Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species.

BACKGROUND: Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. METHODS: For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. RESULTS: Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. CONCLUSIONS: In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps.

Recurrent, ICD-associated L. monocytogenes bacteraemia with multiple septic pulmonary embolisms over a 2-year period.

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.

A citizen science report-Tiger mosquitoes (Aedes albopictus) in allotment gardens in Graz, Styria, Austria.

Aedes albopictus, the Asian tiger mosquito, is an invasive species not native to Europe. Due to its ability to transmit pathogens, such as dengue, chikungunya and Zika viruses, Ae. albopictus is considered a major health threat. In Austria, it was first reported in 2012 in the Western province of Tyrol and was documented in the metropolitan area of Vienna in 2020, demonstrating its ability to colonize urban areas. In July 2021, a garden owner from Graz, Styria, Austria, contacted experts because of the possible presence of tiger mosquitoes in an allotment garden complex. Accordingly, citizen scientists collected adult mosquitoes and set up ovitraps. Adults and eggs were sent to the laboratory for morphological examination and molecular DNA barcoding within the mitochondrial cytochrome c oxidase subunit I gene. In total, 217 eggs of Ae. albopictus were found at the allotment garden as well as at a second location in the city of Graz. In addition, 14 adult Ae. albopictus specimens, of which 7 were molecularly identified as an identical haplotype, were collected at the allotment garden. With its mild climate and numerous parks and gardens, Graz provides the perfect environment for reproduction of tropical/subtropical alien Aedes mosquitoes. The presence of eggs and adult specimens in the current study period indicates that Ae. albopictus is already breeding in Graz. However, monitoring efforts need to be continued to determine whether stable populations of Ae. albopictus can survive there.

Three Cases of Tickborne Francisella tularensis Infection, Austria, 2022.

Tularemia is increasing in Austria. We report Francisella tularensis subspecies holarctica isolated from 3 patients who had been bitten by arthropods. Next-generation sequencing showed substantial isolate similarity. Clinicians should consider bloodstream F. tularensis infections for patients with signs/symptoms of ulceroglandular tularemia, and surveillance of potential vectors should be intensified.

Quantitative relationships among high-throughput sequencing, cyanobacteria toxigenic genotype abundance and microcystin occurrence in bathing waters.

Toxin-producing cyanobacteria pose significant threats to human and animal health if exposed during recreational activities in bathing waters. To better safeguard public health and reduce health risks during the bathing season, an effective monitoring and management strategy is required. Molecular tools used to monitor toxigenic cyanobacteria have been evaluated on the basis of the efficiency and applicability of the method used to (i) establish an early-warning monitoring strategy for EU bathing water sites using both targeted quantitative polymerase chain reaction (qPCR) and non-targeted high-throughput sequencing (HTS) genotype analysis and (ii) to compare the toxigenic potential of cyanobacteria with actual microcystin (MC) occurrence and concentrations. For this purpose, 16 bathing water sites were monitored according to the bathing water directive (BWD) of the European Union (EU) during the bathing season of the summer of 2020 in eastern Austria. The cyanobacterial community composition was analyzed through HTS and qPCR by targeting the microcystin synthetase B gene (mcyB), which indicates MC synthesis within the genera Microcystis and Planktothrix. Within the genus Microcystis, which was identified as the primary MC producer, the mcyB genotypes formed stable subpopulations that increased linearly in correlation with the total Microcystis population. Notably, the HTS cell equivalents assigned to Microcystis and Planktothrix correlated with the corresponding qPCR estimates of genotype abundance, which serves as a confirmation of the suitability of (semi)-quantitative sequencing through HTS. In addition to the elevated trophic state, reduced transparency, increasing water temperatures, as well as cyanobacterial HTS read numbers and Microcystis cell number equivalents per mL estimated through qPCR, were associated with positive MC samples. Therefore, in combination with the monitoring of standard environmental parameters, the use of HTS and qPCR techniques is considered highly useful to ensure the timely identification of health risks to recreational users, as mandated by the BWD.

Vibrio cholerae-An emerging pathogen in Austrian bathing waters?

