Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterize Listeria monocytogenes outbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a new L. monocytogenes typing scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of the L. monocytogenes population based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67 L. monocytogenes isolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated five L. monocytogenes groups. Using all available NCBI RefSeq genomes ( n = 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.

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.

A total of 382 isolates of methicillin-resistant Staphylococcus aureus originating from three Austrian regions and one adjacent Italian region (Vienna, Lower Austria, North Tyrol, and South Tyrol) were typed by DNA sequence analysis of the variable repeat region of the protein A gene ( spa typing). The strain collection consisted of arbitrarily chosen isolates originating from clinical specimens taken in the years 2003 to 2005 at 17 hospitals. The most common spa types found were t001 (28.8% of all isolates), t190 (27.0%), t008 (14.1%), and t041 (11.3%). The 42 remaining spa types accounted for ≤2.4% each. The dominating spa types varied between the different regions. As short sequence DNA repeat units are unstable entities, the 46 spa types were classified into seven spa complexes with respect to short sequence repeat unit composition and organization. Such classification into complexes can provide additional information for the hospital epidemiologist, empowering one to differentiate the introduction of a new strain from mere variation of endemic spa types.

Listeria monocytogenes is causing listeriosis, a rare but severe foodborne infection. Listeriosis affects pregnant women, newborns, older adults, and immunocompromised individuals. Ready-to-eat (RTE) foods are the most common sources of transmission of the pathogen This study explored the virulence factors and antibiotic resistance in L. monocytogenes strains isolated from ready-to-eat (RTE) foods through in vitro and in silico testing by whole-genome sequencing (WGS). The overall positivity of L. monocytogenes in RTE food samples was 3.1% and 14 strains were isolated. L. monocytogenes ST8, ST2763, ST1, ST3, ST5, ST7, ST9, ST14, ST193, and ST451 sequence types were identified by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST. Seven isolates had serotype 1/2a, five 1/2b, one 4b, and one 1/2c. Three strains exhibited in vitro resistance to ampicillin and 100% of the strains carried the fosX, lin, norB, mprF, tetA, and tetC resistance genes. In addition, the arsBC, bcrBC, and clpL genes were detected, which conferred resistance to stress and disinfectants. All strains harbored hlyA, prfA, and inlA genes almost thirty-two the showed the bsh, clpCEP, hly, hpt, iap/cwhA, inlA, inlB, ipeA, lspA, mpl, plcA, pclB, oat, pdgA, and prfA genes. One isolate exhibited a type 11 premature stop codon (PMSC) in the inlA gene and another isolate a new mutation (deletion of A in position 819). The Inc18(rep25), Inc18(rep26), and N1011A plasmids and MGEs were found in nine isolates. Ten isolates showed CAS-Type II-B systems; in addition, Anti-CRISPR AcrIIA1 and AcrIIA3 phage-associated systems were detected in three genomes. These virulence and antibiotic resistance traits in the strains isolated in the RTE foods indicate a potential public health risk for consumers.

Listeria monocytogenes (L. monocytogenes) is a ubiquitous organism that can easily enter the food chain. Infection with L. monocytogenes can cause invasive listeriosis. Since 2014, in Austria, L. monocytogenes isolates from human and food/food-associated samples have been provided on a mandatory basis by food producers and laboratories to the National Reference Laboratory. Since 2017, isolates undergo routinely whole genome sequencing (WGS) and core genome Multilocus Sequence Typing (cgMLST) for cluster analyses. Aims of this study were to characterize isolates and clusters of 2017 by using WGS data and to assess the usefulness of this isolate-based surveillance for generating hypotheses on sources of invasive listeriosis in real-time. WGS data from 31 human and 1744 non-human isolates originating from 2017, were eligible for the study. A cgMLST-cluster was defined as two or more isolates differing by ≤10 alleles. We extracted the sequence types (STs) from the WGS data and analyzed the food subcategories meat, fish, vegetable and diary for associations with the ten most prevalent STs among food, through calculating prevalence ratios (PR) with 95% confidence intervals (CI). The three most frequent STs among the human isolates were ST1 (7/31; 22.6%), ST155 (4/31; 12.9%) and ST451 (3/31; 9.7%) and among the non-human isolates ST451 (614/1744; 35.2%), ST8 (173/1744, 10.0%) and ST9 (117/1744; 6.7%). We found ST21 associated with vegetables (PR: 11.39, 95% CI: 8.32–15.59), ST121 and ST155 with fish (PR: 7.05, 95% CI: 4.88–10.17, PR: 3.29, 95% CI: 1.86–5.82), and ST511, ST7 and ST451 with dairy products (PR: 8.55, 95% CI: 6.65–10.99; PR: 5.05, 95% CI: 3.83–6.66, PR: 3.03, 95% CI: 2.02–4.55). We identified 132 cgMLST-clusters. Six clusters contained human isolates (ST155, ST1, ST101, ST177, ST37 and ST7) and for five of those cgMLST-based cluster analyses solely was able to hypothesize the source: an Austrian meat processing company, two Austrian cheese manufacturers and two vegetable processing companies, one based in Austria and the other in Belgium. Determining routinely STs in food isolates by WGS allows to associate STs with food products. Real-time WGS of L. monocytogenes isolates provided mandatorily, proved to be useful in promptly generating hypotheses on sources of invasive listeriosis.