An appeal for strengthening genomic pathogen surveillance to improve pandemic preparedness and infection prevention: the German perspective.

The SARS-CoV-2 pandemic has highlighted the importance of viable infection surveillance and the relevant infrastructure. From a German perspective, an integral part of this infrastructure, genomic pathogen sequencing, was at best fragmentary and stretched to its limits due to the lack or inefficient use of equipment, human resources, data management and coordination. The experience in other countries has shown that the rate of sequenced positive samples and linkage of genomic and epidemiological data (person, place, time) represent important factors for a successful application of genomic pathogen surveillance. Planning, establishing and consistently supporting adequate structures for genomic pathogen surveillance will be crucial to identify and combat future pandemics as well as other challenges in infectious diseases such as multi-drug resistant bacteria and healthcare-associated infections. Therefore, the authors propose a multifaceted and coordinated process for the definition of procedural, legal and technical standards for comprehensive genomic pathogen surveillance in Germany, covering the areas of genomic sequencing, data collection and data linkage, as well as target pathogens. A comparative analysis of the structures established in Germany and in other countries is applied. This proposal aims to better tackle epi- and pandemics to come and take action from the "lessons learned" from the SARS-CoV-2 pandemic.

[Establishment of genomic pathogen surveillance to strengthen pandemic preparedness and infection prevention in Germany]. / Etablierung der Genomischen Erreger-Surveillance zur Stärkung des Pandemie- und Infektionsschutzes in Deutschland.

The SARS-CoV­2 pandemic has shown a deficit of essential epidemiological infrastructure, especially with regard to genomic pathogen surveillance in Germany. In order to prepare for future pandemics, the authors consider it urgently necessary to remedy this existing deficit by establishing an efficient infrastructure for genomic pathogen surveillance. Such a network can build on structures, processes, and interactions that have already been initiated regionally and further optimize them. It will be able to respond to current and future challenges with a high degree of adaptability.The aim of this paper is to address the urgency and to outline proposed measures for establishing an efficient, adaptable, and responsive genomic pathogen surveillance network, taking into account external framework conditions and internal standards. The proposed measures are based on global and country-specific best practices and strategy papers. Specific next steps to achieve an integrated genomic pathogen surveillance include linking epidemiological data with pathogen genomic data; sharing and coordinating existing resources; making surveillance data available to relevant decision-makers, the public health service, and the scientific community; and engaging all stakeholders. The establishment of a genomic pathogen surveillance network is essential for the continuous, stable, active surveillance of the infection situation in Germany, both during pandemic phases and beyond.

[Therapy-relevant antibiotic resistances in a One Health context]. / Therapierelevante Antibiotikaresistenzen im One-Health-Kontext.

One Health refers to a concept that links human, animal, and environmental health. In Germany, there is extensive data on antibiotic resistance (AMR) and multidrug-resistant (micro)organisms (MDRO) in human and veterinary medicine, as well as from studies in various environmental compartments (soil, water, wastewater). All these activities are conducted according to different specifications and standards, which makes it difficult to compare data. A focus on AMR and MDRO of human therapeutic importance is helpful to provide some guidance. Most data are available across sectors on methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant Enterobacterales such as Escherichia coli and Klebsiella pneumoniae. Here, the trends of resistance are heterogeneous. Antibiotic use leads to MRE selection, which is well documented. Success in minimizing antibiotic use has also been demonstrated in recent years in several sectors and could be correlated with success in containing AMR and MDRO (e.g., decrease in MRSA in human medicine). Sector-specific measures to reduce the burden of MDRO and AMR are also necessary, as not all resistance problems are linked to other sectors. Carbapenem resistance is still rare, but most apparent in human pathogens. Colistin resistance occurs in different sectors but shows different mechanisms in each. Resistance to antibiotics of last resort such as linezolid is rare in Germany, but shows a specific One Health correlation. Efforts to harmonize methods, for example in the field of antimicrobial susceptibility testing and genome-based pathogen and AMR surveillance, are an important first step towards a better comparability of the different data collections.

[The role of infection prevention in the control of antimicrobial resistance : Any avoided infection contributes to the reduction of antibiotic use]. / Die Rolle der Infektionsprävention bei der Eindämmung der Antibiotikaresistenzentwicklung : Jede vermiedene Infektion trägt zur Reduktion des Antibiotikaeinsatzes bei.

