Collaborative genomic and epidemiological investigations identified a tuberculosis outbreak in Vienna as part of a multi-country cluster comprising 57 foreign-born cases of Mycobacterium tuberculosis ST215/Beijing 2.2.1 notified 2021-2025. While 14 of 16 cases in Vienna were considered autochthonous, the diverse geographic origin of clustered cases across nine European countries suggests a common transmission source, possibly linked to migratory routes. Cross-border data exchange and integrated genomic analysis are essential for identifying transmission dynamics in tuberculosis clusters affecting mobile populations.

In May 2018, an outbreak of NDM-5-carrying Escherichia coli (NDM-5-EC) was detected at the hemato-oncology department of a tertiary care center in Austria. This report details the outbreak investigation, control measures and the whole genome sequencing (WGS) data of the outbreak isolates. A total of 15 isolates (seven clinical isolates from allogenic stem cell transplant (SCT) recipients and eight wastewater isolates recovered from patients’ toilets) were analyzed by whole genome sequencing. Genome based typing identified two clusters of the high risk clones ST167/CT12607 and ST617/CT2791. Long-read sequencing of selected isolates from both clusters identified two different plasmids, however with a highly similar genetic context of the blaNDM-5 containing region. Genomic analysis revealed the presence of additional resistance genes, including blaCTX-M-15, and blaOXA-1, and virulence factors. Four patients were colonized with NDM-5-EC, two patients suffered bacteremia caused by the outbreak strain and two deaths were associated with an NDM-5-EC infection. The outbreak source was traced to toilet sewage pipes, which remained persistently contaminated despite extensive cleaning and disinfection. Successful eradication of NDM-5-EC from the installations required disassembly, hot water pressure washing of the sewage pipes and complete replacement of all movable parts. Additionally, colonized patients were instructed to use wheeled commodes instead of toilets, and a pre-admission screening strategy was implemented for all patients undergoing hematologic stem cell transplantation. The outbreak was successfully terminated in November 2020. NDM-5-EC, especially high-risk clones such as ST167 and ST617, can persist in hospital wastewater systems despite cleaning and disinfection efforts and can cause prolonged outbreaks. Therefore, a comprehensive bundle of interventions like the ones applied in our study is essential, especially in clinical settings with heavily immunosuppressed patients.

Objectives: Toscana virus (TOSV) is an emerging sandfly-borne virus, causing meningitis and encephalitis in Mediterranean and Middle Eastern regions. Only sporadic cases have been diagnosed in Austria over the past 20 years. We aim to identify and describe TOSV-associated aseptic meningitis (TOSV-AM) cases in Austria between 2006 and 2023. Methods: We report a series of four imported cases of TOSV-AM diagnosed in Salzburg between July and September 2023, after travel to Italy. In addition, we analyzed all TOSV cases identified by the Austrian National Reference Center (2006-2018). Diagnosis was confirmed by immunoglobulin (Ig)M/G serology. Partial N-gene Sanger sequencing was performed for three cases in total. Results: Before the 2023 case series, we retrospectively identified a total of six TOSV-AM cases (three male and three female; median age was 43 years [22-53]). All four 2023 patients (three male and one female; median age was 45.5 years [37-50]) presented with clinical signs of meningitis, including headache, with three reporting insect bites. Lumbar puncture confirmed the diagnosis in three cases and IgM/IgG serology confirmed infection in all cases. TOSV detection by polymerase chain reaction in serum was possible in one patient, with nucleic acid sequencing identifying a lineage B strain. Previously diagnosed case patients were identified as lineage A. Conclusion: We report a total of 10 serologically confirmed cases of TOSV-AM in Austria since 2006. Despite evidence of growing incidence of TOSV circulation in neighboring countries, this is the first well-characterized case series in Austria. Considering the recent rise in TOSV cases in endemic regions, likely driven by climate change and urbanization, imported TOSV-AM cases in Austria may increase in the future.

