Genomic studies of Borrelia are essential for understanding Lyme borreliosis epidemiology and biology but are limited by slow, labor-intensive culture methods. Here, we introduce an approach that overcomes the need for extended culture by combining a short-term culture step with whole-genome amplification (WGA) of samples derived from field-collected ticks, which can be performed in parallel with classical culturing. The protocol is paired with a tailored bioinformatic pipeline to ensure accurate assembly and robust downstream analyses. Benchmarking on multiple control isolates shows that the method produces high-quality chromosomal assemblies. We further demonstrate applicability by generating five high-quality Borrelia chromosomes from freshly collected ticks (two B. lusitaniae, two B. afzelii, and one B. garinii). By producing sequencing-ready DNA in 5 days rather than months, this workflow streamlines genome generation and reduces reliance on culture-based isolation, facilitating broader genomic representation of understudied Borrelia species and enabling future epidemiological, ecological, and evolutionary studies.

The Borrelia burgdorferi s.l. complex is known for its complex genome and significant genetic variability. We used multilocus sequence typing (MLST) to study the genetic variability of Borrelia in five European countries: Slovakia, Croatia, Ukraine, Finland and Bulgaria. We compared the prevalence, genospecies spectrum and genetic variability of Borrelia in 4013 questing Ixodes ricinus ticks. The highest prevalence of Borrelia was found in Slovakia (26.7 %) and Ukraine (22 %), while in Finland and Bulgaria the prevalence was found to be 12.4 % and 7.2 %, respectively. Genospecies diversity was the highest in Croatia with seven identified genospecies. This was in contrast to Finland and Bulgaria where only three species were detected. In phylogenetic trees Borrelia species clustered according to species designation. Borrelia afzelii and Borrelia garinii were the most prevalent amongst the Borrelia species and were found in all collection regions. Borrelia garinii but also B. afzelii isolates did not show a phylogeographic pattern in minimum spanning trees. Based on MLST, Borrelia lusitaniae isolates from Slovakia, Croatia, Ukraine and Bulgaria belonged to the “northern European” clade, which differs genetically from B. lusitaniae isolates found in southern Portugal and northern Africa. Borrelia bavariensis from the Slovak Republic and Ukraine firmly clustered with Western European isolates. Besides questing ticks, Edible dormice (Glis glis) earlobe biopsies were analyzed as B. afzelii clustering with other European isolates of this species. In this study we report the first evidence of Borrelia genospecies in the following countries: Borrelia spielmanii, B. bavariensis and B. lusitaniae in Ukraine; B. spielmanii in Bulgaria; Borrelia burgdorferi s.s., B. spielmanii, B. lusitaniae and B. bavariensis in Croatia.

OBJECTIVES:Diagnosis of Lyme borreliosis (LB) is mainly based on clinical symptoms, patient's history, and serological testing. Despite a lack of standardization, polymerase chain reaction (PCR) has gained importance for the detection of Borrelia DNA. So far, extensive studies comparing different protocols used in molecular diagnostic or research setting are missing. Here, we describe a European-wide comparison of commercial and in-house PCR protocols using a standardized DNA panel including all relevant Borrelia burgdorferi sensu lato (Bbsl) species to explore variation in PCR results in different laboratories. METHODS:A DNA testing panel composed of 90 DNA samples from 14 Bbsl strains in six different dilutions, plus six specificity controls, was sent blinded to 34 laboratories (33 European plus one in the US). The results from a total of 57 different amplification protocols were collected and compared regarding their detection limits, specificity, and sensitivity over all Bbsl strains for each dilution included in the DNA panel. RESULTS:While the detection limits showed vast differences (>105 genome equivalents (GE)) between different amplification protocols, none of the most commonly used PCR targets (ospA, 16S rRNA, flagellin, 5S-23S intergenic spacer) significantly outperformed other PCR targets. Interestingly, large differences in detection limits were found not only between different protocols, but also when the same protocol was used on different Bbsl species (>105 GE) and even on different strains of the same Bbsl species (>104 GE). Specificity also varied between different protocols, with many protocols recognizing relapsing fever Borreliae. CONCLUSION:A standardization of PCR methods used for confirmation of LB diagnosis is urgently needed.

