Genomic analysis of Vibrio cholerae O1 isolates from cholera cases, Europe, 2022.

BackgroundThe number of cholera cases reported to the World Health Organization (WHO) in 2022 was more than double that of 2021. Nine countries of the WHO European Region reported 51 cases of cholera in 2022 vs five reported cases in 2021.AimWe aimed to confirm that the Vibrio cholerae O1 isolates reported by WHO European Region countries in 2022 belonged to the seventh pandemic El Tor lineage (7PET). We also studied their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships.MethodsWe used microbial genomics to study the 49 V. cholerae O1 isolates recovered from the 51 European cases. We also used > 1,450 publicly available 7PET genomes to provide a global phylogenetic context for these 49 isolates.ResultsAll 46 good-quality genomes obtained belonged to the 7PET lineage. All but two isolates belonged to genomic Wave 3 and were grouped within three sub-lineages, one of which, Pre-AFR15, predominated (34/44). This sub-lineage, corresponding to isolates from several countries in Southern Asia, the Middle East and Eastern or Southern Africa, was probably a major contributor to the global upsurge of cholera cases in 2022. No unusual AMR profiles were inferred from analysis of the AMR gene content of the 46 genomes.ConclusionReference laboratories in high-income countries should use whole genome sequencing to assign V. cholerae O1 isolates formally to the 7PET or non-epidemic lineages. Periodic collaborative genomic studies based on isolates from travellers can provide useful information on the circulating strains and their evolution, particularly as concerns AMR.

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.

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.

Real-time PCR for single-nucleotide polymorphism detection in the 16S rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis.

OBJECTIVES: A real-time PCR screening system was established for rapid detection of single-nucleotide polymorphisms (SNPs) at positions 1401, 1402 and 1484 of the 16S rRNA gene of Mycobacterium tuberculosis leading to resistance to amikacin, kanamycin and capreomycin. Resistances to the respective drugs may indicate the presence of an extensively drug-resistant (XDR) strain of M. tuberculosis. METHODS: Fifty-seven M. tuberculosis isolates that tested phenotypically susceptible or resistant to amikacin, capreomycin or both were subjected to 1401-2/1484 real-time PCR to screen for SNPs in the respective rrs region. RESULTS: 1401-2 and 1484 wild-type and mutant M. tuberculosis strains displayed distinct melting peaks. Of the cross-resistant strains, 86.7% displayed A1401G SNPs, 76.9% of amikacin-resistant strains did not display rrs SNPs and one capreomycin-resistant strain showed a C1402T SNP. CONCLUSIONS: Phenotypic drug susceptibility testing takes several weeks, but with the 1401-2/1484 real-time PCR a preliminary diagnosis can be made within a few hours. SNPs in the rrs region are not exclusively involved in the development of resistances to amikacin and capreomycin. However, 80.0% of XDR-tuberculosis samples tested were detected with the real-time PCR screening assay of the present study.

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.

Outbreak of Yersiniabactin-producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit.

BACKGROUND: The Gram-negative bacterium Klebsiella pneumoniae is a frequent pathogen causing outbreaks in neonatal intensive care units. Some Enterobacteriaceae can acquire the ability to sequester iron from infected tissue by secretion of iron-chelating compounds such as yersiniabactin. Here we describe an outbreak and clinical management of infections because of a highly virulent yersiniabactin-producing, nonmultiresistant K. pneumoniae strain in a neonatal intensive care unit. Outbreak investigation and effectiveness assessment of multidisciplinary infection control measurements to prevent patient-to-patient transmission of highly pathogenic K. pneumoniae were undertaken. METHODS: Outbreak cases were identified by isolation of K. pneumoniae from blood or stool of infants. Clinical data were abstracted from medical charts. K. pneumoniae isolates were genotyped using whole genome sequencing, and yersiniabactin production was evaluated by luciferase assay. RESULTS: Fourteen cases were confirmed with 8 symptomatic and 6 colonized patients. Symptomatic patients were infants of extremely low gestational and chronologic age with fulminant clinical courses including necrotizing enterocolitis and sepsis. Whole genome sequencing for bacterial isolates confirmed the presence of an outbreak. All outbreak isolates produced yersiniabactin. CONCLUSIONS: Yersiniabactin-producing K. pneumoniae can display a high pathogenicity in extremely premature infants with low chronologic age. This outbreak also underlines the considerable potential of today's infection control systems for recognizing and controlling nosocomial infections in highly vulnerable populations.