Clinically relevant infections are the primary indication for the use of antimicrobial agents in human medicine. Consequently, the prevention of infections is the fundament of all measures to rationally reduce the use of antibiotics. A prevented infection must not be treated. For the prevention of several community-acquired infections, vaccines are available. In addition, several infections may be prevented on the basis of knowledge and responsible behavior. However, the prevention of nosocomial infections depends mainly on the responsibility of third parties in the context of medical procedures. Effective preventive measures are described in guidelines carefully prepared by the commission for hospital hygiene and infection prevention in Germany. The consequent implementation of these guidelines contributes to patient safety and the prevention of the spread of multidrug-resistant bacteria. Highly cost-effective measures are a high degree of compliance with the rules for hand hygiene, perioperative antiseptic measures, and guidelines for the use of perioperative antimicrobial prophylaxis. The documentation of decreasing or low rates of infections and antimicrobial resistance helps to verify the success of preventive measures.

Inflammatory response of TLR4 deficient spleen macrophages (CRL 2471) to Brucella abortus S19 and an isogenic ΔmglA deletion mutant.

UNLABELLED: Brucellosis is a worldwide distributed zoonosis caused by members of the genus Brucella. One of them, Brucella abortus, is the etiological agent of bovine brucellosis. With the attenuated strain B. abortus S19 a vaccine is available. However, both, virulence (safety) and the ability to induce a protective B and T cell response (efficacy) have to be tested in suitable assays before successful use in the field. For this purpose, several macrophage cell lines of various origins have been used while splenic macrophages are the preferred host cells in vivo. We here characterized the in vitro response of the murine splenic macrophage cell line CRL 2471(I-13.35) to B. abortus. This cell line still depends on the presence of colony-stimulating factor 1 (CSF1) and is derived from LPS resistant (TLR4 deficient) C3H/HeJ mice. For infection the vaccine strain B. abortus S19A as well as the formerly described isogenic deletion mutant B. abortus S19A ΔmglA 3.14 were used. While numbers of viable bacteria did not differ significantly between the vaccine strain and the deletion mutant at 6h post infection, a higher bacterial load was measured in case of the mutant at 24h and 48h after infection. This was also true, when IFNγ was used for macrophage activation. A comprehensive gene expression profile of macrophages was analysed 6 and 24h after infection by means of an RT-PCR based gene expression array. The mutant strain B. abortus S19A ΔmglA 3.14 elicited a stronger cellular response of the splenic macrophages as compared to the parental vaccine strain. This was most prominent for the pro-inflammatory cytokines IL-1α, IL-1ß, TNF-α and IL6 as well as for the chemokine ligands CXCL1, CXCL2, CXCL10, CCL2, CCL5, CCL7, CCL17 and the co-stimulatory molecules CD40 and ICAM1. While these differences were also present in IFNγ-stimulated macrophages, an addition of IFNγ after infection not only resulted in a dramatic increase of the translation of the afore mentioned genes but also resulted in the translation of IFNß1, IL12ß, MIP1α and ß (CCL3, CCL4), NOS2 (and SOD2) and FAS. CONCLUSION: The TLR4 deficient murine splenic macrophage cell line CRL 2471 was used for the first time for the characterization of macrophage-Brucella interaction to investigate the pre-immune phase of brucellosis in vitro. Typical pro-inflammatory cytokines and certain surface receptors were differentially induced by B. abortus S19 A and an isogenic ΔmglA deletion mutant in vitro. This model may be useful for further studies to characterize the inflammatory response of splenic macrophages to intracellular gram-negative bacteria avoiding cell responses to soluble LPS.

[Update on outbreaks reported from neonatal intensive care units (2010-203): Staphylococcus aureus]. / Literatur-Update zu Staphylococcus-aureus-Ausbrüchen auf neonatologischen Intensivpflegestationen (2010-2013).

In terms of the unique risk profile of the patients and the morbidity associated with S. aureus infections in this vulnerable patient population, the literature on outbreaks of S. aureus (including MRSA) in neonatal intensive care units (NICUs) needs to be analyzed separately from reports derived from other intensive care units. With the objective of updating important information for those involved in outbreak management and fostering preventive efforts, this article summarizes the results of a systematic literature analysis, referring to an earlier publication by Gastmeier et al. It focuses on NICU outbreaks caused by S. aureus (including MRSA) and on controlling them.

[Healthcare-associated infections and antimicrobial use in long term care facilities (HALT-2): German results of the second European prevalence survey]. / Healthcare-associated infections and antimicrobial use in long term care facilities (HALT-2) : Deutsche Ergebnisse der zweiten europäischen Prävalenzerhebung.