The recently described Corynebacterium (C.) silvaticum is a diphtheria toxin (tox-) gene bearing species so far mainly found in wild boar and deers. Due to its close relationship to C. ulcerans a zoonotic potential has been proposed. To date, only two cases of human C. silvaticum infections have been reported in the literature. Here we present the case of a 63-year-old male game-hunting butcher with recurrent and difficult-to-treat axillary lymphadenitis and abscess formation. This case underlines the zoonotic potential of this novel member of the C. diphtheriae species complex and suggests zoonotic transmission from wild boars via direct contact to infectious tissue and possible microtrauma lesions during unprotected animal processing.

In this project, we compared whole-genome Borrelia assemblies using reads produced by Oxford Nanopore Technology (ONT) with R9 and R10 chemistry and flow cells to investigate potential improvements between versions. For this, we sequenced two B. garinii isolates and one B. tillae isolate using ONT (R9 and R10) and reconstructed the genome using three different assemblers (Canu, Flye, Shasta). The generated consensus sequences were compared to references (Illumina for B. garinii, PacBio for B. tillae). A general improvement of genome assemblies between R9 and R10 ONT versions was visible for both B. garinii isolates and B. tillae. This improvement was observed for all assemblers and can be clearly seen in the completeness and the decrease in the number of gaps. Using ONT, it was possible to generate at least one contig for each expected plasmid for both isolates of B. garinii, and even close some gaps that were present in Illumina references (Illumina-REF). Additionally, previously undefined contigs of the Illumina-REF assembly could be identified as parts of plasmid lp28-2, lp28-3, and lp32-10 based on the ONT assemblies. For the more complex genome of B. tillae, ONT was only able to generate complete linear plasmids, but all circular plasmids containing sequence stretches of high similarity described in a PacBio reference assembly resulted in a “wrong fusion”. For all samples the use of different assemblers (Canu and Flye) was necessary to generate a whole genome sequence, with choice of assembler used for the final consensus sequence of individual genome elements varying between Borrelia species. ONT R10 chemistry and flow cells greatly improved assembly of B. garinii genomes lacking complex circular plasmids and even fully resolved incomplete linear plasmids (gap closure) generated using Illumina sequencing. However, circular plasmids of B. tillae having sequence stretches of high similarity were not properly assembled. This implies that additional sequencing methods are needed when dealing with complex bacterial genomes.

BACKGROUND:Recently a Corynebacterium diphtheriae (C. diphtheriae) outbreak was reported in Europe described as the largest upsurge of cases in the last 70 years. Clinically relevant infections and transmission events are not necessarily associated only with toxigenic strains, but they can also be caused by non-toxigenic diphtheria toxin gene-bearing (NTTB) C. diphtheriae. PCR-based identification of potentially toxigenic strains needs to be complemented by testing for diphtheria toxin (DT) production, which is why diagnosis is notoriously difficult. METHODS/FINDINGS:C. diphtheriae was cultivated from an intraoperative swab. The strain although PCR positive for tox gene, lacked DT expression in Elek test and was thus categorized as non-toxigenic tox-bearing (NTTB). Short- and long-read whole-genome sequencing (WGS) revealed a truncated tox gene. At nucleotide position 19, the insertion sequence IS1132 was inserted in the reverse direction. The isolate had a novel sequence type, ST-1111, and core genome MLST (cgST-3956 in BIGSdb-Pasteur's nomenclature) showed it is not related to any of the previously described C. diphtheriae outbreak strains in Germany. INTERPRETATION:Here we report a novel ST of NTTB C. diphtheriae with interruption of its tox gene by IS1132 at nucleotide position 19. Our findings highlight the value of WGS for resolving potentially toxin-positive cases and for reliably assessing the clinical risk and epidemiological context, which are the key prerequisites for infection control measures and public health responses.