Antibiotics have strongly changed the development of humanity by playing a crucial role in the health and wellbeing of the people worldwide. Bacteria are in return, continuously developing mechanisms of antimicrobial resistance. Due to this phenomenon, humanity is now facing the new challenge of fighting antimicrobial resistance. Since the food production system is a part of a larger continuum of trade, communication, and supply, it represents a reservoir of antibiotic resistant bacteria. Therefore, multidisciplinary and joint efforts, involving stakeholders from various sectors, are necessary to be undertaken towards achieving another milestone—suppressing the spread of antimicrobial resistance, while at the same time keeping the antibiotics in the loop for the purpose of human and animal health protection.

A novel, Gram-positive, facultative anaerobe, coccoid and non-motile bacterium, designated as CoE-012-22T was isolated from dried beef sausage (the original name in Montenegro is Govedji Kulen) manufactured in the municipality of Rozaje (Montenegro) in 2021. Cells of this strain were oxidase- and catalase-negative. Growth occurred at 4–50 °C, at pH 5.0–8.0 and with 0–6.5 % (w/v) NaCl in diverse growth media. MALDI-TOF analysis identified the strain as Enterococcus canintestini (log score 2). Phylogenetic analysis of the 16S rRNA gene and whole genome sequences assigned the strain to the genus Enterococcus . The closest relatives were E. canintestini DSM 21207T and E. dispar ATCC 51266T with 16S rRNA gene sequence pairwise similarities of 99.34 and 98.59 %, respectively. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between isolate CoE-012-22T and other enterococci species were below the thresholds for species delineation thresholds (95.0 % ANI; 70.0 % dDDH) with maximum identities of 84.13 % (ANIb), 86.43 % (ANIm) and 28.4 % (dDDH) to E. saigonensis JCM 31193T and 70.97 % (ANIb), 88.99 % (ANIm) and 32.4 % (dDDH) to E. malodoratus ATCC 43197T. Two unknown Enterococcus isolates, Enterococcus sp. MJM12 and Enterococcus SMC-9, showed identities of 99.87 and 99.94 % (16S rRNA), 98.57 and 98.65 % (ANIb), 98.93 and 99.02 % (ANIm), and 89.8 and 90.0 % (dDDH) to strain CoE-012-22T and can therefore be regarded as the same species. Based on the characterization results, strain CoE-012-22T was considered to represent a novel species, for which the name Enterococcus montenegrensis sp. nov. is proposed. The type strain is CoE-012-22T (=DSM 115843T=NCIMB 15468T).

In spring 2022, an increase in metallo-β-lactamase-producing Pseudomonas aeruginosa (MBL-Pa) infections was detected in a hospital in Upper Austria. To identify the source of infection and to stop further transmissions, an epidemiological outbreak investigation including whole-genome sequencing (WGS)-based typing was conducted. The final case definition included cases admitted to the hospital between 2020 and 2023 with an MBL-Pa in one of the three genomic clusters identified. In addition, the investigation was extended to include historical cases from 2017. Core genome multilocus sequence typing was performed to assess the genetic relatedness between the isolates. Fifty-four clinical P. aeruginosa isolates and eight P. aeruginosa isolates from the hospital environment were obtained. All but nine isolates grouped into one of three genomic clusters (ST235/blaVIM-1, ST111/blaVIM-2, or ST621/blaIMP-13), which were considered to be distinct, prolonged outbreaks involving 47 out of 52 cases. The most likely source of infection for cluster 1 (ST111/blaVIM-2) and cluster 2 (ST235/blaVIM-1) was sinks in the intensive care unit (ICU) washroom. Cluster 3 clone (ST621/blaIMP-13) could have originated in the urology ward in 2020 and then spread to the ICU years later. However, the nosocomial origin of this clone could not be proven. In March 2023, following the implementation of control measures (gowning, patient isolation, screening, and daily disinfection), no further MLB-Pa was detected, and the outbreaks were considered to be over. As ICUs play an important role in the transmission of P. aeruginosa, emphasis should be placed on genomic surveillance, infection prevention, and control in such wards. IMPORTANCE:The significance of our work lies in the successful resolution of three prolonged outbreaks of MBL-Pa infections in a hospital in Upper Austria. Through a comprehensive epidemiological investigation coupled with WGS-based typing of P. aeruginosa isolates, the study identified three distinct genomic clusters responsible for prolonged outbreaks involving 47 cases. The investigation pinpointed sinks in the ICU washroom as the likely source of infection for two of the clusters. The study demonstrates the effectiveness of control measures such as hand hygiene, gowning, patient isolation, screening, and disinfection in stopping further transmission and bringing the outbreaks to a close. This underscores the critical role of genomic surveillance and control measures, particularly in high-risk settings like ICUs, in reducing nosocomial transmission of MBL-Pa infections.

