHV genes. Multilocus sequence typing (MLST) was performed for all ESBL-producing Klebsiella pneumoniae and Escherichia coli isolates, and all K. pneumoniae isolates were differentiated by pulsed-field gel electrophoresis (PFGE). Results Among 426 Enterobacteriaceae isolated from blood cultures, non-typhoid Salmonella (n = 215, 50.8%), S. Typhi (n = 110, 26.0%), E. coli (n = 50, 11.8%) and K. pneumoniae (n = 41, 9.7%) were the most frequent. ESBL-producing isolates were restricted to the CTX-M-15 genotype and the species K. pneumoniae (n = 34, 82.9%), Enterobacter cloacae complex (n = 2, 66.7%) and E. coli (n = 5, 10.0%). The rates of ESBL-producers in K. pneumoniae were 55.6% and 90.6% in community-acquired and neonatal bloodstream infections, respectively. MLST and PFGE analysis identified four outbreak clusters among neonates. Conclusions Considering the rural primary health care study setting, the high proportion of ESBL-producing Klebsiella pneumoniae is worrisome and might be devastating in the absence of second line antibiotics. Therefore, enhanced diagnostic laboratories for surveillance purposes and sustainable hospital hygiene measures must be considered to prevent further spread of multidrug resistant bacteria within rural communities.

Chicken meat has been proposed to constitute a source for extended spectrum beta-lactamase (ESBL)-carrying Enterobacteriaceae that colonize and infect humans. In this study the prevalence of ESBL-producing Enterobacteriaceae in stool samples from ambulatory patients who presented in the emergency department of the University Medical Centre Hamburg-Eppendorf with gastrointestinal complains and in chicken meat samples from the Hamburg region were analysed and compared with respect to ESBL-genotypes, sequence types and antibiotic resistance profiles. Twenty-nine (4.1%) of 707 stool samples and 72 (60%) of 120 chicken meat samples were positive for ESBL-producing Enterobacteriaceae. The distribution of ESBL genes in the stool vs. chicken meat isolates (given as % of total isolates from stool vs. chicken meat) was as follows: CTX-M-15 (38% vs. 0%), CTX-M-14 (17% vs. 6%), CTX-M-1 (17% vs. 69%), SHV-12 (3% vs. 18%) and TEM-52 (3% each). Comparison of ESBL- and multilocus sequence type revealed no correlation between isolates of human and chicken. Furthermore, ESBL-producing E. coli from stool samples were significantly more resistant to fluoroquinolones, aminoglycosides and/or trimethoprim-sulfamethoxazole than chicken isolates. The differences in ESBL-genotypes, sequence types and antibiotic resistance patterns indicate that in our clinical setting chicken meat is not a major contributor to human colonization with ESBL-carrying Enterobacteriaceae.

Background. In May–July 2011, Germany experienced a large food-borne outbreak of Shiga toxin 2–producingEscherichia coli (STEC O104:H4) with 3842 cases, including 855 cases with hemolytic uremic syndrome (HUS)and 53 deaths.Methods. A multicenter study was initiated in 5 university hospitals to determine pathogen shedding duration.Diagnostics comprised culture on selective media, toxin enzyme-linked immunosorbent assay, and polymerasechain reaction. Results were correlated with clinical and epidemiologic findings. Testing for pathogen excretion wascontinued after discharge of the patient.Results. A total of 321 patients (104 male, 217 female) were included (median age, 40 years [range, 1–89days]). Median delay from onset of symptoms to hospitalization was 4 days (range, 0–17 days). Two hundred ninepatients presented with HUS. The estimate for the median duration of shedding was 17–18 days. Some patientsremained STEC O104:H4 positive until the end of the observation time (maximum observed shedding duration:157 days). There was no significant influence of sex on shedding duration. Patients presenting with HUS had asignificantly shortened shedding duration (median, 13–14 days) compared to non-HUS patients (median, 33–34days). Antimicrobial treatment was also significantly associated with reduced shedding duration. Children (age ≤15years) had longer shedding durations than adults (median, 35–41 vs 14–15 days).Conclusions. STEC O104:H4 is usually eliminated from the human gut after 1 month, but may sometimes beexcreted for several months. Proper follow-up of infected patients is important to avoid further pathogen spread.Keywords. shedding; STEC; outbreak; hemolytic uremic syndrome.

We analyzed a collection of carbapenem-resistant Gram-negative bacterial isolates and detected VIM-1, VIM-2, and KPC-2 in diverse enterobacterial species and Pseudomonas aeruginosa isolates. Our findings suggest a more widespread dissemination of carbapenemases in Germany than currently appreciated.

Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m/z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.

An outbreak caused by Escherichia coli serotype O104:H4 strains has been affecting northern Germany since May 2011, with 3,222 patients infected and 39 of them dead by 18 June ([4][1]). Around 25% of the patients developed hemolytic-uremic syndrome (HUS) ([1][2], [3][3]), a rate which is much higher