INTRODUCTION:Bile has long been considered sterile. Recent studies show that bacteria can frequently be detected in bile and certain bacterial species are associated with bile duct-associated liver disease.OBJECTIVES:To detect bacterial species and antibiotic resistance in bile in bile duct-associated liver disease.METHODOLOGY:To evaluate microbiological findings of bile samples obtained during ERCP at a tertiary center from 2009 to 2019.RESULTS:There were 1885 bile samples from 992 patients examined by cultural microbiological analysis. Germs were detected in 91% of the samples. Most bile samples (n) were obtained from patients who had undergone liver transplantation (LTX; n = 556), followed by patients with primary sclerosing cholangitis (PSC; n = 287). Enterococci were detected in 67% of samples, followed by E. coli (32.2%) and Klebsiella (28.2%). Of 1151 enterococci detected, 13.1% were vancomycin (VRE)s and of 216 staphylococci detected, 10% were ORSA. The proportion of VRE increased with the number of tests performed during ERCPs ( P  < 0.01; chi-square) and increased 2.5-fold over 10 years, whereas the detection of ORSA remained stable. Patients with cholecystolithiasis were significantly more likely to have evidence of VRE in bile compared to LTX and PSC patients ( P  = 0.02, P  < 0.01; chi-square). The most abundant bacterial genera showed highly statistically significant differences in their levels of liver enzymes and c-reactive protein ( P  < 0.001).CONCLUSION:Knowledge of the bacterial composition of bile in various bile duct-associated liver diseases may allow more targeted antibiotic use in the future.

Background and aims:Bacterial cholangitis is a common complication in patients with ischemic type biliary lesions and/or anastomotic strictures after liver transplantation (LTX). Patients frequently need antibiotics and endoscopic retrograde cholangiography (ERC) to improve the bile flow. Antibiotic treatment is based on findings in standard microbiological cultivation (SMC) of bile. However, the cultivation techniques are limited to a subset of bacteria easy-to-cultivate. Therefore, the aim of our study was to evaluate the value of next generation sequencing as an additional diagnostic tool to SMC in ischemic type biliary lesions and/or anastomotic strictures. Methods:We sequenced the V1-V2 region of the 16S rRNA gene in 242 stored bile samples in patients after LTX and compared the results with findings of SMC. SMC was performed in n = 135 (56%) fresh bile samples in addition to NGS. SMC was part of the clinical routine in these patients. Results:NGS detected bacterial genera in bile samples more often than SMC (P = 5.42 × 10-74). SMC showed insufficient discovery of bacterial genera compared to NGS with better performance in patients receiving antibiotics prior to ERC. SMC missed many bacterial genera detected by NGS. Conclusions:NGS was more sensitive in detecting bacteria in bile than SMC, no clinical parameters could be used to improve discovery rates in SMC and many genera were missed by SMC. Therefore, NGS should be used in a combined approach with SMC for improved diagnostics to achieve more specific and targeted antibiotic treatments.

BACKGROUND:Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS:Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION:Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .

Background:A bundle of infection control measures are implemented in the case of a nosocomial outbreak (NO) in pediatrics and neonatology, including the total closure of the ward for new admissions. This represents a very effective but unfortunately also cost-intensive decision. Thus, knowledge on certain outbreak characteristics that will very probably result in such a closure is important in order to carry this out as early as possible to minimize nosocomial transmissions and subsequent infections as well as financial loss. Objective:To determine the characteristics that are highly associated with a need for closure of the ward or unit. Material and methods:A systematic review of the medical literature on NO in pediatrics and neonatology was carried out in the Worldwide Outbreak Database, which represents the largest collection of literature on NO, and additional international databases on NO with closure were then statistically compared to NO without closure. Results:Overall, 198 NO in pediatrics (including 25 NO with and 173 NO without closure) and a further 413 NO in neonatology (including 52 NO with and 361 NO without closure) were included in the analysis. Intensive care units (ICUs) were involved in 446 of these 611 NO (73.0%). The source of the NO remained unknown in 299 of the 611 NO (48.9%). Among the 16,107 affected patients there were 3155 infections and 1542 fatal cases. The duration of the NOs ranged from 2 to 4380 days. The duration of closure (if applicable) ranged from 2 to 210 days. The most frequently reported pathogens in NO with subsequent closure were Serratia marcescens, Enterobacter cloacae, and Klebsiella pneumoniae. The involvement of an ICU was significantly associated with the decision to finally close the affected location (pediatric ICU: p = 0.026; neonatological ICU: p < 0.001). The same observation was made when a large number of patients were involved in the NO. The top 5 measures in NO in pediatrics and neonatology were screening of patients (387 NO), enforcement of hand hygiene (302 NO), screening of staff (291 NO), isolation precautions in single rooms or cohorts (270 NO) and modification of the antimicrobial treatment (249 NO). Conclusion:Based on these findings one should consider closure of the ward early in the course of events when experiencing a NO on an ICU in pediatrics and especially in neonatology. Furthermore, it would be useful to define a locally established threshold for the number of patients involved in such a NO, which could then trigger the enforcement of new implementation of infection control measures, such as a closure of the entire unit. Finally, we would like to encourage everybody experiencing an NO to share the observations in detail with others. Data from NO are crucial in order to generate infection control recommendations in guidelines, that are appropriate for education of all kinds of medical staff, and it can be very helpful to rely on previous experiences during a new ongoing outbreak investigation.