Microbacterium plantarum (M. plantarum) was recently described as a new species isolated from copper globemallow (Sphaeralcea angustifolia). Here, we report the complete genome of M. plantarum CoE-159-22, which was obtained from traditionally produced Montenegrin cheese.

IntroductionEnterococcus faecium is a widespread acid-lactic bacterium found in the environment, humans, and animal microbiota, and it also plays a role in the production of traditional food. However, the worldwide emergence of multidrug-resistant E. faecium strains represents a major public health threat and is the primary reason that the genus Enterococcus is not recommended for the Qualified Presumption of Safety (QPS) list of the European Food Safety Authority (EFSA), raising concerns about its presence in food products.MethodsIn this study, 39 E. faecium and 5 E. lactis isolates were obtained from artisanal brine cheeses and dry sausages, sourced from 21 different Montenegrin producers. The isolates were collected following the ISO 15214:1998 international method and processed for whole-genome sequencing (WGS).ResultsGenome analysis based on core genome multilocus sequence type (cgMLST) revealed a high diversity among isolates. Furthermore, the isolates carried antimicrobial resistance genes; the virulence genes acm, sgrA, and ecbA; the bacteriocin genes Enterolysin A, Enterocin A, Enterocin P, Duracin Q, Enterocin B, Bacteriocin 31, Enterocin EJ97, Sactipeptides, and Enterocin SEK4; the secondary metabolite genes T3PKS, cyclic lactone autoinducer, RiPP-like, and NRPS and a maximum of eight plasmids.ConclusionThis study highlights the need for careful monitoring of E. faecium and E. lactis strains in food to ensure they do not pose any potential risks to consumer safety.

Abstract Successful treatment of patients with recurring listeria episodes due to colonized foreign material is often challenging, typically requiring a combination of antimicrobial treatment and surgical removal. Here, we present a particularly challenging case of chronic invasive listeriosis with a total of six relapses, confirmed through molecular typing.

Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.

Whole genome sequencing (WGS) has become the new technological standard in public health surveillance and disease control systems worldwide. WGS allows the most accurate identification and detailed characterization of microorganisms and is a significant improvement in outbreak investigation, source identification, tracking and surveillance, infection prevention and control in public health microbiology. This allows assurance and risk assessment of food microorganisms and microbial risk management. The high data quality, the reproducibility, stability and accuracy of WGS technology has been demonstrated. WGS passed its acid test with flying colors during the COVID-19 pandemic where the technology was successfully used for early detection of new virus variants improving risk assessment and public health measurements. With the ongoing development of the entire WGS system i.e. technology, metadata, new bioinformatics and machine learning algorithms, we can expect a paradigm shift from classical/ molecular microbiology towards precision microbiology.

