OXA-48-like carbapenemases in Proteus mirabilis - novel genetic environments and a challenge for detection.

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, blaOXA-48-like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n=9; OXA-181, n=3; OXA-162, n=1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene blaOXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates blaOXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring blaOXA-181 one was located on an IncX3 plasmid and two were located on a novel MOBF plasmid, pOXA-P12, within the new transposon Tn7713. In 5/6 isolates with plasmidic location of blaOXA-48-like, the plasmids could conjugate to E. coli recipients in vitro. Vice versa, blaOXA-48-carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of blaOXA-48-like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of blaOXA-48-like on mobile genetic elements, it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.

Epidemiological trends and susceptibility patterns of bloodstream infections caused by Enterococcus spp. in six German university hospitals: a prospectively evaluated multicentre cohort study from 2016 to 2020 of the R-Net study group.

PURPOSE: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. METHODS: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. RESULTS: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). CONCLUSION: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern.

Viral genome sequencing to decipher in-hospital SARS-CoV-2 transmission events.

The SARS-CoV-2 pandemic has highlighted the need to better define in-hospital transmissions, a need that extends to all other common infectious diseases encountered in clinical settings. To evaluate how whole viral genome sequencing can contribute to deciphering nosocomial SARS-CoV-2 transmission 926 SARS-CoV-2 viral genomes from 622 staff members and patients were collected between February 2020 and January 2021 at a university hospital in Munich, Germany, and analysed along with the place of work, duration of hospital stay, and ward transfers. Bioinformatically defined transmission clusters inferred from viral genome sequencing were compared to those inferred from interview-based contact tracing. An additional dataset collected at the same time at another university hospital in the same city was used to account for multiple independent introductions. Clustering analysis of 619 viral genomes generated 19 clusters ranging from 3 to 31 individuals. Sequencing-based transmission clusters showed little overlap with those based on contact tracing data. The viral genomes were significantly more closely related to each other than comparable genomes collected simultaneously at other hospitals in the same city (n = 829), suggesting nosocomial transmission. Longitudinal sampling from individual patients suggested possible cross-infection events during the hospital stay in 19.2% of individuals (14 of 73 individuals). Clustering analysis of SARS-CoV-2 whole genome sequences can reveal cryptic transmission events missed by classical, interview-based contact tracing, helping to decipher in-hospital transmissions. These results, in line with other studies, advocate for viral genome sequencing as a pathogen transmission surveillance tool in hospitals.

Prognostic Value of Gut Microbiome for Conversion from Mild Cognitive Impairment to Alzheimer's Disease Dementia within 4 Years: Results from the AlzBiom Study.

Alterations in the gut microbiome are associated with the pathogenesis of Alzheimer's disease (AD) and can be used as a diagnostic measure. However, longitudinal data of the gut microbiome and knowledge about its prognostic significance for the development and progression of AD are limited. The aim of the present study was to develop a reliable predictive model based on gut microbiome data for AD development. In this longitudinal study, we investigated the intestinal microbiome in 49 mild cognitive impairment (MCI) patients over a mean (SD) follow-up of 3.7 (0.6) years, using shotgun metagenomics. At the end of the 4-year follow-up (4yFU), 27 MCI patients converted to AD dementia and 22 MCI patients remained stable. The best taxonomic model for the discrimination of AD dementia converters from stable MCI patients included 24 genera, yielding an area under the receiver operating characteristic curve (AUROC) of 0.87 at BL, 0.92 at 1yFU and 0.95 at 4yFU. The best models with functional data were obtained via analyzing 25 GO (Gene Ontology) features with an AUROC of 0.87 at BL, 0.85 at 1yFU and 0.81 at 4yFU and 33 KO [Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog] features with an AUROC of 0.79 at BL, 0.88 at 1yFU and 0.82 at 4yFU. Using ensemble learning for these three models, including a clinical model with the four parameters of age, gender, body mass index (BMI) and Apolipoprotein E (ApoE) genotype, yielded an AUROC of 0.96 at BL, 0.96 at 1yFU and 0.97 at 4yFU. In conclusion, we identified novel and timely stable gut microbiome algorithms that accurately predict progression to AD dementia in individuals with MCI over a 4yFU period.

Forecasting local hospital bed demand for COVID-19 using on-request simulations.

