7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr. med.

Chair

Prof. Dr.

Carbapenemases in Germany in 2023: Report of the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria

National Reference Centers and Consiliary Laboratories

Question:Multidrug-resistance in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii is of utmost therapeutic importance since hardly any innovative antimicrobial drug against gramnegative bacteria will be introduced within the next years. Among all resistance mechanisms the spread of carbapenemases is the most worrisome. However, the correct identification of carbapenemases is still challenging and molecular epidemiology of carbapenemases is required.Methods:The German National Reference Centre for Multidrug-Resistant Gramnegative Bacteria offers the free service of carbapenemase detection in bacterial isolates with elevated carbapenem MICs. All isolates are tested by a wide array of phenotypic and molecular methods. A bioassay based on cell-free extracts and WGS methods allow the detection of still unknown β-lactamases.Results:A total of 8.319 isolates were investigated for carbapenemases at the National Reference Centre in 2023 until November 29th. Carbapenemases were found in 3.513 Enterobacterales strains, 520 of P. aeruginosa and 375 of A. baumannii. The most frequent carbapenemases in Enterobacterales were OXA-48 (n = 612), NDM‑1 (n = 481) and KPC-2 (n = 442), which were also found in various combinations, e.g. NDM-1/OXA-48 (n = 125). Other enzymes like NDM-4, OXA-232 or GIM‑1 were found in less than 60 isolates each. In P. aeruginosa, VIM-2 was the most frequent carbapenemase (n = 291), followed by NDM-1 (n = 61). OXA-23 was again the most frequent carbapenemase in A. baumannii (n = 220), followed by OXA-72 (n = 95). NDM-1, OXA-58 and others were found in less than 10 isolates each.Conclusions:A large variety of different carbapenemases is detected in Germany, especially in Enterobacterales. The molecular epidemiology in Germany still significantly differs from observations made in other countries like Greece, Italy or the USA with a predominance of OXA-48 in Enterobacterales. Compared to previous years, the number of NDM-1 detections remained high. Furthermore, detection of NDM-5 and OXA-244 showed a significant increase compared to 2022.

Oral Presentation

Genomic surveillance identifies stable C. jejuni lineages causing disease in humans

Question: Campylobacter enteritis is a notifiable disease in Germany. More than 40,000 cases are reported annually. The majority of cases is caused by C. jejuni and is considered to be sporadic. In order to gain more insight into the population structure of clinical C. jejuni strains genomic surveillance was established in 2020. Here we describe major genomic lineages of C. jejuni, which play a central role in human disease.Methods: Campylobacter spp. were cultivated on CCDA under microaerophilic conditions followed by PCR-based species determination. Whole genome sequencing was performed with Illumina NextSeq. Analysis of reads was conducted with Ridom SeqSphere+.Results: Four years of surveillance data (2020-2024) reveal the existence of stable C. jejuni lineages as the cause of a significant amount of Campylobacter enteritis cases in Germany. To date, 14 lineages comprising approximately 850 (21%) isolates have been identified. This corresponds to approximately 8400 cases as only 4% of all notified cases are covered by the molecular surveillance program. The majority of isolates (124) belonged to complex type (CT) 2243. Case numbers peaked from February - April 2021 (36 cases) and December - February 2022 (35 cases). A smaller peak occurred in July 2023 (6 cases), indicating that this lineage is recurrent and stable over time. Another remarkable lineage is CT 2151 (ST-7355), which currently comprises 96 isolates, peaking from April – October 2022 (45 cases). This lineage has been associated with clinical cases in Denmark, Sweden and Luxembourg but has also been isolated from German poultry and poultry farms. Many of the lineages belong to CC ST-21 (6/14). Four of the six CC ST-21 lineages belong to ST-50. ST-50 has been described to occur worldwide and evolve regionally (Wallace et al. 2021). All major lineages identified so far have matching isolates from poultry origin.Conclusion: Data from genomic surveillance points to the existence of internationally distributed recurring C. jejuni lineages causing the majority of Campylobacter enteritis cases in Germany. Therefore, an international one health approach is required for achieving reduction of cases.

PD. Dr.

Laboratory confirmation during a large cross-border outbreak of Iatrogenic Botulism in Europe in 2023

Iatrogenic botulism is an adverse event after the injection of botulinum neurotoxin (BoNT) for medical or cosmetic purposes and is usually characterized by mild symptoms frequently involving the muscles adjacent to the injection site. The German national Consultant Laboratory for BoNT-producing Clostridia (Botulism, Tetanus) at the Robert Koch Institute was involved in the investigation of a highly unusual outbreak of travel-associated iatrogenic botulism: 30 out of 33 patients from Germany known to have received intra-gastric injection of BoNT/A for weight reduction in Istanbul (Turkey) developed symptoms of botulism, some of them were admitted to the ICU. The low concentration of BoNT in iatrogenic botulism usually prevents laboratory confirmation in human serum using standard assays (e.g., mouse bioassay). Indeed, state-of-the-art diagnostics of botulism is generally challenging due to the high molecular variability within the BoNT family, its limited time window for detection in serum and the ultimate sensitivity needed. We applied i) an Endopep-MS assay and ii) an Endopep-suspension immunoassay based on neoepitope-specific monoclonal antibodies recognizing the cleavage site in comparison to the classical mouse bioassay. While the mouse bioassay failed to detected any remaining BoNT/A we succeeded in the detection of BoNT/A with both the Endopep-MS and the Endopep-suspension immunoassay. The lessons-learned for botulism diagnostics in this largest outbreak of iatrogenic botulism ever noted will be discussed.