Vibrio cholerae, an important human pathogen, is naturally occurring in specific aquatic ecosystems. With very few exceptions, only the cholera-toxigenic strains belonging to the serogroups O1 and O139 are responsible for severe cholera outbreaks with epidemic or pandemic potential. All other nontoxigenic, non-O1/non-O139 V. cholerae (NTVC) strains may cause various other diseases, such as mild to severe infections of the ears, of the gastrointestinal and urinary tracts as well as wound and bloodstream infections. Older, immunocompromised people and patients with specific preconditions have an elevated risk. In recent years, worldwide reports demonstrated that NTVC infections are on the rise, caused amongst others by elevated water temperatures due to global warming.The aim of this review is to summarize the knowledge gained during the past two decades on V. cholerae infections and its occurrence in bathing waters in Austria, with a special focus on the lake Neusiedler See. We investigated whether NTVC infections have increased and which specific environmental conditions favor the occurrence of NTVC. We present an overview of state of the art methods that are currently available for clinical and environmental diagnostics. A preliminary public health risk assessment concerning NTVC infections related to the Neusiedler See was established. In order to raise awareness of healthcare professionals for NTVC infections, typical symptoms, possible treatment options and the antibiotic resistance status of Austrian NTVC isolates are discussed.

Response to Immunization against SARS-CoV-2 and Risk of Omicron Infection in Dialysis Patients: A Prospective Cohort Study.

It is not well established to what extent previous immunizations offer protection against infections with the SARS-CoV-2 Omicron variant in dialysis patients. We aimed to define the relevant humoral response in dialysis patients using a SARS-CoV-2 IgG chemiluminescence microparticle immunoassay (CMIA) compared to the activity of neutralizing antibodies assessed by a virus neutralization test. Next, we aimed to determine differences in humoral and cellular response levels over time among patients infected or not infected by the Omicron variant of SARS-CoV-2. Immunological parameters of cellular and humoral response to SARS-CoV-2 were analyzed at baseline and after 3 (T3), 6 (T6) and 14 months (T14). In this monocentric cohort study, we followed 110 dialysis patients (mean age 68.4 ± 13.7 years, 60.9% male) for a median of 545 days. We determined an anti-SARS-CoV-2 IgG level of 56.7 BAU/mL as an ideal cut-off value with a J-index of 90.7. Patients infected during the Omicron era had significantly lower (p < 0.001) mean antibody levels at T0 (3.5 vs. 111.2 BAU/mL), T3 (269.8 vs. 699.8 BAU/mL) and T6 (260.2 vs. 513.9 BAU/mL) than patients without Omicron infection. Patients who developed higher antibody levels at the time of the basic immunizations were less likely to become infected with SARS-CoV-2 during the Omicron era. There is a need to adjust the cut-off values for anti-SARS-CoV-2 IgG levels in dialysis patients.

First report on the occurrence of Vibrio cholerae nonO1/nonO139 in natural and artificial lakes and ponds in Serbia: Evidence for a long-distance transfer of strains and the presence of Vibrio paracholerae.

Vibrio cholerae are natural inhabitants of specific aquatic environments. Strains not belonging to serogroups O1 and O139 are usually unable to produce cholera toxin and cause cholera. However, non-toxigenic V. cholerae (NTVC) are able to cause a variety of mild-to-severe human infections (via seafood consumption or recreational activities). The number of unreported cases is considered substantial, as NTVC infections are not notifiable and physicians are mostly unaware of this pathogen. In the northern hemisphere, NTVC infections have been reported to increase due to global warming. In Eastern Europe, climatic and geological conditions favour the existence of inland water-bodies harbouring NTVC. We thus investigated the occurrence of NTVC in nine Serbian natural and artificial lakes and ponds, many of them used for fishing and bathing. With the exception of one highly saline lake, all investigated water-bodies harboured NTVC, ranging from 5.4 × 101 to 1.86 × 104  CFU and 4.5 × 102 to 5.6 × 106 genomic units per 100 ml. The maximum values observed were in the range of bathing waters in other countries, where infections have been reported. Interestingly, 7 out of 39 fully sequenced presumptive V. cholerae isolates were assigned as V. paracholerae, a recently described sister species of V. cholerae. Some clones and sublineages of both V. cholerae and V. paracholerae were shared by different environments indicating an exchange of strains over long distances. Important pathogenicity factors such as hlyA, toxR, and ompU were present in both species. Seasonal monitoring of ponds/lakes used for recreation in Serbia is thus recommended to be prepared for potential occurrence of infections promoted by climate change-induced rise in water temperatures.

