Critical Assessment of Metagenome Interpretation: the second round of challenges.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

Etiology of nosocomial infections in intensive care patients in German hospitals: An analysis of trends between 2008 and 2022.

PURPOSE: Data from the intensive care component of the German hospital infection surveillance system (KISS) was used to investigate the epidemiology of pathogens responsible for the most frequent device-associated infections and their development over time. METHOD: The 10 most common pathogens were identified for ventilator-associated lower respiratory tract infections (VALRTI), catheter associated urinary tract infections (CAUTI), and central venous catheter associated bloodstream infections (CVC-BSI). The development over time was analyzed based on three five-year time periods: 2008-2012, 2013-2017, 2018-2022. RESULTS: Data from 1425 ICUs were included together with 121,762 device-associated infections with 138,299 isolated pathogens. A remarkable and significant increase in the frequency of Klebsiella spp. was found for VALRTI, that was almost twice as high during 2018-2022 compared to 2008-2012. For CAUTI, there was a significant increase of all Enterobacterales with the most prominent increase in Klebsiella spp. With regard to CVC-BSI, the situation for coagulase-negative staphylococci and E. coli was relatively stable; while there was a significant increase in Enterococcus spp. and Klebsiella spp. and a decrease in S. aureus. CONCLUSION: Knowledge about the current frequency of pathogens responsible for nosocomial infections in intensive care units is important for guiding empirical antimicrobial therapy. Data from national nosocomial infection surveillance systems can provide relevant information about the development of pathogens.

Risk factors for transmission of carbapenem-resistant Acinetobacter baumannii in outbreak situations: results of a case-control study.

BACKGROUND: An increase in patients with multidrug-resistant organisms and associated outbreaks during the COVID-19 pandemic have been reported in various settings, including low-endemic settings. Here, we report three distinct carbapenem-resistant Acinetobacter baumannii (CRAB) outbreaks in five intensive care units of a university hospital in Berlin, Germany during the COVID-19 pandemic. METHODS: A case-control study was conducted with the objective of identifying risk factors for CRAB acquisition in outbreak situations. Data utilized for the case-control study came from the investigation of three separate CRAB outbreaks during the COVID-19 pandemic (August 2020- March 2021). Cases were defined as outbreak patients with hospital-acquired CRAB. Controls did not have any CRAB positive microbiological findings and were hospitalized at the same ward and for a similar duration as the respective case. Control patients were matched retrospectively in a 2:1 ratio. Parameters routinely collected in the context of outbreak management and data obtained retrospectively specifically for the case-control study were included in the analysis. To analyze risk factors for CRAB acquisition, univariable and multivariable analyses to calculate odds ratios (OR) and 95% confidence intervals (CI) were performed using a conditional logistic regression model. RESULTS: The outbreaks contained 26 cases with hospital-acquired CRAB in five different intensive care units. Two exposures were identified to be independent risk factors for nosocomial CRAB acquisition by the multivariable regression analysis: Sharing a patient room with a CRAB patient before availability of the microbiological result was associated with a more than tenfold increase in the risk of nosocomial CRAB acquisition (OR: 10.7, CI: 2.3-50.9), while undergoing bronchoscopy increased the risk more than six times (OR: 6.9, CI: 1.3-38.1). CONCLUSIONS: The risk factors identified, sharing a patient room with a CRAB patient and undergoing bronchoscopy, could point to an underperformance of basic infection control measure, particularly hand hygiene compliance and handling of medical devices. Both findings reinforce the need for continued promotion of infection control measures. Given that the outbreaks occurred in the first year of the COVID-19 pandemic, our study serves as a reminder that a heightened focus on airborne precautions should not lead to a neglect of other transmission-based precautions.

The transmission risk of multidrug-resistant organisms between hospital patients and their pets - a case-control study, Germany, 2019 to 2022.