Zusammenfassung Hintergrund Im Fall eines nosokomialen Ausbruchs (NA) in der Pädiatrie und Neonatologie wird neben einer Vielzahl weiterer Maßnahmen mintunter die gesamte Station für Neuaufnahmen geschlossen. Dies ist infektionsprophylaktisch zwar oft effektiv, jedoch ökonomisch oft auch sehr nachteilig. Wichtig sind daher Kenntnisse darüber, wann eine solche Schließung sehr wahrscheinlich unvermeidbar ist, um diese dann frühestmöglich durchzuführen. Ziel der Arbeit Ermittlung von Charakteristika, die eine Schließung letztlich wahrscheinlich machen. Material und Methoden Systematische Literaturrecherche der Worldwide Outbreak Database und weiterer internationaler Datenbanken zu NA mit und ohne Schließung. Ergebnisse Es wurden 198 NA aus der Pädiatrie (darunter 25 NA mit und 173 NA ohne Schließung) sowie weitere 413 NA aus der Neonatologie (darunter 52 NA mit und 361 NA ohne Schließung) eingeschlossen. In 446 dieser 611 NA waren auch Intensivstationen (ITS) betroffen. Insgesamt waren 16.107 Patienten betroffen und es ereigneten sich 3155 Infektionen mit 1542 Todesfällen. Die Gesamtdauer der NA betrug dabei 2 bis 4380 Tage; sofern erfolgt, betrug die Dauer der Schließung 2 bis 210 Tage. Die am häufigsten nachgewiesenen Erreger in Ausbrüchen mit Schließung waren S. marcescens, E. cloacae und K. pneumoniae. Die Beteiligung einer ITS sowie hohe Fallzahlen korrelierten signifikant mit der Wahrscheinlichkeit für eine spätere Schließung. Diskussion Insbesondere für die Neonatologie wird bei der Beteiligung einer ITS an einem NA empfohlen, eine Schließung des Bereiches frühzeitig zu erwägen. Zudem kann in NA die Definition lokal festgelegter Schwellenwerte für Patientenzahlen, bei deren Überschreiten bestimmte Hygienemaßnahmen, wie z. B. die Schließung der Station, initiiert werden, sinnvoll sein.

A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.

This study aims to characterize the biliary microbiome as neglected factor in patients with ischaemic-type biliary lesions (ITBL) after liver transplantation. Therefore, the V1-V2 region of the 16S rRNA gene was sequenced in 175 bile samples. Samples from patients with anastomotic strictures (AS) served as controls. Multivariate analysis and in silico metagenomics were applied cross-sectionally and longitudinally. The microbial community differed significantly between ITBL and AS in terms of alpha and beta diversity. Both, antibiotic treatment and stenting were associated independently with differences in the microbial community structure. In contrast to AS, in ITBL stenting was associated with pronounced differences in the biliary microbiome, whereas no differences associated with antibiotic treatment could be observed in ITBL contrasting the pronounced differences found in AS. Bacterial pathways involved in the production of antibacterial metabolites were increased in ITBL with antibiotic treatment. After liver transplantation, the biliary tract harbours a complex microbial community with significant differences between ITBL and AS. Fundamental changes in the microbial community in ITBL can be achieved with biliary stenting. However, the effect of antibiotic treatment in ITBL was minimal. Therefore, antibiotics should be administered wisely in order to reduce emerging resistance of the biliary microbiome towards external antibiotics.