Complete Reference Genome Sequence of the Extensively Drug-Resistant Strain Neisseria gonorrhoeae AT159, with Ceftriaxone Resistance and High-Level Azithromycin Resistance, Using Nanopore Q20+ Chemistry and Illumina Sequencing.

Extensively drug-resistant Neisseria gonorrhoeae (XDR-NG) strains with resistance to the last remaining first-line treatments (ceftriaxone monotherapy or combined with azithromycin) represent the emerging threat of untreatable gonorrhea. We present the complete reference genome sequence of the XDR-NG strain AT159, with ceftriaxone and high-level azithromycin resistance, from Austria.

Resistance profiles of Neisseria gonorrhoeae isolates in Vienna, Austria: a phenotypic and genetic characterization from 2013 to 2020.

OBJECTIVES: International surveillance data show a constant rise in the number of Neisseria gonorrhoeae infections and an increase in drug resistance of N. gonorrhoeae. As recent N. gonorrhoeae surveillance data in Austria are scarce, this study investigated phenotypic and genotypic antimicrobial resistance in N. gonorrhoeae isolates. METHODS: In total, 440 N. gonorrhoeae samples were collected at the Medical University of Vienna, and the minimal inhibitory concentrations (MICs) for a range of different antibiotics were determined. Sampling sites and treatments were recorded, and whole-genome sequencing of N. gonorrhoeae isolates was performed using allele libraries to determine genotypic resistance. RESULTS: The median MICs for ceftriaxone, cefixime, azithromycin, ciprofloxacin, tetracycline and penicillin were <0.002 µg/mL, <0.016 µg/mL, 0.25 µg/mL, 2.0 µg/mL, 1.5 µg/mL and 0.25 µg/mL, respectively. Annual comparison showed that MICs were generally stable for all antimicrobial agents except azithromycin, for which an increase in median MIC was observed from 2017 (0.25 µg/mL). There was no genetic resistance to ceftriaxone; 8% of samples displayed resistance mutations against cefixime, primarily located in the penA gene. Resistance to azithromycin increased from 2% in 2013 to 12% in 2020. MtrD mosaic had the highest impact on azithromycin susceptibility; 47% of the resistant isolates showed this mutation. The majority of cases of gonorrhoea were treated successfully with either ceftriaxone or a ceftriaxone/azithromycin regime. Two treatment failures occurred under monotherapy with doxycycline. Overall, genotypic resistance corresponded significantly to all respective MICs. CONCLUSIONS: The resistance rate of N. gonorrhoeae to antibiotics has remained stable in Vienna over the last decade, except for azithromycin. The strong correlation found between genetic and phenotypic patterns in this study holds promise for future diagnostics of N. gonorrhoeae resistance based on genotypes.

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.

Rifampicin Resistance Associated with rpoB Mutations in Neisseria gonorrhoeae Clinical Strains Isolated in Austria, 2016 to 2020.

Due to increasing rates of antimicrobial resistance (AMR) in Neisseria gonorrhoeae, alternative treatments should be considered. To assess rifampicin's potential as a gonorrhea treatment, we used rpoB mutations to estimate rifampicin resistance in Austrian N. gonorrhoeae isolates. We found 30% of resistant isolates clustering in three main phylogenomic branches. Rifampicin resistance was associated with resistance to other antibiotics. Therefore, rifampicin cannot be recommended as an alternative gonorrhea treatment in Austria, even in combination therapy. IMPORTANCE Gonorrhea, caused by Neisseria gonorrhoeae, is one of the most common bacterial sexually transmitted infections. It is treated with antibiotics, but an increasing number of N. gonorrhoeae strains are resistant to currently used treatments. In this study, we explored the potential of rifampicin, another antibiotic, as a treatment option for gonorrhea. However, around 30% of Austrian N. gonorrhoeae strains investigated were already resistant to rifampicin, which would limit its benefit as a gonorrhea treatment.

Extensively drug-resistant (XDR) Neisseria gonorrhoeae causing possible gonorrhoea treatment failure with ceftriaxone plus azithromycin in Austria, April 2022.