BackgroundCarriage of multidrug-resistant organisms (MDROs) in humans constitutes an important public health concern. Cross-transmission of bacteria between animals and humans has been demonstrated before.AimOur aim was to quantify the risk factor 'pet ownership' for MDRO colonisation in hospital patients.MethodsWe performed a matched case-control study from 2019 to 2022 in Berlin, Germany and compared MDRO-positive and MDRO-negative patients in terms of contact with pets and other risk factors for MDRO acquisition. Patients completed a questionnaire-based interview and provided nasal and rectal swabs. Pet owners provided swab samples from the throat and stool of their pets (dogs and cats). Phenotypically matching samples of owners and pets were analysed via whole genome sequencing.ResultsThe analyses included 2,891 patients. Reported pet ownership was 17.7% in MDRO-positives (154/871) and 23.4% in MDRO-negatives (472/2,020). Among 397 owner-pet pairs, we identified one pair sharing genotypically indistinguishable pathogens (0.3%). A risk factor analysis of pet ownership was performed for carriers of meticillin-resistant Staphylococcus aureus (MRSA) (OR = 0.662; 95% CI: 0.343-1.277), vancomycin-resistant enterococci (VRE) (OR = 0.764; 95% CI: 0.522-1.118) and multidrug-resistant Gram-negative bacteria (MDR-GNB) (OR = 0.819; 95% CI: 0.620-1.082). Colonisation with MDRO was rare in pets, and dogs were more often colonised than cats (MRSA: 0% vs 0%, VRE: 1.5% vs 1.0%, MDR-GNB: 17.2% vs 3.6%).ConclusionTransmission of MDROs between humans and pets is possible though rare. In an urban living space, neither cat nor dog ownership appears as a relevant risk factor for MDRO carriage in hospital patients.

CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

Relevance of intra-hospital patient movements for the spread of healthcare-associated infections within hospitals - a mathematical modeling study.

The aim of this study is to analyze patient movement patterns between hospital departments to derive the underlying intra-hospital movement network, and to assess if movement patterns differ between patients at high or low risk of colonization. For that purpose, we analyzed patient electronic medical record data from five hospitals to extract information on risk stratification and patient intra-hospital movements. Movement patterns were visualized as networks, and network centrality measures were calculated. Next, using an agent-based model where agents represent patients and intra-hospital patient movements were explicitly modeled, we simulated the spread of multidrug resistant enterobacteriacae (MDR-E) inside a hospital. Risk stratification of patients according to certain ICD-10 codes revealed that length of stay, patient age, and mean number of movements per admission were higher in the high-risk groups. Movement networks in all hospitals displayed a high variability among departments concerning their network centrality and connectedness with a few highly connected departments and many weakly connected peripheral departments. Simulating the spread of a pathogen in one hospital network showed positive correlation between department prevalence and network centrality measures. This study highlights the importance of intra-hospital patient movements and their possible impact on pathogen spread. Targeting interventions to departments of higher (weighted) degree may help to control the spread of MDR-E. Moreover, when the colonization status of patients coming from different departments is unknown, a ranking system based on department centralities may be used to design more effective interventions that mitigate pathogen spread.

Prevalence of third-generation cephalosporin-resistant Enterobacterales colonization on hospital admission and ESBL genotype-specific risk factors: a cross-sectional study in six German university hospitals.

OBJECTIVES: To assess the admission prevalence of third-generation cephalosporin-resistant Enterobacterales (3GCREB) and to assess whether risk factors vary by ß-lactamase genotype. METHODS: Adult patients were recruited within 72 h of admission to general wards of six university hospitals in 2014 and 2015. Rectal swabs were screened for 3GCREB and isolates were analysed phenotypically and genotypically. Patients were questioned on potential risk factors. Multivariable analyses were performed to identify risk factors for 3GCREB colonization and for specific ß-lactamases. RESULTS: Of 8753 patients screened, 828 were 3GCREB positive (9.5%). Eight hundred and thirteen isolates were available for genotyping. CTX-M-15 was the most common ESBL (38.0%), followed by CTX-M-1 (22.5%), CTX-M-14 (8.7%), CTX-M-27 (7.5%) and SHV-ESBL (4.4%). AmpC was found in 11.9%. Interestingly, 18 Escherichia coli isolates were AmpC positive, 12 of which (67%) contained AmpC on a gene of plasmid origin [CMY (n = 10), DHA (n = 2)]. Risk factors for 3GCREB colonization varied by genotype. Recent antibiotic exposure and prior colonization by antibiotic-resistant bacteria were risk factors for all ß-lactamases except CTX-M-14 and CTX-M-27. Travel outside Europe was a risk factor for CTX-M-15 and CTX-M-27 [adjusted OR (aOR) 3.49, 95% CI 2.88-4.24 and aOR 2.73, 95% CI 1.68-4.43]. A previous stay in a long-term care facility was associated with CTX-M-14 (aOR 3.01, 95% CI 1.98-4.59). A preceding hospital stay in Germany increased the risk of CTX-M-15 (aOR 1.27, 95% CI 1.14-1.41), while a prior hospital stay in other European countries increased the risk of SHV-ESBL colonization (aOR 3.85, 95% CI 1.67-8.92). CONCLUSIONS: The detection of different ESBL types is associated with specific risk factor sets that might represent distinct sources of colonization and ESBL-specific dissemination routes.