Background: Lower respiratory tract infections (LRTI) are a significant cause of morbidity and mortality in lung transplant (LTx) recipients. Timely and precise pathogen detection is vital. Rapid-response multiplex PCR-kits (RM-PCR) offer a valuable diagnostic addition. Aim: To assess how RM-PCR compares to conventional microbiology and virological tests of LTx recipients. Methods: A prospective observational cohort study in 60 LTx recipients with suspected LRTI was performed. All patients received RM-PCR testing of bronchoalveolar lavage fluid in addition to conventional tests including microbiological cultures and immunofluorescence testing (IFT) ± conventional PCR for respiratory viruses. Primary outcome was time to test result; secondary outcomes included time-to-treatment modification and test accuracy of RM-PCR. Results: RM-PCR provided results significantly faster than conventional tests (2.3h vs 23.4h, P<.001), allowing for faster clinical decisions (2.3h vs virology 24.2h and microbiology 29.4h, P<.001). Based on all diagnostic modalities, 26 patients were diagnosed with viral LRTI, 9 with non-viral LRTI and 5 with combined viral and non-viral LRTI. These diagnoses could be established by RM-PCR in 92%, 78% and 100%, respectively. The remaining 20 patients received an alternative diagnosis. Preliminary therapies established based on RM-PCR results were upheld in 90%. There were 6 treatment modifications based on pathogen-isolation by conventional testing missed by RM-PCR (fungal n=3). Conclusions: RM-PCR offered significantly faster test results compared to conventional tests with overall good concordance.

Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Background: Serratia marcescens is a well-known and challenging pathogen in neonatal intensive care units. It is responsible for severe infections and can cause nosocomial outbreaks. Methods: We present the infection control response to a Serratia marcescens cluster which occurred in a tertiary neonatal intensive care unit. Results and conclusions: The presented comprehensive and decisive hygiene management response starting with the very first case aims especially at early detection and immediate interruption of nosocomial transmission. Frequent and sensitive microbiological screening, rigorous spatial isolation of colonized infants, and reinforcing adherence to hand hygiene are essential in this response, which comprises eight measures. It prevented a full-blown outbreak.

Einleitung Die Galle wurde lange Zeit als steril betrachtet. Neuere Untersuchungen zeigen, dass Bakterien häufig in der Galle nachgewiesen werden können und bestimmte bakterielle Spezies mit Gallengangs-assoziierten Lebererkrankungen assoziiert sind.

Nocardiosis is a rare but life-threatening infection caused by aerobic Actinomycetes of the genus Nocardia particularly affecting immunocompromised hosts. The identification of Nocardia ssp. and antibiotic susceptibility testing by standard microbiological methods are incomplete and molecular techniques may improve diagnostics. We studied 39 Nocardia strains isolated from 33 patients between 2000 and 2018. Twenty-four patients (72.7 %) were immunocompromised. Whole genome sequencing (WGS) revealed a broad taxonomic range of those isolates spanning 13 different species, including four strains that belonged to three novel species based on average nucleotide identity (ANI < 95 % with currently available genome sequences). 16S rRNA gene analyses mirrored WGS results. Conventional MALDI-TOF analysis correctly identified 29 isolates at the species level (74.4 %). Our advanced protocol with formic acid and acetonitrile treatment increased identification to 35 isolates (89.7 %). Antibiotic resistance was tested using both a microdilution method and MIC strip testing. Results were in good concordance with an overall trimethoprim-sulfamethoxazole (SXT) resistance rate of 13.5 %. WGS of a SXT resistant N. farcinica isolate showed a deletion of several amino acids in a homolog of dihydropteroate synthase (FolP2) that was not seen in sensitive members of this species. Diversity of Nocardia isolates was high and involved many different species, suggesting that this taxon has broadly distributed mechanisms for infecting individuals. Widely applicable diagnostic methods including MALDI-TOF and 16S rRNA gene analyses correctly identified most strains. WGS additionally revealed molecular insights into SXT resistance mechanisms of clinical Nocardia isolates highlighting the potential application of (meta)genomic-based diagnostics in the future.