ABSTRACT The Corynebacterium (C.) diphtheriae species complex (CdSC) comprises the closely related pathogenic species C. diphtheriae, C. belfantii, C. rouxii, C. ulcerans, C. silvaticum, C. ramonii, and C. pseudotuberculosis according to the taxonomic revisions after 2018. Due to their close relationship, reliable species identification within the CdSC is also challenging using MALDI-TOF mass spectrometry (MS) for fast and reliable identification of bacteria. This is relevant for human and veterinary medicine. However, the Bruker MALDI-Biotyper (MBT), a commonly used MALDI-TOF MS system, does not reflect the current CdSC taxonomy, which includes C. belfantii, C. ramonii, C. rouxii, and C. silvaticum as additional Corynebacterium species in the CdSC. Therefore, the objective of the present study is to expand the commercial Bruker database version K (MBTK) in order to achieve reliable identification for all currently known Corynebacterium species within the CdSC. Furthermore, the database expansions were verified with a systematic formal validation procedure in accordance with the German guideline for species identification by MALDI-TOF MS. For this, 328 spectra made from 321 strains including all seven valid species of the CdSC were identified using the MBTK database and a customer-supplemented modified MBTK database. This custom database version included 33 reference spectra of the CdSC covering all member species. The results of the formal validations for both the commercial and the extended database, combined partly with a single signal analysis, provide a substantiated basis for the identification of bacteria of the CdSC. A key element for a fast up-to-date identification has been the exchange of user-generated sets of main spectrum profiles (MSPs) on the open access MALDI User Platform (MALDI-UP) catalog.IMPORTANCEBacteria belonging to the Corynebacterium (C.) diphtheriae complex are relevant pathogens for humans and animals with zoonotic potential. Identification of the novel species of this complex described since 2018, that is, C. belfantii, C. rouxii, C. ramonii, and C. silvaticum, is possible using the widely approved MALDI-TOF mass spectrometry. This is important for targeted therapies, preventive measures, and epidemiological studies. Precise species identification has been achieved by using a supplemented Bruker MALDI reference database. Comprehensive validation of database entries meets the requirements for accurate pathogen identification in accordance with recognized guidelines. This approach to community-driven database expansion and validation is certainly a model for other bacterial species of interest to react to current taxonomic revisions in a short time frame.

Zusammenfassung Die Diagnose der Lyme-Borreliose basiert primär auf charakteristischen klinischen Kriterien, das bedeutet: Die mikrobiologische Diagnostik ist nur bei Vorliegen charakteristischer Symptome angezeigt. Wichtigste Methode ist der Antikörpernachweis, nachgeordnet sind Polymerase-Kettenreaktion (PCR) und Anzucht – wobei das typische Erythema migrans keiner mikrobiologischen Diagnostik bedarf. Um eine hohe Spezifität sicherzustellen, wird der Antikörpernachweis als 2-Stufen-Diagnostik durchgeführt, wobei der als zweite Stufe eingesetzte Immunoblot auch Aussagen bzgl. des Erkrankungsstadiums erlaubt: Frühe Manifestationen zeigen ein enges Bandenspektrum (OspC, VlsE, p41), späte Formen dagegen ein breites Spektrum (u. a. p83, p58, p39). Bei späten Erkrankungsformen sind praktisch immer IgG-Antikörper (Ig: Immunglobulin) nachweisbar, IgM-Antikörper sind hier diagnostisch nicht relevant. Nur positives IgM (bei negativem IgG) spricht gegen die Diagnose einer späten Erkrankungsform.

Lyme borreliosis is caused by species of the Borrelia burgdorferi sensu lato complex. Genospecies vary in human pathogenicity, reservoir host and tick vector adaptation. What causes these differences is currently unknown, but some answers are likely to be found within the fragmented genomes containing linear and circular plasmids (and even fusions). These plasmids are highly variable and challenging to reconstruct completely. We aimed to analyze plasmid presence/absence and to identify trends in plasmid numbers and/or types associated with specific ecological adaptations or pathogenic potentials. For this, we used the most complete, reliable, and currently available 86 B. burgdorferi s.l. genomes (30 newly sequenced, 56 publicly available) belonging to 21 of the 28 known species. The plasmid types per isolate vary from 6-21 (median: 12) and linear plasmid numbers (3-12, median: 7) are significantly higher than circular plasmids (2-11, median: 5). The only plasmids found in all isolates are lp54 and cp26 and only a few species- or population-specific plasmids are identified, demonstrating the high variability. Species with smaller plasmid counts (median < 11) tend to have fewer lp28 and cp32 plasmids than larger plasmid counts species (median ≥ 15). Significantly higher total and circular plasmid numbers are found in rodents-only compared to birds-only associated species. Interestingly, plasmid numbers are not significantly reduced in species associated with a single reservoir host/vector compared to multiple. Analyses of the plasmid-location of the vls/vlsE antigenic variation system highlighted the dynamic nature of B. burgdorferi s.l. plasmids. Surprisingly, whilst the plasmid repertoire of B. burgdorferi s.l. is very plastic and variable, our analyses rarely reveal plasmids associated with specific ecological adaptations or pathogenic potentials. However, some plasmids were more frequently found in one of the groups and will be of interest for gene-based analyses that will be required for further progress in this research area.