Vancomycin-resistant Enterococcus faecium colonizing patients on hospital admission in Germany: prevalence and molecular epidemiology.

OBJECTIVES: To analyse the rectal carriage rate and the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) recovered from patients upon hospital admission. METHODS: Adult patients were screened at six German university hospitals from five different federal states upon hospital admission for rectal colonization with VREfm between 2014 and 2018. Molecular characterization of VREfm was performed by WGS followed by MLST and core-genome MLST analysis. RESULTS: Of 16350 patients recruited, 263 were colonized with VREfm, with increasing prevalence rates during the 5 year study period (from 0.8% to 2.6%). In total, 78.5% of the VREfm were vanB positive and 20.2% vanA positive, while 1.2% harboured both vanA and vanB. The predominant ST was ST117 (56.7%) followed by ST80 (15%), ST203 (10.9%), ST78 (5.7%) and ST17 (3.2%). ST117/vanB VREfm isolates formed a large cluster of 96 closely related isolates extending across all six study centres and four smaller clusters comprising 13, 5, 4 and 3 isolates each. In contrast, among the other STs inter-regional clonal relatedness was rarely observed. CONCLUSIONS: To our knowledge, this is the largest admission prevalence and molecular epidemiology study of VREfm. These data provide insight into the epidemiology of VREfm at six German university hospitals and demonstrate the remarkable inter-regional clonal expansion of the ST117/vanB VREfm clone.

Emergence of ESBL-producing Escherichia coli ST131-C1-M27 clade colonizing patients in Europe.

Background: The ST131 Escherichia coli clone is associated with the global dissemination of ESBLs. It has been hypothesized that ST131 could take advantage of better colonizing abilities. However, the data on colonization prevalence of ESBL-ST131 in European hospitals are scarce. Objectives: To assess the prevalence of the ST131 clone and its microbiological characteristics among colonizing ESBL-producing E. coli (ESBL-Ec) from hospitalized patients in four European hospitals (Berlin, Geneva, Madrid and Utrecht) during the R-GNOSIS study. Methods: ESBL-Ec isolates (n = 688) were obtained from rectal swabs of hospitalized patients from March 2014 to February 2015 using selective media. The ST131 clone and its subclones were sought using PCR and positive isolates were further studied. blaESBL genes were characterized (PCR and sequencing), antibiotic susceptibility testing was performed, clonal relationships were studied by PFGE and fimH allele and O type (PCR) were assessed. Results: ST131 prevalence was 20.5% (141/688); C1/H30R1 isolates were significantly more prevalent in Geneva (49%) and C2/H30Rx in Madrid (67%). C1/H30R1 isolates showed less resistance to amikacin than C2/H30Rx (4% versus 35%) and all were susceptible to penicillin/inhibitor combinations. CTX-M-15 was the most common enzyme (49%) followed by CTX-M-27 (27%). C1/H30R1 isolates were significantly associated with CTX-M-27 (72%) and all of these isolates belonged to the C1-M27 clade. Moreover, C2/H30Rx isolates and CTX-M-15 were also significantly related (88%). Conclusions: The predominance of C2/H30Rx-CTX-M-15 in Madrid and C1/H30R1-CTX-M-27 in Geneva demonstrates a changing epidemiology of ESBLs in Europe caused by ST131 subclones; in particular, the emergence of the C1-M27 clade in Europe.

Survey of Clostridium difficile infection surveillance systems in Europe, 2011.

To develop a European surveillance protocol for Clostridium difficile infection (CDI), existing national CDI surveillance systems were assessed in 2011. A web-based electronic form was provided for all national coordinators of the European CDI Surveillance Network (ECDIS-Net). Of 35 national coordinators approached, 33 from 31 European countries replied. Surveillance of CDI was in place in 14 of the 31 countries, comprising 18 different nationwide systems. Three of 14 countries with CDI surveillance used public health notification of cases as the route of reporting, and in another three, reporting was limited to public health notification of cases of severe CDI. The CDI definitions published by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Centre for Disease Prevention and Control (ECDC) were widely used, but there were differing definitions to distinguish between community- and healthcare-associated cases. All CDI surveillance systems except one reported annual national CDI rates (calculated as number of cases per patient-days). Only four surveillance systems regularly integrated microbiological data (typing and susceptibility testing results). Surveillance methods varied considerably between countries, which emphasises the need for a harmonised European protocol to allow consistent monitoring of the CDI epidemiology at European level. The results of this survey were used to develop a harmonised EU-wide hospital-based CDI surveillance protocol.