Accurate forecasting of hospital bed demand is crucial during infectious disease epidemics to avoid overwhelming healthcare facilities. To address this, we developed an intuitive online tool for individual hospitals to forecast COVID-19 bed demand. The tool utilizes local data, including incidence, vaccination, and bed occupancy data, at customizable geographical resolutions. Users can specify their hospital's catchment area and adjust the initial number of COVID-19 occupied beds. We assessed the model's performance by forecasting ICU bed occupancy for several university hospitals and regions in Germany. The model achieves optimal results when the selected catchment area aligns with the hospital's local catchment. While expanding the catchment area reduces accuracy, it improves precision. However, forecasting performance diminishes during epidemic turning points. Incorporating variants of concern slightly decreases precision around turning points but does not significantly impact overall bed occupancy results. Our study highlights the significance of using local data for epidemic forecasts. Forecasts based on the hospital's specific catchment area outperform those relying on national or state-level data, striking a better balance between accuracy and precision. These hospital-specific bed demand forecasts offer valuable insights for hospital planning, such as adjusting elective surgeries to create additional bed capacity promptly.

Association of ward-level antibiotic consumption with healthcare-associated Clostridioides difficile infections: an ecological study in five German university hospitals, 2017-2019.

OBJECTIVES: To analyse the influence of antibiotic consumption on healthcare-associated healthcare onset (HAHO) Clostridioides difficile infection (CDI) in a German university hospital setting. METHODS: Monthly ward-level antibiotic consumption measured in DDD/100 patient days (pd) and CDI surveillance data from five university hospitals in the period 2017 through 2019 were analysed. Uni- and multivariable analyses were performed with generalized estimating equation models. RESULTS: A total of 225 wards with 7347 surveillance months and 4 036 602 pd participated. With 1184 HAHO-CDI cases, there was a median incidence density of 0.17/1000 pd (IQR 0.03-0.43) across all specialties, with substantial differences among specialties. Haematology-oncology wards showed the highest median incidence density (0.67/1000 pd, IQR 0.44-1.01), followed by medical ICUs (0.45/1000 pd, IQR 0.27-0.73) and medical general wards (0.32/1000 pd, IQR 0.18-0.53). Multivariable analysis revealed carbapenem (mostly meropenem) consumption to be the only antibiotic class associated with increased HAHO-CDI incidence density. Each carbapenem DDD/100 pd administered increased the HAHO-CDI incidence density by 1.3% [incidence rate ratio (IRR) 1.013; 95% CI 1.006-1.019]. Specialty-specific analyses showed this influence only to be valid for haematological-oncological wards. Overall, factors like ward specialty (e.g. haematology-oncology ward IRR 2.961, 95% CI 2.203-3.980) or other CDI cases on ward had a stronger influence on HAHO-CDI incidence density (e.g. community-associated CDI or unknown association case in same month IRR 1.476, 95% CI 1.242-1.755) than antibiotic consumption. CONCLUSIONS: In the German university hospital setting, monthly ward-level carbapenem consumption seems to increase the HAHO-CDI incidence density predominantly on haematological-oncological wards. Furthermore, other patient-specific factors seem to be equally important to control HAHO-CDI.

Increasing numbers and complexity of Staphylococcus aureus bloodstream infection-14 years of prospective evaluation at a German tertiary care centre with multi-centre validation of findings.

OBJECTIVES: Staphylococcus aureus bloodstream infection (SAB) is a common and severe infection. This study aims to describe temporal trends in numbers, epidemiological characteristics, clinical manifestations, and outcomes of SAB. METHODS: We performed a post-hoc analysis of three prospective SAB cohorts at the University Medical Centre Freiburg between 2006 and 2019. We validated our findings in a large German multi-centre cohort of five tertiary care centres (R-Net consortium, 2017-2019). Time-dependent trends were estimated using Poisson or beta regression models. RESULTS: We included 1797 patients in the mono-centric and 2336 patients in the multi-centric analysis. Overall, we observed an increasing number of SAB cases over 14 years (6.4%/year and 1000 patient days, 95% CI: 5.1% to 7.7%), paralleled by an increase in the proportion of community-acquired SAB (4.9%/year [95% CI: 2.1% to 7.8%]) and a decrease in the rate of methicillin-resistant-SAB (-8.5%/year [95% CI: -11.2% to -5.6%]). All of these findings were confirmed in the multi-centre validation cohort (6.2% cases per 1000 patient cases/year [95% CI: -0.6% to 12.6%], community-acquired-SAB 8.7% [95% CI: -1.2% to 19.6%], methicillin-resistant S. aureus-SAB -18.6% [95% CI: -30.6 to -5.8%]). Moreover, we found an increasing proportion of patients with multiple risk factors for complicated/difficult-to-treat SAB (8.5%/year, 95% CI: 3.6% to 13.5%, p < 0.001), alongside an overall higher level of comorbidities (Charlson comorbidity score 0.23 points/year, 95% CI: 0.09 to 0.37, p 0.005). At the same time, the rate of deep-seated foci such as osteomyelitis or deep-seated abscesses significantly increased (6.7%, 95% CI: 3.9% to 9.6%, p < 0.001). A reduction of in-hospital mortality by 0.6% per year (95% CI: 0.08% to 1%) was observed in the subgroup of patients with infectious diseases consultations. DISCUSSION: We found an increasing number of SAB combined with a significant increase in comorbidities and complicating factors in tertiary care centres. The resulting challenges in securing adequate SAB management in the face of high patient turnover will become an important task for physicians.

High incidence of carbapenemase-producing Pseudomonas aeruginosa clinical isolates from Lagos, Nigeria.

Background: Carbapenem-resistant Pseudomonas aeruginosa strains are on the rise worldwide. This study characterized clinical isolates of P. aeruginosa from three Nigerian hospitals for carbapenem resistance. Methods: Strains isolated from wounds (n = 88), urine/catheter tips (n = 25), sputum/tracheotomy aspirates (n = 5), ear swabs (n = 4) and vaginal swabs (n = 1) were identified by MALDI-TOF and antibiotic susceptibility testing was performed using the VITEK 2 system. The genomic DNA of each isolate was subject to sequencing using Illumina and Oxford nanopore technology. Bioinformatics analyses were performed to detect antimicrobial resistance genes, clonal affiliations and phylogenetic relations of 123 non-duplicate P. aeruginosa isolates, whereas assembly of the nanopore reads using the plasmIDent pipeline enabled the identification of plasmids. Results: Forty-three percent of the isolates were resistant to all antibiotic categories tested. More than 40% of the isolates were resistant to the carbapenems imipenem and/or meropenem (39% and 44%, respectively). Among the meropenem-resistant isolates, 48 (89%) carried at least one carbapenemase gene. The predominant one was bla NDM-1 (n = 34), which conferred resistance to all five antibiotic categories and highly increased the MICs of both meropenem and imipenem. The other recurrent carbapenemase genes were bla VIM-2 (n = 4), and bla VIM-5-like (n = 11), which co-existed with bla NDM-1 in two isolates. Conclusions: The study revealed a high rate of carbapenem resistance and conjugative, broad host range plasmids carrying carbapenemase-encoding genes, especially the NDM-1 type, among isolates of P. aeruginosa. This may forebode the emergency of ubiquitous carbapenem resistance urging the implementation of infection control and antimicrobial stewardship strategies in Nigerian hospitals.

Resolving colistin resistance and heteroresistance in Enterobacter species.

Species within the Enterobacter cloacae complex (ECC) include globally important nosocomial pathogens. A three-year study of ECC in Germany identified Enterobacter xiangfangensis as the most common species (65.5%) detected, a result replicated by examining a global pool of 3246 isolates. Antibiotic resistance profiling revealed widespread resistance and heteroresistance to the antibiotic colistin and detected the mobile colistin resistance (mcr)-9 gene in 19.2% of all isolates. We show that resistance and heteroresistance properties depend on the chromosomal arnBCADTEF gene cassette whose products catalyze transfer of L-Ara4N to lipid A. Using comparative genomics, mutational analysis, and quantitative lipid A profiling we demonstrate that intrinsic lipid A modification levels are genospecies-dependent and governed by allelic variations in phoPQ and mgrB, that encode a two-component sensor-activator system and specific inhibitor peptide. By generating phoPQ chimeras and combining them with mgrB alleles, we show that interactions at the pH-sensing interface of the sensory histidine kinase phoQ dictate arnBCADTEF expression levels. To minimize therapeutic failures, we developed an assay that accurately detects colistin resistance levels for any ECC isolate.

Vancomycin-resistant Enterococcus faecium: admission prevalence, sequence types and risk factors-a cross-sectional study in seven German university hospitals from 2014 to 2018.