We describe a gonorrhoea case with ceftriaxone plus high-level azithromycin resistance. In April 2022, an Austrian heterosexual male was diagnosed with gonorrhoea after sexual intercourse with a female sex worker in Cambodia. Recommended treatment with ceftriaxone (1 g) plus azithromycin (1.5 g) possibly failed. Worryingly, this is the second strain in an Asian Neisseria gonorrhoeae genomic sublineage including high-level azithromycin-resistant strains that developed ceftriaxone resistance by acquisition of mosaic penA-60.001. Enhanced resistance surveillance and actions are imperative to prevent spread.

A Yersiniabactin-producing Klebsiella aerogenes Strain Causing an Outbreak in an Austrian Neonatal Intensive Care Unit.

BACKGROUND: Yersiniabactin, a siderophore with a high affinity to iron, has been described as a potential virulence factor in Enterobacteriaceae. Klebsiella aerogenes is a Gram-negative rod known to cause invasive infection in very low birth weight infants but is an unusual pathogen to cause outbreaks in neonatal intensive care units (NICU). METHODS: We performed a retrospective analysis of all patients colonized with K. aerogenes in our NICU from September to December 2018. Each infant with an occurrence of K. aerogenes in any microbiological culture was defined as a case. Clinical data were taken from medical charts. K. aerogenes isolates were genotyped using whole-genome sequencing combined with core genome multilocus sequencing type analysis. Yersiniabactin production was evaluated by luciferase assay. RESULTS: In total 16 patients were colonized with K. aerogenes over the 3-month period and 13 patients remained asymptomatic or developed late-onset neonatal sepsis from another pathogen. Three patients developed necrotizing enterocolitis, 2 complicated by sepsis and 1 of them died. All symptomatic patients were premature infants with low birth weight. Genetic sequencing confirmed an outbreak with the same strain, all samples expressed the high-pathogenicity island, necessary for the production of yersiniabactin. Six exemplary cases were proven to produce yersiniabactin in vitro. CONCLUSION: This is the first report of an outbreak of a yersiniabactin-producing K. aerogenes strain causing invasive infection in preterm infants. We hypothesize that, due to improved iron uptake, this strain was associated with higher virulence than non-yersiniabactin-producing strains. Extended search for virulence factors and genetic sequencing could be pivotal in the management of NICU outbreaks in the future.

Comparison of SARS-CoV-2 Antibody Response 4 Weeks After Homologous vs Heterologous Third Vaccine Dose in Kidney Transplant Recipients: A Randomized Clinical Trial.

Importance: Fewer than 50% of kidney transplant recipients (KTRs) develop antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Preliminary data suggest that a heterologous vaccination, combining mRNA and viral vector vaccines, may increase immunogenicity. Objective: To assess the effectiveness of a third dose of an mRNA vs a vector vaccine in KTRs who did not have antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Design, Setting, and Participants: This was a single center, single-blinded, 1:1 randomized clinical trial of a third dose of vaccine against SARS-CoV-2, conducted from June 15 to August 16, 2021, in 201 KTRs who had not developed SARS-CoV-2 spike protein antibodies after 2 doses of an mRNA vaccine. Data analyses were performed from August 17 to August 31, 2021. Interventions: mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as a third dose of a SARS-CoV-2 vaccine. Main Outcomes and Measures: The primary study end point was seroconversion after 4 weeks (29-42 days) following the third vaccine dose. Secondary end points included neutralizing antibodies and T-cell response assessed by interferon-γ release assays (IGRA). In addition, the association of patient characteristics and vaccine response was assessed using logistic regression, and the reactogenicity of the vaccines was compared. Results: Among the study population of 197 kidney transplant recipients (mean [SD] age, 61.2 [12.4] years; 82 [42%] women), 39% developed SARS-CoV-2 antibodies after the third vaccine. There was no statistically significant difference between groups, with an antibody response rate of 35% and 42% for the mRNA and vector vaccines, respectively. Only 22% of seroconverted patients had neutralizing antibodies. Similarly, T-cell response assessed by IGRA was low with only 17 patients showing a positive response after the third vaccination. Receiving nontriple immunosuppression (odds ratio [OR], 3.59; 95% CI, 1.33-10.75), longer time after kidney transplant (OR, 1.44; 95% CI, 1.15-1.83, per doubling of years), and torque teno virus plasma levels (OR, 0.92; 95% CI, 0.88-0.96, per doubling of levels) were associated with vaccine response. The third dose of an mRNA vaccine was associated with a higher frequency of local pain at the injection site compared with the vector vaccine, while systemic symptoms were comparable between groups. Conclusions and Relevance: This randomized clinical trial found that 39% of KTRs without an immune response against SARS-CoV-2 after 2 doses of an mRNA vaccine developed antibodies against the SARS-CoV-2 spike protein 4 weeks after a third dose of an mRNA or a vector vaccine. The heterologous vaccination strategy with a vector-based vaccine was well tolerated and safe but not significantly better than the homologous mRNA-based strategy. Trial Registration: EudraCT Identifier: 2021-002927-39.