Background Carbapenem resistant (CR) Klebsiella pneumoniae (Kp) and Acinetobacter baumannii (Ab) are emerging multidrug resistant bacteria with very limited treatment options in case of infection. Both are well-known causes of nosocomial infections and outbreaks in healthcare facilities. Methods A retrospective study was conducted to investigate the epidemiology of inpatients with CR Kp and CR Ab in a 1500-bed German university hospital from 2015 to 2019. We present our infection control concept including a weekly microbiologic screening for patients who shared the ward with a CR Kp or CR Ab index patient. Results Within 5 years, 141 CR Kp and 60 CR Ab cases were hospitalized corresponding to 118 unique patients (74 patients with CR Kp, 39 patients with CR Ab and 5 patients with both CR Ab and CR Kp). The mean incidence was 0.045 (CR Kp) and 0.019 (CR Ab) per 100 inpatient cases, respectively. Nosocomial acquisition occurred in 53 cases (37.6%) of the CR Kp group and in 12 cases (20.0%) of the CR Ab group. Clinical infection occurred in 24 cases (17.0%) of the CR Kp group and in 21 cases (35.0%) of the CR Ab group. 14 cases (9.9%) of the CR Kp group and 29 cases (48.3%) of the CR Ab group had a history of a hospital stay abroad within 12 months prior to admission to our hospital. The weekly microbiologic screening revealed 4 CR Kp cases caused by nosocomial transmission that would have been missed without repetitive screening. Conclusions CR Kp and CR Ab cases occurred infrequently. A history of a hospital stay abroad, particularly in the CR Ab group, warrants pre-emptive infection control measures. The weekly microbiologic screening needs further evaluation in terms of its efficiency.

BACKGROUND:Lower respiratory tract infections (LRTIs) are a significant cause of morbidity and mortality in lung transplant (LTx) recipients. Timely and precise pathogen detection is vital to successful treatment. Multiplex PCR kits with short turnover times like the BioFire Pneumonia Plus (BFPPp) (manufactured by bioMérieux) may be a valuable addition to conventional tests. METHODS:We performed a prospective observational cohort study in 60 LTx recipients with suspected LRTI. All patients received BFPPp testing of bronchoalveolar lavage fluid in addition to conventional tests including microbiological cultures and conventional diagnostics for respiratory viruses. Primary outcome was time-to-test-result; secondary outcomes included time-to-clinical-decision and BFPPp test accuracy compared to conventional tests. RESULTS:BFPPp provided results faster than conventional tests (2.3 h [2-2.8] vs. 23.4 h [21-62], p < 0.001), allowing for faster clinical decisions (2.8 [2.2-44] vs. virology 28.1 h [23.1-70.6] and microbiology 32.6 h [4.6-70.9], both p < 0.001). Based on all available diagnostic modalities, 26 (43%) patients were diagnosed with viral LRTI, nine (15 %) with non-viral LRTI, and five (8 %) with combined viral and non-viral LRTI. These diagnoses were established by BFPPp in 92%, 78%, and 100%, respectively. The remaining 20 patients (33 %) received a diagnosis other than LRTI. Preliminary therapies based on BFPPp results were upheld in 90% of cases. There were six treatment modifications based on pathogen-isolation by conventional testing missed by BFPPp, including three due to fungal pathogens not covered by the BFPPp. CONCLUSION:BFPPp offered faster test results compared to conventional tests with good concordance. The absence of fungal pathogens from the panel is a potential weakness in a severely immunosuppressed population.

INTRODUCTION:Vancomycin-resistant enterococci (VRE) are emerging multidrug-resistant bacteria. They can cause serious nosocomial infections, especially in immunocompromised patients.OBJECTIVES AND METHODS:In this study, we aimed to determine the burden of intestinal VRE colonization and clinically relevant infection in adult hematologic and oncologic patients at a tertiary care clinic in Germany based on prospective infection surveillance and an active screening program.RESULTS:In a 12 month period, 132 of 555 patients had intestinal VRE-colonization (23.8%) and four patients (0.7% of the entire cohort, and 3.0% of those colonized with VRE) developed a nosocomial infection with VRE.CONCLUSIONS:The prospective surveillance and active screening for VRE was very useful to determine the true ratio of intestinal colonization to infection and thus helps to shape infection control management.

Background. Asymptomatic C. difficile colonization is believed to predispose to subsequent C. difficile infection (CDI). While emerging insights into the role of the commensal microbiota in mediating colonization resistance against C. difficile have associated CDI with specific microbial components, corresponding prospectively collected data on colonization with C. difficile are largely unavailable. Methods. C. difficile status was assessed by GDH EIA and real-time PCR targeting the toxin A (tcdA) and B (tcdB) genes. 16S V3 and V4 gene sequencing results from fecal samples of patients tested positive for C. difficile were analyzed by assessing alpha and beta diversity, LefSe, and the Piphillin functional inference approach to estimate functional capacity. Results. 1506 patients were recruited into a prospective observational study (DRKS00005335) upon admission into one of five academic hospitals. 936 of them provided fecal samples on admission and at discharge and were thus available for longitudinal analysis. Upon hospital admission, 5.5% (83/1506) and 3.7% (56/1506) of patients were colonized with toxigenic (TCD) and nontoxigenic C. difficile (NTCD), respectively. During hospitalization, 1.7% (16/936) acquired TCD. Risk factors for acquisition of TCD included pre-existing lung diseases, lower GI endoscopy and antibiotics. Species protecting against hospital-related C. difficile acquisition included Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococcus spp. Metagenomic pathway analysis identified steroid biosynthesis as the most underrepresented metabolic pathway in patients who later acquire C. difficile colonization. Conclusions. Gemmiger spp., Odoribacter splanchnicus, Ruminococcus bromii and other Ruminococci were associated with a decreased risk of C. difficile acquisition.