OBJECTIVES: Assessment of vancomycin-resistant Enterococcus faecium (VREfm) prevalence upon hospital admission and analysis of risk factors for colonization. METHODS: From 2014 to 2018, patients were recruited within 72 hours of admission to seven participating German university hospitals, screened for VREfm and questioned for potential risk factors (prior multidrug-resistant organism detection, current/prior antibiotic consumption, prior hospital, rehabilitation or long-term care facility stay, international travel, animal contact and proton pump inhibitor [PPI]/antacid therapy). Genotype analysis was done using cgMLST typing. Multivariable analysis was performed. RESULTS: In 5 years, 265 of 17,349 included patients were colonized with VREfm (a prevalence of 1.5%). Risk factors for VREfm colonization were age (adjusted OR [aOR], 1.02; 95% CI, 1.01-1.03), previous (aOR, 2.71; 95% CI, 1.87-3.92) or current (aOR, 2.91; 95% CI, 2.60-3.24) antibiotic treatment, prior multidrug-resistant organism detection (aOR, 2.83; 95% CI, 2.21-3.63), prior stay in a long-term care facility (aOR, 2.19; 95% CI, 1.62-2.97), prior stay in a hospital (aOR, 2.91; 95% CI, 2.05-4.13) and prior consumption of PPI/antacids (aOR, 1.29; 95% CI, 1.18-1.41). Overall, the VREfm admission prevalence increased by 33% each year and 2% each year of life. 250 of 265 isolates were genotyped and 141 (53.2%) of the VREfm were the emerging ST117. Multivariable analysis showed that ST117 and non-ST117 VREfm colonized patients differed with respect to admission year and prior multidrug-resistant organism detection. DISCUSSION: Age, healthcare contacts and antibiotic and PPI/antacid consumption increase the individual risk of VREfm colonization. The VREfm admission prevalence increase in Germany is mainly driven by the emergence of ST117.

Surveillance and Genomic Analysis of Third-Generation Cephalosporin-Resistant and Carbapenem-Resistant Klebsiella pneumoniae Complex in Germany.

To analyse the epidemiology and population structure of third-generation cephalosporin-resistant (3GCR) and carbapenem-resistant (CR) Klebsiella pneumoniae complex isolates, patients were screened for rectal colonisation with 3GCR/CR K. pneumoniae complex on admission to six German university hospitals (2016-2019). Also collected were 3GCR/CR and susceptible K. pneumoniae isolates from patients with bloodstream infections (2016-2018). Whole-genome sequencing was performed followed by multilocus sequencing typing (MLST), core-genome MLST, and resistome and virulome analysis. The admission prevalence of 3GCR K. pneumoniae complex isolates during the 4-year study period was 0.8%, and 1.0 bloodstream infection per 1000 patient admissions was caused by K. pneumoniae complex (3GCR prevalence, 15.1%). A total of seven K. pneumoniae complex bloodstream isolates were CR (0.8%). The majority of colonising and bloodstream 3GCR isolates were identified as K. pneumoniae, 96.7% and 98.8%, respectively; the remainder were K. variicola and K. quasipneumoniae. cgMLST showed a polyclonal population of colonising and bloodstream isolates, which was also reflected by MLST and virulome analysis. CTX-M-15 was the most prevalent extended-spectrum beta-lactamase, and 29.7% of the colonising and 48.8% of the bloodstream isolates were high-risk clones. The present study provides an insight into the polyclonal 3GCR K. pneumoniae population in German hospitals.

SARS-CoV-2 B.1.214.1, B.1.214.2 and B.1.620 are predominant lineages between December 2020 and July 2021 in the Republic of Congo.