Historically, a considerable number of diphtheria outbreaks, mainly in the UK and the USA, have been epidemiologically or bacteriologically linked to the consumption of milk and dairy products. However, with the introduction of milk pasteurization and hygienic improvements in livestock farming and animal welfare, no cases of milk-linked human diphtheria outbreaks or bovine mastitis due to toxigenic corynebacterial have been reported in the recent decades. Here we report the first isolation of a toxigenic Corynebacterium ulcerans strain from the milk of a cow with acute mastitis within nearly 40 years and outside of UK or Finland. The isolated strain was analysed by state-of-the-art bacteriological methods including toxigenicity testing by a novel Lateral Flow Immunoassay and Elek test, molecular typing was done by whole genome sequencing and MLST/cgMLST analysis. Due to the rarity of our finding, an extensive historical review of milk-associated diphtheria outbreaks was performed. The obtained sequence type ST-331 is also found in human isolates of cutaneous diphtheria. cgMLST analysis, however, found no close relationship to 43 human ST-331 isolates from our German strain collection or to two animal samples from a zoonic cluster of this ST. The risk of milk-associated diphtheria due to C. ulcerans, although today extremely rare, should be avoided by milk pasteurization and the respective hygienic standards.

In the Northern Hemisphere, annual waves of influenza disease with varying degrees of spread and severity are observed each winter. With wastewater-based surveillance (WBS), including both centralized (one wastewater treatment plant, WWTP) and decentralized (three sewers) sampling, we aimed to detect differences in influenza viral copy numbers in wastewater over time, to investigate (sub)-community transmission within a city. A total of 313 grab/spot and composite samples were collected in Munich, Germany, during three consecutive influenza seasons (2022/23, 2023/24, and 2024/25) and were analyzed for influenza A virus (IAV) and influenza B virus (IBV) nucleic acids using digital droplet PCR (ddPCR). IAV and IBV wastewater copy numbers and citywide reported influenza cases showed strong correlations in both sampling approaches, suggesting the decentralized approach to be a reliable indicator of infection trends across the city. The three influenza seasons analyzed differed significantly in terms of their seasonal distribution, for example, exhibiting a strong co-circulation of IAV and IBV only in the 2024/25 season. Only with wastewater analysis, we reveal a reporting delay of influenza A cases at the beginning of the 2023/24 season. Higher influenza copy numbers were detected in sewer samples compared to the WWTP influent, likely due to viral decay. The study underscores the potential of influenza WBS to enable detection of seasonal onset early, identify local transmission patterns, and reveal underreporting in routine surveillance systems.

We report 2 human Corynebacterium silvaticum infections in Germany with axillary lymphadenitis and abscess formation; in 1 case the infection likely originated from a slaughtered wild boar. This recently described member of the diphtheria toxin gene-bearing C. diphtheriae species complex might be a new zoonotic pathogen.

Within a Corynebacterium diphtheriae ST-574 outbreak comprising mostly migration-associated cases since 2022, Germany detected two sub-clusters in 2025. Among sub-cluster cases with travel information (24/26), 22 were acquired autochthonously. One of two imported cases came from a Polish voivodeship having an additional case. Sub-cluster 1 included 16 cutaneous cases, sub-cluster 2 included six cutaneous and five respiratory cases. One unvaccinated child and two older adults died. Of 27 cases, 12 experienced homelessness. Strengthened immunisation, improved wound care and international surveillance are needed.

Bacteria of the Borrelia burgdorferi sensu lato (sl) species complex can cause Lyme borreliosis (LB) in humans. PCR plays an important role in the diagnosis of many infectious diseases but it is used auxiliary in LB diagnostics. Here, we re-analysed a previously published real-time PCR targeting the multicopy gene of the large subunit of phage terminase (terL) in Borrelia. We analysed cultured material of Borrelia burgdorferi sl species, serum and clinical tissue samples of LB patients. PCR conditions were as previously described by Shan et al. 2021 but we also investigated PCR modifications. PCR on cultured specimens showed that whilst all samples of B. burgdorferi sensu stricto (ss) gave a positive result, not all isolates of Borrelia species causing LB in Europe (i.e. B. afzelii, B. garinii) were detected by the terL PCR. Only slight differences in Ct values were detected between PCR runs using the original ZEN/IFBQ double quencher probe compared to other double quencher probes or single quencher probes. Contrary to the hypothesis expressed by the authors of the original paper that the PCR could detect phage DNA in serum, our data show that the terL PCR was negative on all tested serum samples of individuals diagnosed with proven LB. Furthermore, using patient's tissue samples not all infections with B. afzelii or B. garinii were detected, similar to the results obtained with cultured material or serial DNA dilutions of Borrelia species. We conclude, that the terL PCR in its current form is unsuitable for LB diagnosis in Europe.

