BackgroundListeria (L.) monocytogenes is primarily transmitted via contaminated food and can cause listeriosis, an infection often associated with sepsis and meningitis in at-risk individuals. Accurate outbreak detection relies on whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), which use allele thresholds to identify related strains.MethodsThis study investigated mutation rates in L. monocytogenes, focusing on isolates with DNA repair deficiencies. Serial subcultivations were performed, comparing a repair-deficient isolate with a wild-type control. Genetic variability was assessed using WGS and cgMLST.ResultsMutation rates were significantly higher in repair-deficient isolates, exceeding typical cgMLST thresholds currently used in Listeria outbreak investigations, leading to a misclassification of related isolates as unrelated. An additional analysis of the Austrian Listeria database revealed that such deficiencies are rare among isolates.ConclusionsThe standard 7-allele cgMLST threshold effectively identifies related strains in most cases, but may require adjustments for hypermutator strains. Incorporating DNA repair data could improve the accuracy of outbreak investigations, ensuring reliable public health responses.

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.

Here, we report the complete genome of Bordetella parapertussis strain 400431-b isolated from a nasopharyngeal swab from a 4-year-old patient presenting with a protracted course of whooping cough, vaccinated with three doses of diphtheria-tetanus-acellular pertussis-hepatitis B-poliomyelitis-Haemophilus influenzae type b vaccine.

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.

BACKGROUND:Men who have sex with men (MSM) are more vulnerable to acquiring sexually transmitted infections (STIs). In 2019, for instance, 74% of European Neisseria gonorrhoeae (Ng) cases among males affected MSM. A recent report by the World Health Organization showed that most of the 2020' interim targets to end STIs by 2030 had not been met. A broadened understanding of STI transmission networks could guide future elimination strategies and reduce the STI burden. Therefore, we used whole-genome sequencing (WGS) to determine Ng-clusters and assess sexual mixing. METHODS:WGS was performed on Ng-isolates collected at the Medical University of Vienna, Austria and was used for core genome multi-locus sequencing typing cluster analysis. Epidemiologic and infection-specific details were extracted from medical records. RESULTS:Genomic analysis and demographic data were available for 415 isolates, and 43.9% (182/415) were allocated to 31 Ng-clusters. Nine clusters comprised samples from heterosexual individuals only (women N = 4, human immunodeficiency virus (HIV)-negative men N = 49, HIV-positive man N = 1), nine clusters included MSM only (HIV-negative N = 22, HIV-positive N = 13) and 13 clusters included both heterosexuals and MSM (HIV-negative N = 75, HIV-positive N = 18). Current use of HIV pre-exposure prophylaxis (PrEP) was reported by 22.8% of MSM. In multivariate analysis, only 'MSM' predicted clustering with isolates from HIV-positive individuals (adjusted odds ratio 10.24 (95% CI 5.02-20.90)). CONCLUSIONS:Sexual mixing of HIV-positive, HIV-negative MSM and non-MSM was frequently observed. Furthermore, HIV-serodiscordant clustering highlights the importance of PrEP rollout to avert HIV transmission. Our findings can inform future STI prevention strategies and continuous surveillance efforts are required to keep up with transmission dynamics.

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.

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.

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.

SUMMARY The molecular mechanisms by which the gut microbiome influences human health remain largely unknown. Pseudomonadota is the third most abundant phylum in normal gut microbiomes. Several pathogens in this phylum can inject so-called virulence effector proteins into host cells. We report the identification of intact type 3 secretion systems (T3SS) in 5 - 20% of commensal Pseudomonadota in normal human gut microbiomes. To understand their functions, we experimentally generated a high-quality protein-protein meta-interactome map consisting of 1,263 interactions between 289 bacterial effectors and 430 human proteins. Effector targets are enriched for metabolic and immune functions and for genetic variation of microbiome-influenced traits including autoimmune diseases. We demonstrate that effectors modulate NF-κB signaling, cytokine secretion, and adhesion molecule expression. Finally, effectors are enriched in metagenomes of Crohn’s disease, but not ulcerative colitis patients pointing toward complex contributions to the etiology of inflammatory bowel diseases. Our results suggest that effector-host protein interactions are an important regulatory layer by which the microbiome impacts human health.