Background: : SARS-CoV-2 variants have been emerging and are shown to increase transmissibility, pathogenicity, and decreased vaccine efficacies. The objective of this study was to determine the distribution, prevalence, and dynamics of SARS-CoV-2 variants circulating in Brazzaville, the Republic of Congo (ROC). Methods: : Between December 2020 and July 2021, a total of n=600 oropharyngeal specimens collected in the community were tested for COVID-19. Of the samples tested, 317 (53%) were SARS-CoV-2 positive. All samples that had a threshold of Ct <30 (n=182) were sequenced by next-generation sequencing (NGS), and all complete sequenced genomes were submitted to GISAID; lineages were assigned using pangolin nomenclature and a phylogenetic tree was reconstructed. In addition, the global prevalence of the predominant lineages was analysed using data from GISAID and Outbreak databases. Results: : A total of 15 lineages circulated with B.1.214.2 (26%), B.1.214.1 (19%) and B.1.620 (18%) being predominant. The variants of concern (VOC) alpha (B.1.1.7) (6%) and for the first time in June delta (B.1.617.2) (4%) were observed. In addition, the B.1.214.1 lineage first reported from ROC was observed to be spreading locally and regionally. Phylogenetic analysis suggests that the B.1.620 variant (VUM) under observation may have originated from either Cameroon or the Central African Republic. SARS-CoV-2 lineages were heterogeneous, with the densely populated districts of Poto-Poto and Moungali likely the epicenter of spread. Conclusion: : Longitudinal monitoring and molecular surveillance across time and space are critical to understanding viral phylodynamics, which could have important implications for transmissibility and impact infection prevention and control measures.

Evaluation of a laboratory-based high-throughput SARS-CoV-2 antigen assay.

OBJECTIVES: Antigen tests are an essential part of SARS-CoV-2 testing strategies. Rapid antigen tests are easy to use but less sensitive compared to nucleic acid amplification tests (NAT) and less suitable for large-scale testing. In contrast, laboratory-based antigen tests are suitable for high-throughput immunoanalyzers. Here we evaluated the diagnostic performance of the laboratory-based Siemens Healthineers SARS-CoV-2 Antigen (CoV2Ag) assay. METHODS: In a public test center, from 447 individuals anterior nasal swab specimens as well as nasopharyngeal swab specimens were collected. The nasal swab specimens were collected in sample inactivation medium and measured using the CoV2Ag assay. The nasopharyngeal swab specimens were measured by RT-PCR. Additionally, 9,046 swab specimens obtained for screening purposes in a tertiary care hospital were analyzed and positive CoV2Ag results confirmed by NAT. RESULTS: In total, 234/447 (52.3%) participants of the public test center were positive for SARS-CoV-2-RNA. Viral lineage B1.1.529 was dominant during the study. Sensitivity and specificity of the CoV2Ag assay were 88.5% (95%CI: 83.7-91.9%) and 99.5% (97.4-99.9%), respectively. Sensitivity increased to 93.7% (97.4-99.9%) and 98.7% (97.4-99.9%) for swab specimens with cycle threshold values <30 and <25, respectively. Out of 9,046 CoV2Ag screening tests from hospitalized patients, 21 (0.2%) swab specimens were determined as false-positive by confirmatory NAT. CONCLUSIONS: Using sample tubes containing inactivation medium the laboratory-based high-throughput CoV2Ag assay is a very specific and highly sensitive assay for detection of SARS-CoV-2 antigen in nasal swab specimens including the B1.1.529 variant. In low prevalence settings confirmation of positive CoV2Ag results by SARS-CoV-2-RNA testing is recommended.

Host genetic loci LZTFL1 and CCL2 associated with SARS-CoV-2 infection and severity of COVID-19.

OBJECTIVES: Host genetic factors contribute to the variable severity of COVID-19. We examined genetic variants from genome-wide association studies and candidate gene association studies in a cohort of patients with COVID-19 and investigated the role of early SARS-CoV-2 strains in COVID-19 severity. METHODS: This case-control study included 123 COVID-19 cases (hospitalized or ambulatory) and healthy controls from the state of Baden-Wuerttemberg, Germany. We genotyped 30 single nucleotide polymorphisms, using a custom-designed panel. Cases were also compared with the 1000 genomes project. Polygenic risk scores were constructed. SARS-CoV-2 genomes from 26 patients with COVID-19 were sequenced and compared between ambulatory and hospitalized cases, and phylogeny was reconstructed. RESULTS: Eight variants reached nominal significance and two were significantly associated with at least one of the phenotypes "susceptibility to infection", "hospitalization", or "severity": rs73064425 in LZTFL1 (hospitalization and severity, P <0.001) and rs1024611 near CCL2 (susceptibility, including 1000 genomes project, P = 0.001). The polygenic risk score could predict hospitalization. Most (23/26, 89%) of the SARS-CoV-2 genomes were classified as B.1 lineage. No associations of SARS-CoV-2 mutations or lineages with severity were observed. CONCLUSION: These host genetic markers provide insights into pathogenesis and enable risk classification. Variants which reached nominal significance should be included in larger studies.