Background:Classified by the WHO as one of the 19 most dangerous fungal pathogens, Pneumocystis jirovecii has been associated with increasing outbreaks of Pneumocystis pneumonia (PCP) among solid organ transplant (SOT) recipients worldwide. Mycophenolic acid (MPA), an inosine monophosphate dehydrogenase (IMPDH) inhibitor commonly used as an immunosuppressant to prevent organ rejection, is a risk factor for PCP. However, MPA also displays antifungal activity, potentially protecting against PCP, despite not being used to treat it. Therefore the underlying factors driving these outbreaks remain undefined. Methods:In this international multicenter retrospective observational study, P. jirovecii samples were collected from 96 SOT patients (including 94 from nine separate outbreaks and 84 on MPA therapy) and 67 non-transplant controls (none on MPA), between 1986 and 2020 across six countries in Europe, North America and Asia. All samples underwent extensive targeted sequencing of the P. jirovecii inosine monophosphate dehydrogenase (impdh) gene and multiple genetic markers, with selected samples further analyzed for complete mitogenome and restriction fragment length polymorphisms. Computational modeling was employed to predict the effects of IMPDH mutations on protein structure and MPA binding. Results:Six impdh mutations (including one previously reported) were identified, with frequencies of 4-21% each in SOT patients and 0-1% in controls. These mutations were strongly associated with prior MPA exposure and showed marked geographic segregation and temporal shifts. Four mutations were each linked to multiple distinct genotype profiles, representing separate P. jirovecii strains. Structure modeling predicted that these four mutations reduced protein stability and binding affinity to MPA. Conclusions:This study suggests that the widespread use of MPA in SOT recipients has unexpectedly driven the emergence of multiple impdh mutations in P. jirovecii, each presumably arising independently in multiple strains worldwide. These mutations likely confer drug resistance and provide a selective survival advantage to P. jirovecii in SOT recipients exposed to MPA, thereby facilitating transmission and outbreaks. These findings have significant implications for the prevention and clinical management of PCP in SOT recipients, highlighting a rare example of how antimicrobial resistance can emerge through unexpected pathways, transcending conventional antimicrobial use and emphasizing the need for increased vigilance and strategic adaptation in clinical practice.

In September 2024, a school-aged child (P1), unvaccinated against diphtheria, was hospitalised with severe respiratory diphtheria caused by toxigenic Corynebacterium diphtheriae. P1 subsequently died from the disease. The child’s mother (P2) had pharyngitis 9 days before the onset of symptoms of P1 and subsequently tested positive for C. diphtheriae. In multilocus sequence typing (MLST), the C. diphtheriae isolates from P1 and P2 were of sequence type (ST) 574. In core genome (cg)MLST, they were clonal, suggesting recent human-to-human transmission. This indicates the first autochthonous respiratory diphtheria outbreak by toxigenic C. diphtheriae in Germany since 1984 with epidemiologically- and molecularly-confirmed transmission. Furthermore, the isolates were close to isolates from patients with cutaneous diphtheria among people experiencing homelessness in two major German cities in 2023 and 2024, and to isolates from an outbreak among newly arriving migrants across several European countries, including Germany, detected in 2022. This indicates transmission beyond vulnerable groups. Our findings illustrate the potential of C. diphtheriae spreading further from patients with cutaneous diphtheria and even causing outbreaks of respiratory diphtheria. Given the potentially serious complications of respiratory diphtheria, including death, equitably achieving and maintaining high vaccination coverage among the whole population, especially among vulnerable people is essential.

PURPOSE:Diphtheria is a re-emerging vaccine-preventable disease, mainly caused by toxigenic Corynebacterium diphtheriae. METHODS:Here, we report a fatal respiratory diphtheria infection in a Polish woman travelling to Germany possibly linked to a cutaneous diphtheria infection in a homeless man living in the same geographic area. Laboratory diagnostics involving MALDI-TOF MS, tox-gene PCR, Lateral Flow Immuno Assay, a modified Elek test and Next Generation Sequencing (NGS) identified the causative strain as cotrimoxazole-resistant toxigenic Corynebacterium diphtheriae biotype mitis, sequence type ST574. Moreover, a review on the diphtheria situation in Poland is presented. RESULTS:NGS data suggest a common source of infection in Poland and a possible link to the Europe-wide outbreak of imported diphtheria with C. diphtheriae since 2022. This is the first diphtheria case in Poland since 2000.