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.

Oxazolidinone Resistance Mediated by optrA in Clinical Enterococcus faecalis Isolates in Upper Austria: First Report and Characterization by Whole Genome Sequencing.

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.

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

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

Draft Genome Sequences of Six Corynebacterium ulcerans Strains Isolated from Humans and Animals in Austria, 2013 to 2019.

Corynebacterium ulcerans is an emerging pathogen responsible for severe diseases in humans and animals. Here, we present the draft genome of six C. ulcerans strains isolated in Austria. These draft genomes have 2,446,822 to 2,551,141 bp encoding 57 to 60 RNAs.

Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant Klebsiella pneumoniae isolates from Austrian rivers and clinical isolates from hospitals.

In 2016, the Austrian Agency for Health and Food Safety started a pilot project to investigate antimicrobial resistance in surface water. Here we report on the characterisation of carbapenem resistant and ESBL-producing K. pneumoniae isolates from Austrian river water samples compared to 95 clinical isolates recently obtained in Austrian hospitals. Ten water samples were taken from four main rivers, collected upstream and downstream of major cities in 2016. For subtyping and comparison, public core genome multi locus sequence typing (cgMLST) schemes were used. The presence of AMR genes, virulence genes and plasmids was extracted from whole genome sequence (WGS) data. In total three ESBL-producing strains and two carbapenem resistant strains were isolated. WGS based comparison of these five water isolates to 95 clinical isolates identified three clusters. Cluster 1 (ST11) and cluster 2 (ST985) consisted of doublets of carbapenem resistant strains (one water and one clinical isolate each). Cluster 3 (ST405) consisted of three ESBL-producing strains isolated from one water sample and two clinical specimens. The cities, in which patient isolates of cluster 2 and 3 were collected, were in concordance with the water sampling locations downstream from these cities. The genetic concordance between isolates from river water samples and patient isolates raises concerns regarding the release of wastewater treatment plant effluents into surface water. From a public health perspective these findings demand attention and strategies are required to minimize the spread of multiresistant strains to the environment.

Limited value of routine spa typing: a cross-sectional study of methicillin-resistant Staphylococcus aureus-positive patients in an Austrian hospital.

BACKGROUND: We investigated whether spa typing is useful for indicating the setting of methicillin-resistant Staphylococcus aureus (MRSA) acquisition (community or health care acquired), the clinical relevance (colonization or infection), the type of infection (invasive or noninvasive), and the clinical outcome. METHODS: Between August 2006 and December 2009, 381 routinely diagnosed culture-confirmed MRSA-positive patients were included into a cross-sectional study at an 800-bed hospital. RESULTS: Out of 159 patients with colonization, 27 (17%) acquired MRSA in the community (CA-MRSA) and 123 (77.4%) in health care settings (HA-MRSA), and, of the 222 patients with infections, 119 (53.6%) had HA-MRSA and 103 (46.4%) had CA-MRSA. The 10 most frequent spa types accounted for 68.2% of the 346 typed MRSA isolates: t190 (28.3%), t032 (16.5%), t041 (9.4%), t008 (8.4%), t001 (3.4%), t002 (2.9%), t044 (3.1%), t223 (2.1%), t015 (2.1%), t127 (1.3%). CONCLUSION: Spa typing of routinely identified MRSA isolates is unsuitable to predict the likeliness of an infection, of an invasive infection, and the clinical outcome. Molecular criteria such as spa type or Panton-Valentine leukocidin positivity used for classifying MRSA as either belonging to a community or hospital clone are of limited value to indicate the setting, where the MRSA strain was actually acquired according to epidemiologic criteria.

Detection of virulence-associated and regulatory protein genes in association with phage typing of human Vibrio cholerae from several geographical regions of the world.

Vibrio cholerae O1, O139 and occasionally non-O1/non-O139 serogroups are most often responsible for epidemic and pandemic cholera. This study used genotypic patterns of PCR-based detection of virulence-associated and regulatory protein genes, along with phage typing, to characterize 86 V. cholerae strains. Thirty-eight of 53 O1 biotype El Tor strains harboured both tcpA classical and tcpA El Tor genes, and three El Tor strains lacked the V. cholerae O1-specific gene (Vc-O1); three O139 strains contained both Vc-O1 and Vc-O139 genes and seven out of ten non-O1/non-O139 strains possessed the Vc-O1 gene. The latter strains all harboured the virulence-associated genes ctxA, zot, ace, RS1, hlyA, ompU, rtxA and sxt. Two phage types, T27 and T25, were predominant in strains from different geographical regions of India, whereas more variation in phage susceptibility was observed for tetracycline-resistant strains from Kolkata. These results suggest that the pattern and distribution of virulence genes and phage types of V. cholerae are equally useful and discriminatory in tracing the origin of newly emerging strains.

Occurrence of Vibrio cholerae serogroups other than O1 and O139 in Austria.

From 2000 to 2005, 13 infections due to non-O1/non-O139 Vibrio cholerae were documented in Austria. Twelve patients (8 years to 65 years old; 7 male) had symptomatic infections: diarrhea x 5, otitis x 6, septicemia once. All 5 patients who acquired their infections abroad, suffered from diarrhea. The 8 persons without travel history outside of Austria had otitis media (n = 4) or otitis externa (n = 2); the lethal case of septicemia affected a fisherman with underlying malignancy. One isolate was from an asymptomatic child. Detailed data on travel history inside Austria was available for 5 of these 8 patients: all 5 had visited or lived near Austria's largest lake. The concentration of salt in this westernmost steppe lake in Europe is approximately one-twentieth of that of sea water. Why otitis and not diarrhea is the dominating manifestation of non-O1/non-O139 infection acquired in Austria remains to be elucidated. We hypothesize that diarrhea due to Vibrio cholerae serogroups other than O1 and O139 acquired in Austria may simply be unrecognized by the standard operating procedures employed in clinical microbiology laboratories. Testing for Vibrio cholerae is not considered necessary for domestically acquired diarrhea. Only in patients who acquired diarrhea abroad, do physicians sometimes consider cholera as a differential diagnosis, thereby prompting the laboratory to use thiosulfate citrate bile salt sucrose (TCBS) agar plates.