High prevalence of Pseudomonas aeruginosa carriage in residents of French and German long-term care facilities.

OBJECTIVES: To determine prevalence, incidence, and factors associated with Pseudomonas aeruginosa (PA) intestinal carriage in residents of long-term care facilities (LTCFs) and to understand the population structure of this pathogen in LTCFs from two European countries. METHODS: We assessed the prevalence of PA intestinal carriage and the incidence of acquisition by collecting fecal samples from 403 residents of 20 LTCFs. We collected 289 environmental samples from sinks and drinking water. Factors associated with carriage and acquisition of intestinal PA were identified. All PA isolates had their antibiotic phenotypic resistance profile determined and their genome sequenced, from which we assessed the population structure of the collection and identified resistance determinants. RESULTS: We found a high proportion of residents with PA intestinal carriage (51.6%) over the entire study period. Over the follow-up period, 28.6% of the residents acquired intestinal PA. Older age (OR, 1.29; 95% CI, 1.09-1.52; p = 0.002), urinary incontinence (OR, 2.56; 95% CI, 1.37-4.88; p = 0.003), and male sex (OR, 2.55; 95% CI, 1.05-6.18; p = 0.039) were associated with higher probability of carriage. Wheelchair usage (OR, 4.56; 95% CI, 1.38-15.05; p = 0.013) and a body mass index >25 (OR, 3.71; 95% CI, 1.17-11.82; p = 0.026) were associated with higher risk of PA acquisition. Population structure of our isolates was mainly non-clonal with 112 different STs among the 241 isolates. Most represented STs were high risk clones ST253 (n = 26), ST17 (n = 11), ST244 (n = 11), ST309 (n = 10), and ST395 (n = 10). Most PA isolates (86.3%) were susceptible to antibiotics, with no acquired genes conferring resistance to antipseudomonal agents. DISCUSSION: We found an unexpected high prevalence of PA intestinal carriage in LTCF residents mainly associated with individual-level factors. Our study revealed a polyclonal PA population structure suggesting that individual acquisition is more frequent than resident-to-resident transmission.

Signature of Alzheimer's Disease in Intestinal Microbiome: Results From the AlzBiom Study.

Background: Changes in intestinal microbiome composition have been described in animal models of Alzheimer's disease (AD) and AD patients. Here we investigated how well taxonomic and functional intestinal microbiome data and their combination with clinical data can be used to discriminate between amyloid-positive AD patients and cognitively healthy elderly controls. Methods: In the present study we investigated intestinal microbiome in 75 amyloid-positive AD patients and 100 cognitively healthy controls participating in the AlzBiom study. We randomly split the data into a training and a validation set. Intestinal microbiome was measured using shotgun metagenomics. Receiver operating characteristic (ROC) curve analysis was performed to examine the discriminatory ability of intestinal microbiome among diagnostic groups. Results: The best model for discrimination of amyloid-positive AD patients from healthy controls with taxonomic data was obtained analyzing 18 genera features, and yielded an area under the receiver operating characteristic curve (AUROC) of 0.76 in the training set and 0.61 in the validation set. The best models with functional data were obtained analyzing 17 GO (Gene Ontology) features with an AUROC of 0.81 in the training set and 0.75 in the validation set and 26 KO [Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog] features with an AUROC of 0.83 and 0.77, respectively. Using ensemble learning for these three models including a clinical model with the 4 parameters age, gender, BMI and ApoE yielded an AUROC of 0.92 in the training set and 0.80 in the validation set. Discussion: In conclusion, we identified a specific Alzheimer signature in intestinal microbiome that can be used to discriminate amyloid-positive AD patients from healthy controls. The diagnostic accuracy increases from taxonomic to functional data and is even better when combining taxonomic, functional and clinical models. Intestinal microbiome represents an innovative diagnostic supplement and a promising area for developing novel interventions against AD.

Surveillance of Enterobacter cloacae complex colonization and comparative analysis of different typing methods on a neonatal intensive care unit in Germany.