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food.

Meat products are prone to adulteration by the replacement of meat from more expensive animal species with meat from cheaper sources. We present a DNA metabarcoding method allowing the identification and differentiation of 15 mammalian and six poultry species in foodstuffs. The method, developed on the MiSeq® platform, targets a mitochondrial 16S rDNA region recently found to be suitable for the differentiation of 300 mammalian species. We designed a novel primer pair for poultry and applied it in combination with the primer pair for mammalian species in a duplex assay. The applicability of the method was investigated by analysing DNA extracts from muscle, DNA extract mixtures and extracts from model sausages. Our results indicated that the species of interest can be identified, differentiated and detected down to a proportion of 0.1%. Since 96 samples can be sequenced in one run, the method has high potential for application in routine analysis.

Babesia species occurring in Austrian Ixodes ricinus ticks.

Babesiosis is a tick-transmitted disease of veterinary and medical importance. The first Austrian case of human babesiosis was recently recorded. In the current study, ticks at all life cycle stages (instars), including 853 Ixodes ricinus and 11 Haemaphysalis concinna ticks, from sampling sites throughout Austria were examined for the presence of Babesia spp. by using 18S rRNA gene PCR and sequencing, and the overall mean infection rate was 51.04%. The infection rates for sampling sites were highly variable, ranging from 0% to almost 100%. Different instars and different sexes were infected almost equally. Babesia isolates occurring in Austrian ticks were identified as Babesia divergens, Babesia divergens-like, and Babesia sp. strain DD by sequencing a fragment of the heat shock protein 70 gene and internal transcribed spacer regions 1 and 2. To our knowledge, this is the first investigation of Babesia spp. in Austrian ticks.

Draft Genome Sequences of Interpatient and Intrapatient Epidemiologically Linked Neisseria gonorrhoeae Isolates.

Neisseria gonorrhoeae is the causative agent of gonorrhea and was identified by the World Health Organization as an urgent public health threat due to emerging antibiotic resistance. Here, we report 13 draft genome sequences of N. gonorrhoeae isolates derived from two epidemiologically linked cases from Austria.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope.

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Draft Genome Sequence of a Vancomycin-Resistant and Vancomycin-Dependent Enterococcus faecium Isolate.

Vancomycin-resistant enterococci have emerged as major nosocomial pathogens worldwide. While antimicrobial pressure promotes nosocomial colonization with these enterococci, prolonged exposure to vancomycin may foster the transition from vancomycin resistance to vancomycin dependence. Here, we report the draft genome sequence of a vancomycin-dependentEnterococcus faeciumisolate showing partial teicoplanin dependence.

Draft Genome Sequence of Legionella jamestowniensis Isolated from a Patient with Chronic Respiratory Disease.

Legionella jamestowniensis can be found in the environment in various water samples, in wet soil, and in compost facilities, but evidence of its human pathogenicity has not yet been demonstrated. Here, we report the first draft genome sequence of an L. jamestowniensis isolate, derived from a patient suffering from a chronic respiratory disease.

Whole genome sequence-based serogrouping of Listeria monocytogenes isolates.