Professor

Genomic Antibiotic Resistance Prediction in Tuberculosis: A Comprehensive Evaluation of Whole Genome Sequencing for Routine Diagnostics in Germany

Phenotypic drug susceptibility testing (DST) remains gold standard for evaluating drug resistance in Germany, despite limitations. Since 2020, the National Reference Center (NRC) for Mycobacteria, has integrated whole genome sequencing of tuberculosis pathogens as part of the Robert Koch Institute (RKI) coordinated IMS (integrated molecular surveillance)-TB project. Accordingly, a comprehensive data set is available to test the potential of genomic resistance prediction.This study aims to evaluate the efficacy of genome-based antibiotic resistance prediction using the expanded WHO catalog, within a low-burden setting for routine diagnostics.Between 2020 and 2022, over 3,000 isolates underwent phenotypic DST at the NRC using the proportion method at critical concentration in the MGIT system. As part of the IMS-TB project, each of these isolates was analyzed using next generation sequencing (NGS). Genomic identification of resistance-causing variants from the extended WHO catalogue (2021) predicted resistance against first-line drugs and group A second-line drugs (Levofloxacin, Moxifloxacin, Bedaquiline and Linezolid). The results were compared with phenotypic outcome.For 3,148 isolates, a complete phenotypic and genotypic first-line resistance profile was produced. Among these 2,279 (72.4%) were fully susceptible against first-line drugs. NGS prediction demonstrated sensitivities above 95 % for each drug except pyrazinamide (90.4%), specificities exceeding 97 % for all four first-line drugs. The negative predictive value exceeded 99% for each of all first-line antibiotics. Group A drug analysis included 341 full datasets including 66 Fluorquinolone, 11 Bedaquiline and 5 Linezolid resistant isolates. Revealing high sensitivity and specificity for the detection of Fluorquinolone resistance (95.5% and 99.6%), while Bedaquiline and Linezolid resistant isolates must be evaluated individually due to limited numbers.NGS-based genomic DST reliably predicts first-line susceptibility. Standard sequencing of all culture-positive isolates may replace phenotypic first-line testing, given that the technical and quality management requirements can be met.

Identification of hemolysis-related pathogenicity factors of Bartonella bacilliformis

QuestionBartonella bacilliformis is the causative agent of Carrion's disease, a vector-borne illness restricted to the South American Andes. In the acute phase, bacteria infect erythrocytes causing severe hemolytic anemia with case-fatality rates as high as 90% in untreated patients. Erythrocyte invasion is the most important step in the pathogenesis of Carrion's disease and results in its subsequent destruction. Exact knowledge of this process is crucial for drug development.MethodsTo identify genes involved in hemolysis, a Tn5 transposon library was generated and screened for hemolytic activity. Hemolysis mutants were identified and systematically analyzed by loss of function/gain of function experiments. For this, markerless deletion was newly established for B. bacilliformis, and the generated deletion and complementation mutants were tested for hemolytic activity in a novel in vitro based hemolysis assay using human erythrocytes. To study B. bacilliformis hemolysis and to test potential therapeutic drugs in vivo, we currently establish a (humanized) murine infection model.ResultsTwo hemolysis-related genes, a porin and a phospholipase, were identified. The loss of one of the two genes led to the complete inhibition of hemolysis whereas the hemolytic activity was restored by plasmid-based complementation. Although B. bacilliformis is believed to be a human-specific pathogen, we were still interested in whether murine erythrocytes can nevertheless be infected by B. bacilliformis in vitro. It turned out that murine erythrocytes undergo hemolysis upon B. bacilliformis infection. Murine infection experiments are ongoing to analyze the course of infection in detail.ConclusionTwo hemolysis-related genes/proteins of B. bacilliformis have been identified and were functionally characterized. Ongoing work aims to establish a mouse model to study hemolysis in vivo and to evaluate potential anti-virulence strategies.

Invasive meningococcal disease in Germany 2023: Requirement of an adjusted vaccination recommendation?

IntroductionDuring the COVID-19 pandemic not only a reduction of invasive meningococcal disease (IMD) cases occurred in Germany, but also changes of the affected age-groups and the serogroup distribution. At the end of 2022, the number of IMD started to increase.GoalsTo present laboratory surveillance data of IMD in Germany 2023Materials and MethodsData on IMD isolates and clinical samples of suspected IMD cases sent to the National Reference Center for meningococci and Haemophilus influenzae (NRZMHi) were analysed according to antimicrobial susceptibility, age, serogroup and whole genome sequence (WGS).ResultsIn 2023, 227 Neisseria meningitidis (Nm) positive samples were submitted to the NRZMHi, of which 193 were viable isolates. Penicillin resistance was detected in 17 (8.8%) isolates of which one harboured a rarely occurring β-lactamase.The age distribution of the cases was comparable to the pre-pandemic years 2018 and 2019. Most prevalent serogroups were MenB (n=107, 47,1%) and MenY (n=99, 43,6%). MenC (n=7, 3,1%) and MenW (n=10, 4,4%) cases were rare. In comparison to pre-pandemic years, MenY cases increased in the age-groups 15-19 yrs, 20-29 yrs and 80-89 yrs.WGS data were available for 120 of the 193 meningococcal isolates. cgMLST analysis identified two genetic MenB clusters and ten genetic MenY clusters. The latter belonged to five different sequence type / finetype combinations. The genetic clusters comprised no spatio-temporally related cases.SummaryThe number of IMD cases analysed at the NRZMHi in 2023 was at a level comparable to 2018 and 2019. A major change was the increase of MenY cases from 18.4% in 2019 to 43.6% in 2023. This trend needs to be monitored and considered regarding future vaccination recommendations.

Oral Session
National Reference Centers and Consiliary Laboratories

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Oral SessionNational Reference Centers and Consiliary Laboratories

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Oral Session
National Reference Centers and Consiliary Laboratories