BACKGROUND:Listeria monocytogenes is a bacterial pathogen known for causing listeriosis, a foodborne illness with a wide spectrum of clinical presentations ranging from mild gastroenteritis to severe invasive disease, particularly affecting immunocompromised individuals, pregnant women, newborns, and the elderly. Successful treatment of patients with recurring listeria episodes due to colonised foreign material is often challenging, typically requiring a combination of antimicrobial treatment and surgical removal. CASE PRESENTATION:Here, we present a particularly complex case of chronic invasive listeriosis with a total of six relapses. After extensive investigations, the patient's ICD device was identified as the focus of infection. CONCLUSION:The confirmation of relapses through cgMLST analysis highlights the persistence of Listeria monocytogenes and the potential for recurrence even after apparent resolution of symptoms in patients with foreign material. It emphasises the necessity for a comprehensive assessment to identify and mitigate the risk of relapses, thereby ensuring optimal management and outcomes.

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches.

Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain.

Journal Article Special Issue ‘One Health meets Omics: The way forward to investigate zoonosis’ Get access Adriana Cabal, Adriana Cabal Institute for Medical Microbiology and Hygiene Austrian Agency for Health and Food Safety Vienna Austria https://orcid.org/0000-0001-9346-1612 Search for other works by this author on: Oxford Academic Google Scholar Aleksandra Martinovic Aleksandra Martinovic Faculty of Food Technology, Food Safety and Ecology University of Donja Gorica, Donja Gorica bb Podgorica Montenegro https://orcid.org/0000-0002-3177-9868 Search for other works by this author on: Oxford Academic Google Scholar Journal of Applied Microbiology, Volume 133, Issue 3, 1 September 2022, Pages 1144–1145, https://doi.org/10.1111/jam.15768 Published: 01 September 2022 Article history Received: 15 July 2022 Revision received: 28 July 2022 Accepted: 07 August 2022 Published: 01 September 2022

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

In many dairy products, Leuconostoc spp. is a natural part of non-starter lactic acid bacteria (NSLAB) accounting for flavor development. However, data on the genomic diversity of Leuconostoc spp. isolates obtained from cheese are still scarce. The focus of this study was the genomic characterization of Leuconostoc spp. obtained from different traditional Montenegrin brine cheeses with the aim to explore their diversity and provide genetic information as a basis for the selection of strains for future cheese production. In 2019, sixteen Leuconostoc spp. isolates were obtained from white brine cheeses from nine different producers located in three municipalities in the northern region of Montenegro. All isolates were identified as Ln. mesenteroides. Classical multilocus sequence tying (MLST) and core genome (cg) MLST revealed a high diversity of the Montenegrin Ln. mesenteroides cheese isolates. All isolates carried genes of the bacteriocin biosynthetic gene clusters, eight out of 16 strains carried the citCDEFG operon, 14 carried butA, and all 16 isolates carried alsS and ilv, genes involved in forming important aromas and flavor compounds. Safety evaluation indicated that isolates carried no pathogenic factors and no virulence factors. In conclusion, Ln. mesenteroides isolates from Montenegrin traditional cheeses displayed a high genetic diversity and were unrelated to strains deposited in GenBank.

Lactococcus garvieae and Lactococcus petauri cause lactococcosis in fish. Both species have also been isolated from various food products and are considered emerging zoonotic pathogens. Here, we report the genomes of L. garvieae INF126 and L. petauri INF110, obtained from traditional Montenegrin brine cheeses.

Pertussis is a vaccine-preventable disease, and its recent resurgence might be attributable to the emergence of strains that differ genetically from the vaccine strain. We describe a novel pertussis isolate-based surveillance system and a core genome multilocus sequence typing scheme to assess Bordetella pertussis genetic variability and investigate the increased incidence of pertussis in Austria. During 2018-2020, we obtained 123 B. pertussis isolates and typed them with the new scheme (2,983 targets and preliminary cluster threshold of <6 alleles). B. pertussis isolates in Austria differed genetically from the vaccine strain, both in their core genomes and in their vaccine antigen genes; 31.7% of the isolates were pertactin-deficient. We detected 8 clusters, 1 of them with pertactin-deficient isolates and possibly part of a local outbreak. National expansion of the isolate-based surveillance system is needed to implement pertussis-control strategies.