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.

Enterococcus dispar was isolated for the first time from synovial fluid and stool cultures and described as a new species in 1991. Here, we report the genome of E. dispar CoE-457-22, which was obtained from traditionally produced Montenegrin dry sausage (sudzuk).

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.

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.

The genetic mechanisms associated with acquisition of linezolid (LZD) resistance are diverse, including point mutations in the V domain of the 23S rRNA and the 50S ribosomal proteins as well as cfr, optrA, and/or poxtA genes, which may be plasmid- or chromosomally encoded. The aim of this study was to investigate through Whole Genome Sequencing (WGS)-based typing the presence and location of genes and point mutations associated with LZD resistance in two Enterococcus faecalis isolates from Upper Austrian patients. The isolates were retrieved during screening by LZD disk diffusion test of a total of 911 clinical E. faecalis isolates in 2017. The two E. faecalis isolates had LZD minimum inhibitory concentrations of 8 and 32 mg/L and were optrA-positive (ST476 and ST585). Bioinformatic analysis revealed the presence of optrA located in the chromosome of both isolates. One isolate carried the optrA gene in the transposon 6674, previously reported as chromosomally encoded, and the second isolate in fragments originating from the integrative plasmid pEF10748. Additional mechanisms of LZD resistance on the 23S rRNA and the 50S ribosomal proteins were detected. None of the patients reported travels to geographical areas with high LZD resistance or previous LZD treatments. This is the first report of optrA carrying E. faecalis, including characterization by WGS from Austria. LZD resistance in a low-prevalence setting is of concern and should be further monitored.

Abstract Motivation Protein orthologous group databases are powerful tools for evolutionary analysis, functional annotation or metabolic pathway modeling across lineages. Sequences are typically assigned to orthologous groups with alignment-based methods, such as profile hidden Markov models, which have become a computational bottleneck. Results We present DeepNOG, an extremely fast and accurate, alignment-free orthology assignment method based on deep convolutional networks. We compare DeepNOG against state-of-the-art alignment-based (HMMER, DIAMOND) and alignment-free methods (DeepFam) on two orthology databases (COG, eggNOG 5). DeepNOG can be scaled to large orthology databases like eggNOG, for which it outperforms DeepFam in terms of precision and recall by large margins. While alignment-based methods still provide the most accurate assignments among the investigated methods, computing time of DeepNOG is an order of magnitude lower on CPUs. Optional GPU usage further increases throughput massively. A command-line tool enables rapid adoption by users. Availabilityand implementation Source code and packages are freely available at https://github.com/univieCUBE/deepnog. Install the platform-independent Python program with $pip install deepnog. Supplementary information Supplementary data are available at Bioinformatics online.

Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (PPpAOX1). The HpFMD promoter even surpassed PPpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced PPpAOX1. These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris.

The increasing emergence of multi-resistant bacteria in healthcare settings, in the community and in the environment represents a major health threat worldwide. In 2016, we started a pilot project to investigate antimicrobial resistance in surface water. Bacteria were enriched, cultivated on selective chromogenic media and species identification was carried out by MALDI-TOF analysis. From a river in southern Austria a methicillin resistant Staphylococcus aureus (MRSA) was isolated. Whole genome sequence analysis identified the isolate as ST8, spa type t008, SCCmecIV, PVL and ACME positive, which are main features of CA-MRSA USA300. Whole genome based cgMLST of the water isolate and comparison to 18 clinical MRSA USA300 isolates from the Austrian national reference laboratory for coagulase positive staphylococci originating from 2004, 2005 and 2016 and sequences of 146 USA300 isolates arbitrarily retrieved from the Sequence Read Archive revealed a close relatedness to a clinical isolate from Austria. The presence of a CA-MRSA USA300 isolate in an aquatic environment might pose a public health risk by serving as a potential source of infection or a source for emergence of new pathogenic MRSA clones.