Prevention of infections and strategies for the prudent use of antimicrobials in long-term care facilities have gained importance in view of the demographic changes, not only in Germany. To generate appropriate data and to identify relevant aspects of infection prevention in this field, the European Centre for Disease Prevention and Control (ECDC) launched the second point prevalence survey of healthcare-associated infections and antimicrobial use in European long-term care facilities in 2013 (HALT-2). Despite methodical adjustments in the collection of data on healthcare-associated infections, in this second survey healthcare workers in the participating facilities were intensively trained in methodology and data collection. Overall, 221 German facilities participated and collected data from 17,208 residents. Well-established structures of regional networks facilitated the recruitment of participants as well as the preparations for training and survey. The median prevalence of residents receiving at least one antimicrobial agent was 1.1% (95 %-CI 0,7-1,6)), which is remarkably low. However, the most frequently used antimicrobials in German facilities beside beta-lactams (penicillins 18.2%, other beta-lactams 17.2%) were quinolones (28.2%). Data collection of infections was performed based on signs and symptoms in detailed decision algorithms according to the recently updated McGeer surveillance criteria and yielded a median prevalence of 1.7% (95 %-CI 1,1-2,2). Symptomatic urinary tract infections (28.4%), skin and soft tissue infection (27.9%), and respiratory tract infections (24.7%) were identified both as the most common types of infections and the most common indications for the use of systemic antimicrobials. Clinical implications evolve mainly from the high use of quinolones. In terms of infection prevention measures, compliance of health care workers with a hand hygiene regimen revealed further potential for improvement.

[Update on outbreaks reported from neonatal intensive care units: Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa]. / Literatur-Update zu Ausbrüchen auf neonatologischen Intensivpflegestationen - Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii und Pseudomonas aeruginosa.

In terms of the unique risk profile, the clinical course of nosocomial infections, and the most prevalent bacterial pathogens, literature on outbreaks of potentially pathogenic bacteria on neonatal intensive care units (NICUs) needs to be analyzed separately from reports derived from other intensive care units. With the purpose of updating important information for those involved in outbreak management and fostering preventive efforts, this article summarizes the results of a systematic literature analysis, referring to an earlier publication by Gastmeier et al. This review focuses on NICU outbreaks caused by Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

Inguinal skin colonization with multidrug-resistant bacteria among residents of elderly care facilities: frequency, persistence, molecular analysis and clinical impact.

Frequency, persistence and molecular characteristics of multidrug resistant bacteria colonizing inhabitants of long term care facilities are topics of current concern. We performed a point-prevalence survey of 402 residents in 7 elderly care facilities in Berlin, Germany. Inguinal swabs were analyzed for the presence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multidrug-resistant gram-negative bacteria. Three and six months following the initial investigation, all colonized residents were sampled again and the occurrence of intercurrent infections, hospital admissions and use of antimicrobials were registered. Genetic relatedness of the bacteria was investigated using multi-locus sequence typing (MLST), spa-typing and SmaI/XbaI-macrorestriction analysis. 33 (8.2%) residents were skin-colonized with multidrug-resistant bacteria. MRSA were found in 19 (4.7%) and ESBL-producing Enterobacteriaceae in 16 residents (3.98%). Independent risk factors for colonization with multidrug-resistant bacteria were a high level of care and the presence of chronic wounds. A large proportion of the observed bacteria persisted up to six months and showed a high degree of inter-individual diversity. Outcome analysis revealed that infections tend to occur slightly more often in residents colonized by multiresistant pathogens. We assume that a perceptible population of residents in nursing homes is at risk for individual colonization with multidrug-resistant bacteria as well as healthcare associated infections.

Infection prevention in a connected world: the case for a regional approach.

Results from microbiological and epidemiological investigations, as well as mathematical modelling, show that the transmission dynamics of nosocomial pathogens, especially of multiple antibiotic-resistant bacteria, is not exclusively amenable to single-hospital infection prevention measures. Crucially, their extent of spread depends on the structure of an underlying "healthcare network", as determined by inter-institutional referrals of patients. The current trend towards centralized healthcare systems favours the spread of hospital-associated pathogens, and must be addressed by coordinated regional or national approaches to infection prevention in order to maintain patient safety. Here we review recent advances that support this hypothesis, and propose a "next-generation" network-approach to hospital infection prevention and control.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-ß-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-ß-lactamases (MBL) such as New Delhi metallo-ß-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential ß-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.

During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers.

Outbreaks of virulent diarrheagenic Escherichia coli--are we in control?

Shiga toxin-producing Escherichia coli (STEC) are the most virulent diarrheagenic E. coli known to date. They can be spread with alarming ease via food as exemplified by a large sprout-borne outbreak of STEC O104:H4 in 2011 that was centered in northern Germany and affected several countries. Effective control of such outbreaks is an important public health task and necessitates early outbreak detection, fast identification of the outbreak vehicle and immediate removal of the suspected food from the market, flanked by consumer advice and measures to prevent secondary spread.In our view, opportunities to improve control of STEC outbreaks lie in early clinical suspicion for STEC infection, timely diagnosis of all STEC at the serotype-level and integrating molecular subtyping information into surveillance systems. Furthermore, conducting analytical studies that supplement patients' imperfect food history recall and performing, as an investigative element, product tracebacks, are pivotal but underutilized tools for successful epidemiologic identification of the suspected vehicle in foodborne outbreaks. As a corollary, these tools are amenable to tailor microbiological testing of suspected food. Please see related article: http://www.biomedcentral.com/1741-7015/10/12.