Standardised surveillance of Clostridium difficile infection in European acute care hospitals: a pilot study, 2013.

Clostridium difficile infection (CDI) remains poorly controlled in many European countries, of which several have not yet implemented national CDI surveillance. In 2013, experts from the European CDI Surveillance Network project and from the European Centre for Disease Prevention and Control developed a protocol with three options of CDI surveillance for acute care hospitals: a 'minimal' option (aggregated hospital data), a 'light' option (including patient data for CDI cases) and an 'enhanced' option (including microbiological data on the first 10 CDI episodes per hospital). A total of 37 hospitals in 14 European countries tested these options for a three-month period (between 13 May and 1 November 2013). All 37 hospitals successfully completed the minimal surveillance option (for 1,152 patients). Clinical data were submitted for 94% (1,078/1,152) of the patients in the light option; information on CDI origin and outcome was complete for 94% (1,016/1,078) and 98% (294/300) of the patients in the light and enhanced options, respectively. The workload of the options was 1.1, 2.0 and 3.0 person-days per 10,000 hospital discharges, respectively. Enhanced surveillance was tested and was successful in 32 of the hospitals, showing that C. difficile PCR ribotype 027 was predominant (30% (79/267)). This study showed that standardised multicountry surveillance, with the option of integrating clinical and molecular data, is a feasible strategy for monitoring CDI in Europe.

Deep transcriptome profiling of clinical Klebsiella pneumoniae isolates reveals strain and sequence type-specific adaptation.

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiella pneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait.

The mother as most important risk factor for colonization of very low birth weight (VLBW) infants with extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E).

OBJECTIVES: This study aimed to determine the prevalence of and risk factors for colonization with extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) and methicillin-resistant Staphylococcus aureus (MRSA) in very low birth weight (VLBW; <1500 g) infants and their mothers. METHODS: This investigation was conducted in the perinatal centre at the Charité Berlin between May 2012 and June 2013. VLBW infants and their mothers were screened for colonization with ESBL-E and MRSA. Demographic and clinical data were obtained from the German nationwide surveillance system for nosocomial infections in VLBW infants (NEO-KISS) and used to perform univariate and multivariate analyses. RESULTS: Of 209 VLBW infants, 12 (5.7%) were colonized with ESBL-E. Eighteen of 209 (8.6%) ESBL-E-tested neonates were related to an ESBL-E-positive mother. Univariate analysis, strain typing and multivariate analysis (OR 7.4, 95% CI 2.1-26.7, P = 0.002) identified an ESBL-E-positive mother and maternal-neonatal transmission as a main source of colonization. The prevalence of MRSA was 2.3% (5 of 221) among VLBW infants. One of the 221 (0.5%) MRSA-tested neonates was related to an MRSA-positive mother. No risk factors for transmission of MRSA could be detected in this study. CONCLUSIONS: Our study demonstrated that maternal-neonatal transmission of ESBL-E from mother to child is an important risk factor for colonization of VLBW infants. As a consequence, routine ESBL-E screening of neonates and mothers should be considered as a means of reducing neonatal morbidity and mortality.

CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence?

OBJECTIVES: To discern the relevance of ST648 extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. METHODS: ESBL-producing E. coli isolates (n = 1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typing was performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. RESULTS: Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1, -3, -14, -15 and -61 versus CTX-M-1,-2,-14,-15,-27 and -55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-ß-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. CONCLUSIONS: Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.

The genetic relationship between human and pet isolates: a core genome multilocus sequence analysis of multidrug-resistant bacteria.