BACKGROUND: Enterobacter cloacae complex is a group of common opportunistic pathogens on neonatal intensive care units. Active microbiological screening to guide empirical antimicrobial treatment or to detect transmission events is recommended in high-risk preterm neonates. A rise in colonization with E. cloacae complex was observed in a German perinatal centre. The aim of this study was to evaluate the performance of different typing techniques using whole genome sequencing (WGS) as a reference. METHODS: Enterobacter cloacae complex isolates from clinical and screening specimens with an epidemiological link to the neonatal intensive care units were further assessed. Identification and antibiotic susceptibility testing was performed by a combination of VITEK2 (bioMérieux) and MALDI-TOF (Bruker Daltonics), followed by RAPD/rep-PCR and PFGE (XbaI). Retrospectively, all isolates were analyzed by Fourier-transform infrared (FTIR) spectroscopy (IR Biotyper, Bruker Daltonics). Whole genome sequencing with SNP-based clustering was used as the reference method. Furthermore, resistome analysis, sequence type and species identification were derived from the WGS data. Transmission analysis was based on epidemiological and typing data. RESULTS: Between September 2017 and March 2018 32 mostly preterm neonates were found to be colonized with E. cloacae complex and 32 isolates from 24 patients were available for further typing. RAPD/rep-PCR and PFGE showed good concordance with WGS whereas FTIR displayed mediocre results [adjusted rand index (ARI) = 0.436]. A polyclonal increase and two dominant and overlapping clonal clusters of two different E. hormaechei subspecies were detected. Overall, four different species were identified. Genotyping confirmed third-generation cephalosporin resistance development in isolates of the same patient. During the six-month period several infection prevention interventions were performed and no E. cloacae complex isolates were observed during the following months. CONCLUSIONS: Interpretation of the microbiological results alone to detect transmission events is often challenging and bacterial typing is of utmost importance to implement targeted infection control measures in an epidemic occurrence of E. cloacae complex. WGS is the most discriminatory method. However, traditional methods such as PFGE or RAPD/rep-PCR can provide reliable and quicker results in many settings. Furthermore, research is needed to quickly identify E. cloacae complex to the species level in the microbiological laboratory.

A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes.

Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. KEY POINTS: • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin.

A chemical reinvestigation of the Indonesian strain Streptomyces sp. SHP 22-7 led to the isolation of three new pyrimidine nucleosides, along with six known analogues and zincphyrin. The structures of the new compounds (6, 7, 10) were elucidated by employing spectroscopic techniques (NMR, MS, CD, and IR) as well as enantioselective analyses of methyl branched side chain configurations. Application of the precursor-directed feeding approach led to the production and partial isolation of nine further pyrimidine analogues. The new compounds 6, 7, and 11 and three of the known compounds (2-4) were found to possess antimycobacterial and cytotoxic properties.

Impact of single-room contact precautions on acquisition and transmission of vancomycin-resistant enterococci on haematological and oncological wards, multicentre cohort-study, Germany, January-December 2016.

BackgroundEvidence supporting the effectiveness of single-room contact precautions (SCP) in preventing in-hospital acquisition of vancomycin-resistant enterococci (haVRE) is limited.AimWe assessed the impact of SCP on haVRE and their transmission.MethodsWe conducted a prospective, multicentre cohort study in German haematological/oncological departments during 2016. Two sites performed SCP for VRE patients and two did not (NCP). We defined a 5% haVRE-risk difference as non-inferiority margin, screened patients for VRE, and characterised isolates by whole genome sequencing and core genome MLST (cgMLST). Potential confounders were assessed by competing risk regression analysis.ResultsWe included 1,397 patients at NCP and 1,531 patients at SCP sites. Not performing SCP was associated with a significantly higher proportion of haVRE; 12.2% (170/1,397) patients at NCP and 7.4% (113/1,531) patients at SCP sites (relative risk (RR) 1.74; 95% confidence interval (CI): 1.35-2.23). The difference (4.8%) was below the non-inferiority margin. Competing risk regression analysis indicated a stronger impact of antimicrobial exposure (subdistribution hazard ratio (SHR) 7.46; 95% CI: 4.59-12.12) and underlying disease (SHR for acute leukaemia 2.34; 95% CI: 1.46-3.75) on haVRE than NCP (SHR 1.60; 95% CI: 1.14-2.25). Based on cgMLST and patient movement data, we observed 131 patient-to-patient VRE transmissions at NCP and 85 at SCP sites (RR 1.76; 95% CI: 1.33-2.34).ConclusionsWe show a positive impact of SCP on haVRE in a high-risk population, although the observed difference was below the pre-specified non-inferiority margin. Importantly, other factors including antimicrobial exposure seem to be more influential.