The mglA gene and its flanking regions in Brucella: the role of mglA in tolerance to hostile environments, Fe-metabolism and in vivo persistence.

We previously demonstrated that a spontaneous smooth small-colony variant of Brucella abortus S19 is characterized by increased in vivo persistence and the differential expression of a gene predicted to encode a galactoside transport ATP binding protein (mglA). In order to further investigate the role of this gene in the context of its flanking regions, we analyzed the respective DNA sequences from the formerly described B. abortus S19 as well as from avirulent B. neotomae 5K33 and compared these with published data from other Brucella species. Deletion mutagenesis of mglA in the large-colony variant of B. abortus S19 resulted in increased tolerance of the deletion mutant to a hyperosmotic (toxic), galactose-containing medium as well as to oxidative stress (H(2)O(2)). Whilst the deletion mutant is characterized by reduced growth on solid Fe(3+)-containing minimal medium (small-colony morphology), in vivo studies in mice demonstrated statistical significant differences in the bacterial load of spleens in the pre-immune, but not in the late phase of the infection.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Trends in respiratory virus circulation following COVID-19-targeted nonpharmaceutical interventions in Germany, January - September 2020: Analysis of national surveillance data.

BACKGROUND: During the initial COVID-19 response, Germany's Federal Government implemented several nonpharmaceutical interventions (NPIs) that were instrumental in suppressing early exponential spread of SARS-CoV-2. NPI effect on the transmission of other respiratory viruses has not been examined at the national level thus far. METHODS: Upper respiratory tract specimens from 3580 patients with acute respiratory infection (ARI), collected within the nationwide German ARI Sentinel, underwent RT-PCR diagnostics for multiple respiratory viruses. The observation period (weeks 1-38 of 2020) included the time before, during and after a far-reaching contact ban. Detection rates for different viruses were compared to 2017-2019 sentinel data (15350 samples; week 1-38, 11823 samples). FINDINGS: The March 2020 contact ban, which was followed by a mask mandate, was associated with an unprecedented and sustained decline of multiple respiratory viruses. Among these, rhinovirus was the single agent that resurged to levels equalling those of previous years. Rhinovirus rebound was first observed in children, after schools and daycares had reopened. By contrast, other nonenveloped viruses (i.e. gastroenteritis viruses reported at the national level) suppressed after the shutdown did not rebound. INTERPRETATION: Contact restrictions with a subsequent mask mandate in spring may substantially reduce respiratory virus circulation. This reduction appears sustained for most viruses, indicating that the activity of influenza and other respiratory viruses during the subsequent winter season might be low,whereas rhinovirus resurgence, potentially driven by transmission in educational institutions in a setting of waning population immunity, might signal predominance of rhinovirus-related ARIs. FUNDING: Robert Koch-Institute and German Ministry of Health.

Fast, broad-range disinfection of bacteria, fungi, viruses and prions.

Effective disinfectants are of key importance for the safe handling and reprocessing of surgical instruments. This study tested whether new formulations containing SDS, NaOH and 1-propanol (n-propanol) are simultaneously active against a broad range of pathogens including bacteria, fungi, non-enveloped viruses and prions. Inactivation and disinfection were examined in suspension and on carriers, using coagulated blood or brain homogenate as an organic contaminant. Coomassie blue staining was used to assess whether the formulations undesirably fixed proteins to rough surfaces. A mixture of 0.2 % SDS and 0.3 % NaOH in 20 % n-propanol achieved potent decontamination of steel carriers contaminated with PrP(TSE), the biochemical marker for prion infectivity, from 263K scrapie hamsters or from patients with sporadic or variant Creutzfeldt-Jakob disease. 263K scrapie infectivity on carriers was decreased by > or =5.5 logs. Furthermore, the formulation effectively inactivated poliovirus, hepatitis A virus and caliciviruses (including murine norovirus) in suspension tests. It also yielded significant titre reductions of bacteria (Enterococcus faecium, Mycobacterium avium; >6 logs), fungi (spores of Aspergillus niger; > or =5 logs) and poliovirus (>4 logs) embedded in coagulated blood on carriers. The formulation was not found to fix proteins more than was observed with water as the cleaning reagent. In conclusion, SDS, NaOH and n-propanol can synergistically achieve fast, broad-range disinfection.