INTRODUCTION: The global increase of multidrug-resistant organisms (MDROs) is one of the most urgent public health threats affecting both humans and animals. The One Health concept emphasizes the interconnectedness of human, animal and environmental health and highlights the need for integrated approaches to combat antimicrobial resistance (AMR). Although the sharing of environments and antimicrobial agents between companion animals and humans poses a risk for MDRO transmission, companion animals have been studied to a lesser extent than livestock animals. This study therefore used core genome multilocus sequence typing (cgMLST) to investigate the genetic relationships and putative transmission of MDROs between humans and pets. METHODS: This descriptive integrated typing study included 252 human isolates, 53 dog isolates and 10 cat isolates collected from 2019 to 2022 at the Charité University Hospital in Berlin, Germany. CgMLST was performed to characterize methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant gram-negative bacteria. The genetic diversity of the MDROs of the different host populations was determined and compared based on sequence type and core genome complex type. RESULTS: Within this study the majority of samples from pets and humans was genetically distinct. However, for some isolates, the number of allelic differences identified by cgMLST was low. Two cases of putative household transmission or shared source of VR E. faecium and MDR E. coli between humans and pets were documented. CONCLUSIONS: The interaction between humans and their pets appears to play a minor role in the spread of the MDROs studied. However, further research is needed. This study emphasizes the importance of comprehensive molecular surveillance and a multidisciplinary One Health approach to understand and contain the spread of MDROs in human and animal populations. TRIAL REGISTRATION: The study is registered with the German Clinical Trials Register (DRKS00030009).

Split k-mer analysis compared to cgMLST and SNP-based core genome analysis for detecting transmission of vancomycin-resistant enterococci: results from routine outbreak analyses across different hospitals and hospitals networks in Berlin, Germany.

The increase of Vancomycin-resistant Enterococcus faecium (VREfm) in recent years has been partially attributed to the rise of specific clonal lineages, which have been identified throughout Germany. To date, there is no gold standard for the interpretation of genomic data for outbreak analyses. New genomic approaches such as split k-mer analysis (SKA) could support cluster attribution for routine outbreak investigation. The aim of this project was to investigate frequent clonal lineages of VREfm identified during suspected outbreaks across different hospitals, and to compare genomic approaches including SKA in routine outbreak investigation. We used routine outbreak laboratory data from seven hospitals and three different hospital networks in Berlin, Germany. Short-read libraries were sequenced on the Illumina MiSeq system. We determined clusters using the published Enterococcus faecium-cgMLST scheme (threshold ≤20 alleles), and assigned sequence and complex types (ST, CT), using the Ridom SeqSphere+ software. For each cluster as determined by cgMLST, we used pairwise core-genome SNP-analysis and SKA at thresholds of ten and seven SNPs, respectively, to further distinguish cgMLST clusters. In order to investigate clinical relevance, we analysed to what extent epidemiological linkage backed the clusters determined with different genomic approaches. Between 2014 and 2021, we sequenced 693 VREfm strains, and 644 (93 %) were associated within cgMLST clusters. More than 74 % (n=475) of the strains belonged to the six largest cgMLST clusters, comprising ST117, ST78 and ST80. All six clusters were detected across several years and hospitals without apparent epidemiological links. Core SNP analysis identified 44 clusters with a median cluster size of three isolates (IQR 2-7, min-max 2-63), as well as 197 singletons (41.4 % of 475 isolates). SKA identified 67 clusters with a median cluster size of two isolates (IQR 2-4, min-max 2-19), and 261 singletons (54.9 % of 475 isolates). Of the isolate pairs attributed to clusters, 7 % (n=3064/45 596) of pairs in clusters determined by standard cgMLST, 15 % (n=1222/8500) of pairs in core SNP-clusters and 51 % (n=942/1880) of pairs in SKA-clusters showed epidemiological linkage. The proportion of epidemiological linkage differed between sequence types. For VREfm, the discriminative ability of the widely used cgMLST based approach at ≤20 alleles difference was insufficient to rule out hospital outbreaks without further analytical methods. Cluster assignment guided by core genome SNP analysis and the reference free SKA was more discriminative and correlated better with obvious epidemiological linkage, at least recently published thresholds (ten and seven SNPs, respectively) and for frequent STs. Besides higher overall discriminative power, the whole-genome approach implemented in SKA is also easier and faster to conduct and requires less computational resources.

Validation of a New PCR-Based Screening Method for Prevention of Serratia marcescens Outbreaks in the Neonatal Intensive Care Unit.