Clostridium difficile infection (CDI) poses an increased risk to patients with inflammatory bowel disease. Preconditions are colonisation with C. difficile, but also a breakdown of the colonisation resistance, of which secondary bile acids are believed to constitute a decisive component. 7α-dehydroxylation is one of the key steps in primary to secondary bile acid transformation, and required genes have been located in a single bile acid inducible (baiCD) operon in C. scindens as well as in C. hiranonis. The prevalence of these species in human fecal samples—particularly with regard to samples tested positive for C. difficile—and in the setting of fecal microbiota transfer (FMT) in CDI patients has not been studied. A species-specific qPCR for detection of baiCD in C. scindens and C. hiranonis was established. Fecal samples of patients with CDI, toxigenic C. difficile colonisation (TCD), non-toxigenic C. difficile colonisation (NTCD), of C. difficile negative (NC) patients, and of two patients before and after fecal microbiota transfer (FMT) for recurrent CDI (rCDI) were tested for the presence of the baiCD gene cluster. In NC samples, the prevalence of the baiCD gene cluster was significantly higher than in CDI samples, with 72.5% (100/138) vs. 35.9% (23/64; p < 0.0001), respectively. No differences were seen between NC compared with NTCD samples or TCD samples. Both rCDI patients were baiCD negative at baseline, but one patient turned positive after successful FMT from a baiCD positive donor. BaiCD/16S rDNA ratio calculation showed no significant difference between the relative abundances in both groups (NC vs. CDI; p = 0.3244). The median of the NC group was 0.006% (IQR 0.0005%-0.376%) and for the CDI group 0.009% (IQR 0.0002%-7.8%). In addition, stool consistency did not measurably influence the relative abundance of bacterial species in this context of complex microbial communities. BaiCD gene positive species are reduced in fecal samples of patients with C. difficile infection as compared with asymptomatic carriers or C. difficile negative fecal samples. Furthermore, we present a case of baiCD gene positivity observed after successful fecal microbiota transfer for recurrent CDI.

Background Clostridium difficile infection (CDI) is a major cause of hospital-acquired diarrhea. Secondary bile acids were shown to confer resistance to colonization by C. difficile. 7α-dehydroxylation is a key step in transformation of primary to secondary bile acids and required genes have been located in a single bile acid-inducible (bai) operon in C. scindens as well as in C. hiranonis, two Clostridium sp. recently reported to protect against C. difficile colonization. Aim To analyze baiCD gene abundance in C. difficile positive and negative fecal samples. Material & methods A species-specific qPCR for detecting baiCD genes was established. Fecal samples of patients with CDI, asymptomatic toxigenic C. difficile colonization (TCD), non-toxigenic C. difficile colonization (NTCD), of C. difficile negative (NC) patients, and of two patients before and after fecal microbiota transplantation (FMT) for recurrent CDI (rCDI) were tested for the presence of the baiCD genes. Results The prevalence of the baiCD gene cluster was significantly higher in C. difficile negative fecal samples than in samples of patients diagnosed with CDI (72.5% (100/138) vs. 35.9% (23/64; p<0.0001). No differences in baiCD gene cluster prevalence were seen between NC and NTCD or NC and TCD samples. Both rCDI patients were baiCD-negative at baseline, but one of the two patients turned positive after successful FMT from a baiCD-positive donor. Conclusion Fecal samples of CDI patients are less frequently baiCD-positive than samples from asymptomatic carriers or C. difficile-negative individuals. Furthermore, we present a case of baiCD positivity observed after successful FMT for rCDI.

Enrollment and outcomes of Clostridium Difficile infection surveillence in critically ill, tubefed patients with feedings that contain carbohydrates >150 g/ml, protein >50 g/ml, or fat >60 g/ml, and a matched cohort of feedings that contain carbohydrates <150 g/ml, protein <50 g/ml, or fat <60 g/ml. Mo1926