Can We Protect Those We Care for in A Pandemic? - Prevalence of Neutralizing Antibodies against SARS-CoV-2 in Nursing Homes.

In December 2019, the People's Republic of China and the World Health Organization first reported on a cluster of pneumonia with an unknown cause. Nine months later more than 1.4 million people have died from COVID 19. In this work, the effects of the COVID 19 pandemic on five nursing homes in Austria, which cared for 889 residents in the first half of 2020, were examined. The research question was whether the measures taken were appropriate to prevent an outbreak within the individual facilities. To detect previously unrecognized infections, the present study evaluated the prevalence of neutralizing antibodies against the SARS-CoV-2 virus in residents and employees of the nursing homes. Following the analysis of blood samples, the prospectively collected data was connected to data from screening examinations and data from contact tracing. The present study demonstrated an overall prevalence of neutralizing antibodies against the SARS-CoV-2 virus in nursing homes of 3.7%. Whereas the prevalence in those facilities that have never been hit by an outbreak is 0%, the prevalence in those facilities with an outbreak is up to 4.9%. Neutralizing antibodies against SARS-CoV-2 were detected in 35 persons. A retrospective analysis of all 5 included nursing homes demonstrated that upon regular clinical screening in combination with PCRs an infection with SARS-COV-2 was detected in 66 residents and 24 employees from different professional groups. In only 25 of the 35 persons with neutralizing antibodies against SARS-CoV-2 an infection was proven in advance. This study suggests that specific measures can prevent transmission within a health care facility. Nevertheless, the results also show that a risk reduction to 0% cannot be achieved. In preparation for further pandemic waves there is still the need to reduce the probability of a transmission in nursing homes with specific test strategies.

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile.

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission.

Tracking the Origin of Austrian Human Brucellosis Cases Using Whole Genome Sequencing.

Brucellosis is a zoonotic disease caused by Brucella spp. and a major concern for livestock. Most human cases are caused by B. melitensis and clinical presentation is usually a mild febrile illness. However, treatment failure is frequent and more severe complications can occur. In Austria, every human brucellosis is investigated to determine whether it was imported from endemic areas or is the sign of an undetected autochthonous transmission. For this study, 21 B. melitensis strains isolated in Austria between 2005 and 2019 were collected, 17 strains from 15 different patients and four strains from cattle. Whole genome sequencing combined with core-genome MLST analysis was used to characterize these strains. A cluster of seven isolates from 2018 (three human and four cattle isolates) was identified, with fewer than two allelic differences. They corresponded to the only Austrian B. melitensis outbreak that happened over the past 15 years. The other 12 Austrian brucellosis cases were single cases, and geographical origins were available for 8/12. Genomic data was used to locate probable geographical origins and compared with the results of the epidemiological investigations. Austrian strains were compared with 67 published B. melitensis sequences available on NCBI. The result of genomic analysis matched for 7/8 cases with documented conclusion of the epidemiological investigation. Genome analysis also pointed to the geographical origin for three of the four cases with missing epidemiological data. Strains from six cases were grouped together (<40 allelic differences) with 4/6 cases imported from the Balkans. Additional B. melitensis isolates from Serbian animals were analyzed and grouped with this branch, suggesting frequent importation from Balkan countries to Austria. Overall, this study highlights the specificities of human brucellosis in Austria. It also underlines the value of whole genome sequencing as a tool to investigate brucellosis cases, allowing to identify and investigate outbreaks but also to support epidemiological investigation of imported cases. However, the reliability of such methods depends on the number of strains for comparison, which can be challenging in low incidence countries. Increasing the availability of published sequences with documented geographical origins would help establishing genomic-based methods for investigating brucellosis cases.

A publicly accessible database for Clostridioides difficile genome sequences supports tracing of transmission chains and epidemics.

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile.