Genomic surveillance of SARS-CoV-2 evolution by a centralised pipeline and weekly focused sequencing, Austria, January 2021 to March 2023.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

Association of Phylogenomic Relatedness among Neisseria gonorrhoeae Strains with Antimicrobial Resistance, Austria, 2016-2020.

We investigated genomic determinants of antimicrobial resistance in 1,318 Neisseria gonorrhoeae strains isolated in Austria during 2016-2020. Sequence type (ST) 9363 and ST11422 isolates had high rates of azithromycin resistance, and ST7363 isolates correlated with cephalosporin resistance. These results underline the benefit of genomic surveillance for antimicrobial resistance monitoring.

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus).

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.

First records of Aedes pulcritarsis (Rondani, 1872) (Diptera: Culicidae) in Austria.

Aedes pulcritarsis is a tree-hole breeding species with its main distribution in the Mediterranean area. Within the scope of two independent monitoring programmes, this mosquito species was detected for the first time in Austria, in the province of Lower Austria (2018, districts Mistelbach and Gaenserndorf; 2020, district Bruck an der Leitha). As the climatic and habitat situation in Central Europe seems to be generally suitable for this species, the most likely explanation for the species not being recorded previously is that it might have been overlooked in the past due to its specialized breeding habitat. However, further research on the distribution of Ae. pulcritarsis in Austria would be needed to support this hypothesis. The results from this study will contribute to the investigation of the northern distribution limit of Ae. pulcritarsis in Europe and possible changes thereof.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections.

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.

Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria.

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.

Isolate-Based Surveillance of Bordetella pertussis, Austria, 2018-2020.

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.

Assessing the Genetic Diversity of Austrian Corynebacterium diphtheriae Clinical Isolates, 2011 to 2019.

Diphtheria is a vaccine-preventable disease with a high potential for reemergence. One of its causative agents is Corynebacterium diphtheriae, with some strains producing diphtheria toxin. From 2011 to 2019, 57 clinical C. diphtheriae strains were isolated in Austria, either from the respiratory tract or from skin infections. The aim of this study was to investigate the genetic diversity of these C. diphtheriae isolates using whole-genome sequencing. Isolates were characterized by genome-wide comparisons using single nucleotide polymorphism analysis or core genome multilocus sequence typing and by searching sequence data for antimicrobial resistance genes and genes involved in diphtheria toxin production. The genetic diversity among the isolates was high, with no clear distribution over time or place. Corynebacterium belfantii isolates were separated from other strains and were strongly associated with respiratory infections (odds ratio [OR] = 57). Two clusters, limited in time and space, were identified. Almost 40% of strains carried resistance genes against tetracycline or sulfonamides, mostly from skin infections. Microbiological tests showed that 55% of isolates were resistant to penicillin but did not carry genes conferring ß-lactam resistance. A diphtheria toxin gene with no nonsynonymous mutation was found in three isolates only. This study showed that sequencing can provide valuable information complementing routine microbiological and epidemiological investigations. It allowed us to identify unknown clusters, evaluate antimicrobial resistance more broadly, and support toxigenicity results obtained by PCR. For these reasons, C. diphtheriae surveillance could strongly benefit from the routine implementation of whole-genome sequencing.

SARS-CoV-2 re-infection risk in Austria.

BACKGROUND: A key question concerning coronavirus disease 2019 (COVID-19) is how effective and long lasting immunity against this disease is in individuals who were previously infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to evaluate the risk of SARS-CoV-2 re-infections in the general population in Austria. METHODS: This is a retrospective observational study using national SARS-CoV-2 infection data from the Austrian epidemiological reporting system. As the primary outcome, we aim to compare the odds of SARS-CoV-2 re-infections of COVID-19 survivors of the first wave (February to April 30, 2020) versus the odds of first infections in the remainder general population by tracking polymerase chain reaction (PCR)-confirmed infections of both groups during the second wave from September 1 to November 30, 2020. Re-infection counts are tentative, since it cannot be excluded that the positive PCR in the first and/or second wave might have been a false positive. RESULTS: We recorded 40 tentative re-infections in 14 840 COVID-19 survivors of the first wave (0.27%) and 253 581 infections in 8 885 640 individuals of the remaining general population (2.85%) translating into an odds ratio (95% confidence interval) of 0.09 (0.07 to 0.13). CONCLUSIONS: We observed a relatively low re-infection rate of SARS-CoV-2 in Austria. Protection against SARS-CoV-2 after natural infection is comparable with the highest available estimates on vaccine efficacies. Further well-designed research on this issue is urgently needed for improving evidence-based decisions on public health measures and vaccination strategies.

Presence of ß-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant ß-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-ß-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

Large Nationwide Outbreak of Invasive Listeriosis Associated with Blood Sausage, Germany, 2018-2019.

Invasive listeriosis is a severe foodborne infection in humans and is difficult to control. Listeriosis incidence is increasing worldwide, but some countries have implemented molecular surveillance programs to improve recognition and management of listeriosis outbreaks. In Germany, routine whole-genome sequencing, core genome multilocus sequence typing, and single nucleotide polymorphism calling are used for subtyping of Listeria monocytogenes isolates from listeriosis cases and suspected foods. During 2018-2019, an unusually large cluster of L. monocytogenes isolates was identified, including 134 highly clonal, benzalkonium-resistant sequence type 6 isolates collected from 112 notified listeriosis cases. The outbreak was one of the largest reported in Europe during the past 25 years. Epidemiologic investigations identified blood sausage contaminated with L. monocytogenes highly related to clinical isolates; withdrawal of the product from the market ended the outbreak. We describe how epidemiologic investigations and complementary molecular typing of food isolates helped identify the outbreak vehicle.

Multicenter Study of Cronobacter sakazakii Infections in Humans, Europe, 2017.

Cronobacter sakazakii has been documented as a cause of life-threating infections, predominantly in neonates. We conducted a multicenter study to assess the occurrence of C. sakazakii across Europe and the extent of clonality for outbreak detection. National coordinators representing 24 countries in Europe were requested to submit all human C. sakazakii isolates collected during 2017 to a study center in Austria. Testing at the center included species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, subtyping by whole-genome sequencing (WGS), and determination of antimicrobial resistance. Eleven countries sent 77 isolates, including 36 isolates from 2017 and 41 historical isolates. Fifty-nine isolates were confirmed as C. sakazakii by WGS, highlighting the challenge of correctly identifying Cronobacter spp. WGS-based typing revealed high strain diversity, indicating absence of multinational outbreaks in 2017, but identified 4 previously unpublished historical outbreaks. WGS is the recommended method for accurate identification, typing, and detection of this pathogen.

Listeriosis outbreak likely due to contaminated liver pâté consumed in a tavern, Austria, December 2018.

In late December 2018, an outbreak of listeriosis occurred after a group of 32 individuals celebrated in a tavern in Styria, Austria; traditional Austrian food (e.g. meat, meat products and cheese) was served. After the celebration, 11 individuals developed gastrointestinal symptoms, including one case with severe sepsis. Cases had consumed mixed platters with several meat products and pâtés originating from a local production facility (company X). Human, food and environmental samples taken from the tavern and company X were tested for L. monocytogenes. Whole genome sequence-based typing detected a novel L. monocytogenes strain of serotype IVb, sequence type 4 and CT7652 in 15 samples; 12 human, two food and one environmental sample from company X with an allelic difference of 0 to 1. Active case finding identified two further cases who had not visited the tavern but tested positive for the outbreak strain. In total, 13 cases (seven females and six males; age range: 4-84 years) were identified. Liver pâté produced by company X was identified as the likely source of the outbreak. Control measures were implemented and since the end of December 2018, no more cases were detected.

Petting zoos as sources of Shiga toxin-producing Escherichia coli (STEC) infections.

Despite their general low incidence, Shiga toxin-producing Escherichia (E.) coli (STEC) infections are considered an important public health issue due to the severity of illness that can develop, particularly in young children. We report on two Austrian petting zoos, one in Tyrol (2015) and one in Vorarlberg (2016), which were identified as highly likely infection sources of STEC infections. The petting zoo related cases involved a case of hemolytic uremic syndrome (HUS) due to STEC O157:HNM in 2015 and an outbreak of STEC O157:H7 infections affecting five young children and two adults in 2016. The HUS case accounted for 2.8% of the 36 STEC O157:HNM/H7 infections notified in Austria in 2015 (5,9% of 17 HUS cases). The seven cases described for 2016 accounted for 4.0% of the 177 human STEC infections documented for Austria in 2016, and for 19.4% of the 36 STEC O157:HNM/H7 infections notified that year. The evaluation of the STEC infections described here clearly underlines the potential of sequence-based typing methods to offer suitable resolutions for public health applications. Furthermore, we give a state-of-the-art mini-review on the risks of petting zoos concerning exposure to the zoonotic hazard STEC and on proper measures of risk-prevention.

Shiga toxin-producing Escherichia coli O103:H2 outbreak in Germany after school trip to Austria due to raw cow milk, 2017 - The important role of international collaboration for outbreak investigations.

Following a school ski-trip to Austria from 10 to 18/02/2017, nine of 25 participants of the group from Lower Saxony (Germany) developed gastroenteritis. The students and teachers (17-41 years) shared meals in a hotel. Active case finding revealed further cases among German school groups from North Rhine-Westphalia and Schleswig-Holstein, staying at the same hotel in February 2017. We conducted two retrospective cohort studies using self-administered questionnaires on clinical symptoms and food consumption. We defined a case as a trip participant in February 2017, staying at the aforementioned hotel and developing diarrhoea, vomiting or abdominal pain during or within ten days after the trip and/or who had a stool sample tested positive for STEC within four weeks after the trip. During the outbreak investigation, Austrian authorities detected that unlabeled raw cow milk delivered by a dairy farm had been offered at the hotel for breakfast during January and February 2017. Stool samples of participants, samples of milk served in the hotel and fecal samples of various animals kept at the milk-delivering farm were examined by culture and polymerase chain reaction. STEC isolates were typed using Pulsed-field Gel Electrophoresis (PFGE) and Whole-Genome Sequencing (WGS). All 25 participants from Lower Saxony completed the questionnaire on symptoms and milk consumption; 14 were cases (56%). Thirteen of 20 participants who had consumed cold milk fell ill (risk ratio (RR): 3.25; 95%-confidence interval (CI): 0.55-19.32). Of 159 trip participants from North Rhine-Westphalia, 81 completed the questionnaire (51%), 25 were cases (31%); RR for cold milk was 2.11 (CI: 0.89-5.03). The combined RR for cold milk in both groups was 2.49 (CI: 1.16-5.35). Shiga toxin 1a-gene and eaeA-gene positive STEC O103:H2 were detected in nine of 32 patients' stool samples and in two of 18 dairy farm cattle. Nine isolates from human stool samples and two isolates from cattle fecal samples yielded the same strain with an almost identical PFGE-pattern and WGS-profile. Microbiological and epidemiological evidence identified raw cow milk as the vehicle. Results may have been compromised by misclassification of cases due to a recall bias and mild symptoms. As a result of this outbreak investigation, the Austrian authorities enforced Austrian law in the hotel, to provide milk only when pasteurized. We recommend re-emphasizing the risk of raw milk consumption to providers.

Adult Hyalomma marginatum tick positive for Rickettsia aeschlimannii in Austria, October 2018.

We report on a non-indigenous adult Hyalomma marginatum tick in Austria carrying the human pathogenic Rickettsia aeschlimannii; presumably introduced as a nymph via migratory birds and completed the moulting within the same year. It was negative for Crimean-Congo haemorrhagic fever virus, but the finding of R. aeschlimannii represents a potential threat for humans due to its zoonotic character. Awareness of invasive tick species and carried pathogens should be improved in central and northern Europe.

Detection of the mcr-1 Gene in a Multidrug-Resistant Escherichia coli Isolate from an Austrian Patient.

Since colistin resistance based on the plasmid-encoded mcr-1 gene was first described, this resistance gene in Enterobacteriaceae has been found worldwide. These organisms are typically of heterogeneous genetic background and show exceptional clonal diversity. We describe the first confirmation of mcr-1 in a human Escherichia coli strain cultured from a surveillance stool sample of an Austrian oncology patient.

Usutu virus infections among blood donors, Austria, July and August 2017 - Raising awareness for diagnostic challenges.

Between July and August 2017, seven of 12,047 blood donations from eastern Austria, reacted positive to West Nile virus (WNV) in the cobas test (Roche). Follow-up investigations revealed Usutu virus (USUV) nucleic acid in six of these. Retrospective analyses of four blood donors diagnosed as WNV-infected in 2016 showed one USUV positive. Blood transfusion services and public health authorities in USUV-endemic areas should be aware of a possible increase of human USUV infections.

Re-evaluation of a 2014 multi-country European outbreak of Salmonella Enteritidis phage type 14b using recent epidemiological and molecular data.

A European multi-country outbreak of Salmonella Enteritidis phage type (PT) 14b occurred from March to November 2014 associated with the consumption of eggs. The outbreak involved more than 400 human cases from France, Luxembourg, Austria and the United Kingdom. In 2016-2017, it has been re-evaluated combining recent epidemiological results with latest molecular data. The outbreak was traced back to one large Bavarian egg producer with four distinct premises, three located in Bavaria, one in the Czech Republic. The outbreak isolates of S. Enteritidis PT 14b were grouped into three closely related clades by whole genome sequencing. Two of these clades could be referred to two Bavarian premises of the egg producer on the basis of epidemiological and molecular data, while epidemiological data presumably linked the third clade to another premises of the egg producer. Interestingly and in contrast to the situation in other European countries where several outbreaks were documented, all notified 91 laboratory-confirmed cases of S. Enteritidis PT 14b from Bavaria were sporadic, singular cases not belonging to any epidemiological outbreaks. In conclusion, as demonstrated here, the resolution of food-related outbreaks with such a high discriminatory power is rare in outbreak investigation.

West Nile virus surveillance in Europe: moving towards an integrated animal-human-vector approach.

This article uses the experience of five European countries to review the integrated approaches (human, animal and vector) for surveillance and monitoring of West Nile virus (WNV) at national and European levels. The epidemiological situation of West Nile fever in Europe is heterogeneous. No model of surveillance and monitoring fits all, hence this article merely encourages countries to implement the integrated approach that meets their needs. Integration of surveillance and monitoring activities conducted by the public health authorities, the animal health authorities and the authorities in charge of vector surveillance and control should improve efficiency and save resources by implementing targeted measures. The creation of a formal interagency working group is identified as a crucial step towards integration. Blood safety is a key incentive for public health authorities to allocate sufficient resources for WNV surveillance, while the facts that an effective vaccine is available for horses and that most infected animals remain asymptomatic make the disease a lesser priority for animal health authorities. The examples described here can support other European countries wishing to strengthen their WNV surveillance or preparedness, and also serve as a model for surveillance and monitoring of other (vector-borne) zoonotic infections.