Whole genome sequencing (WGS) is currently becoming the method of choice for characterization of Listeria monocytogenes isolates in national reference laboratories (NRLs). WGS is superior with regards to accuracy, resolution and analysis speed in comparison to several other methods including serotyping, PCR, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable number tandem repeat analysis (MLVA), and multivirulence-locus sequence typing (MVLST), which have been used thus far for the characterization of bacterial isolates (and are still important tools in reference laboratories today) to control and prevent listeriosis, one of the major sources of foodborne diseases for humans. Backward compatibility of WGS to former methods can be maintained by extraction of the respective information from WGS data. Serotyping was the first subtyping method for L. monocytogenes capable of differentiating 12 serovars and national reference laboratories still perform serotyping and PCR-based serogrouping as a first level classification method for Listeria monocytogenes surveillance. Whole genome sequence based core genome MLST analysis of a L. monocytogenes collection comprising 172 isolates spanning all 12 serotypes was performed for serogroup determination. These isolates clustered according to their serotypes and it was possible to group them either into the IIa, IIc, IVb or IIb clusters, respectively, which were generated by minimum spanning tree (MST) and neighbor joining (NJ) tree data analysis, demonstrating the power of the new approach.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the ß-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Rifaximin disc diffusion test for in vitro susceptibility testing of Clostridium difficile.

Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these, 184 isolates from a strain collection were tested by an in-house rifaximin disc (40 µg) diffusion test, by an in-house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ≥32 µg ml(-1) as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n = 1) to ≥1024 µg ml(-1) (n = 61); 62 isolates showed a reduced susceptibility (MIC ≥32 µg ml(-1)). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of >1024 µg ml(-1), rifaximin end-point MICs were determined by broth dilution: 4096 µg ml(-1) (n = 2), 8192 µg ml(-1) (n = 6), 16,384 µg ml(-1) (n = 2) and 32,678 µg ml(-1) (n = 1). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ≤0.002 (n = 117) to ≥32 µg ml(-1) (n = 59). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ≥32 µg ml(-1). There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ≥32 µg ml(-1) as breakpoint for reduced rifampicin susceptibility and a <38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin--despite its water-insolubility--may be a suitable candidate for disc diffusion testing.

Mechanisms behind variation in the Clostridium difficile 16S-23S rRNA intergenic spacer region.

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S-23S rRNA intergenic spacer region (16S-23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S-23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S-23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S-23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S-23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNA(Ala) gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S-23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.

First detection of Rickettsia helvetica in Ixodes ricinus ticks in Austria.

A total of 853 Ixodes ricinus ticks collected from the nine federal states of Austria were examined by molecular methods for possible infections with Rickettsia spp. It was shown that roughly one-third of the ticks were infected with Rickettsia spp. Moreover, Rickettsia helvetica was detected in Austria for the first time.

Detection of a serine proteinase gene in Acanthamoeba genotype T6 (Amoebozoa: Lobosea).

Cytopathic proteins are assumed to contribute to the pathogenicity of Acanthamoeba spp. due to their degrading capacity that is required for tissue invasion. In this study, a serine proteinase gene was demonstrated in a highly virulent Acanthamoeba keratitis causing strain with genotype T6. This gene was detected in both, the genomic DNA and the cDNA by PCR and subsequent sequencing. The gene fragment comprises about 500 bp and exhibits high sequence similarity to the serine proteinases of Acanthamoeba strains with genotype T4 and T12. The detection of a serine proteinase in this Acanthamoeba T6 strain is significant, because while T4 is the most common genotype among pathogenic Acanthamoeba strains and also T12 is known to be associated with disease, this is the only virulent Acanthamoeba T6 strain known to date. Obviously, this serine proteinase represents a common tool in pathogenic processes during Acanthamoeba infection.

ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa).

The subgenus classification of the ubiquitously spread and potentially pathogenic acanthamoebae still poses a great challenge. Fifteen 18S rDNA sequence types (T1-T15) have been established, but the vast majority of isolates fall into sequence type T4, and so far, there is no means to reliably differentiate within T4. In this study, the first internal transcribed spacer (ITS1), a more variable region than the 18S rRNA gene, was sequenced, and the sequences of 15 different Acanthamoeba isolates were compared to reveal if ITS1 sequence variability correlates with 18S rDNA sequence typing and if the ITS1 sequencing allows a differentiation within T4. It was shown that the variability in ITS1 is tenfold higher than in the 18S rDNA, and that ITS1 clusters correlate with the 18S rDNA clusters and thus corroborate the Acanthamoeba sequence type system. Moreover, high sequence dissimilarities and distinctive microsatellite patterns could enable a more detailed differentiation within T4.