BACKGROUND: Serratia marcescens may cause severe nosocomial infections, mostly in very low birth weight infants. Since S. marcescens exhibits by far the highest adjusted incidence rate for horizontal transmission, it can cause complex outbreak situations in neonatal intensive care units. OBJECTIVE: The aim of this study was to establish a fast and highly sensitive colonization screening for prompt cohorting and barrier nursing strategies. METHODS: A probe-based duplex PCR assay targeting the 16S rRNA gene of S. marcescens was developed and validated by using 36 reference strains, 14 S. marcescens outbreak- and nonoutbreak isolates, defined by epidemiological linkage and molecular typing, and applied in 1,347 clinical specimens from 505 patients. RESULTS AND CONCLUSIONS: The novel PCR assay proved to be highly specific and had an in vitro sensitivity of 100 gene copies per reaction (∼15 bacteria). It showed a similar (in laryngeal/tracheal specimens) or even higher (in rectal/stoma swabs) in vivo sensitivity in comparison to routine microbial culture and was much quicker (<24 h vs. 2 days). By combining different oligonucleotide primers, there was robust detection of genetic variants of S. marcescens strains. PCR inhibition was low (1.6%) and observed with rectal swabs only. Cohort analysis illustrated applicability of the PCR assay as a quick tool to prevent outbreak scenarios by allowing rapid decisions on cohorting and barrier nursing. In summary, this novel molecular screening for colonization by S. marcescens is specific, highly sensitive, and substantially accelerates detection.

Screening for Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become less common in Germany in recent years. In this paper, we report data from the MRSA module of the Hospital Infection Surveillance System (Krankenhaus-Infektionen- Surveillance-System, KISS) for the years 2006-2021. We also describe the association of MRSA rates with the frequency of patient screening for MRSA and discuss the findings. METHODS: Participation in the MRSA KISS module is voluntary. Once a year, the participating hospitals submit structural data, information on cases in which MRSA was detected (both colonizations and infections; both detected on admission and nosocomially acquired), and the number of nasal swabs taken for the detection of MRSA to the German National Reference Center for the Surveillance of Nosocomial Infections. Statistical analyses were performed with R software. RESULTS: The number of hospitals participating in the MRSA module rose from 110 in 2006 to 525 in 2021. From 2006 onward, the overall MRSA prevalence in German hospitals increased, reaching a maximum of 1.04 cases per 100 patients in 2012. The prevalence on admission fell by 44% from 0.96 in 2016 to 0.54 in 2021. The incidence density of nosocomial MRSA fell by an average of 12% per year, from 0.27 per 1000 patient-days in 2006 to 0.06 in 2021, while MRSA screening frequency increased sevenfold by 2021. The nosocomial incidence density was stable, independently of the screening frequency. CONCLUSION: MRSA rates in German hospitals fell markedly from 2006 to 2021, reflecting a general trend. The incidence density was no higher in hospitals with a low or moderate screening frequency than in those with a high one. Thus, a targeted, riskadapted MRSA screening strategy on hospital admission can be recommended.

Quantification of time delay between screening and subsequent initiation of contact isolation for carriers of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales: A post hoc subgroup analysis of the R-GNOSIS WP5 Trial.

OBJECTIVES: The aim of this study was to quantify the time delay between screening and initiation of contact isolation for carriers of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-E). METHODS: This study was a secondary analysis of contact isolation periods in a cluster-randomized controlled trial that compared 2 strategies to control ESBL-E (trial no. ISRCTN57648070). Patients admitted to 20 non-ICU wards in Germany, the Netherlands, Spain, and Switzerland were screened for ESBL-E carriage on admission, weekly thereafter, and on discharge. Data collection included the day of sampling, the day the wards were notified of the result, and subsequent ESBL-E isolation days. RESULTS: Between January 2014 and August 2016, 19,122 patients, with a length of stay ≥2 days were included. At least 1 culture was collected for 16,091 patients (84%), with a median duration between the admission day and the day of first sample collection of 2 days (interquartile range [IQR], 1-3). Moreover, 854 (41%) of all 2,078 ESBL-E carriers remained without isolation during their hospital stay. In total, 6,040 ESBL-E days (32% of all ESBL-E days) accrued for patients who were not isolated. Of 2,078 ESBL-E-carriers, 1,478 ESBL-E carriers (71%) had no previous history of ESBL-E carriage. Also, 697 (34%) were placed in contact isolation with a delay of 4 days (IQR, 2-5), accounting for 2,723 nonisolation days (15% of ESBL-E days). CONCLUSIONS: Even with extensive surveillance screening, almost one-third of all ESBL-E days were nonisolation days. Limitations in routine culture-based ESBL-E detection impeded timely and exhaustive implementation of targeted contact isolation.

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.