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.

On June 13, 2007, the public health authority informed the Austrian Agency for Health and Food Safety about 40 children from two neighboring elementary schools who had fallen ill with abdominal cramps and vomiting on June 8. School milk products consumed on June 8 were suspected as the source of the outbreak. On June 8, the milk products provided by local dairy X to eight elementary schools and two nurseries. The short incubation period - all cases fell ill on the day on which the products were consumed - and the short duration of illness (1-2 days) strongly suggested intoxication. In order to identify the causative pathogen, its reservoir and the mode of transmission, a descriptive-epidemiological and microbiological investigation and a retrospective cohort study were conducted. Six of the 10 institutions served by dairy X completed questionnaires on demographics and food consumption. One school had a 79% response rate (203/258) and was chosen as the basis for our cohort study. A total of 166 of the 1025 children (16.2%) at the 10 institutions fulfilled the case definition. Consumption of milk, cacao milk or vanilla milk originating from dairy X was associated with a 37.8 times higher risk of becoming a case (95% CI: 2.3-116.5). Unopened milk products left over at the affected institutions yielded staphylococcal enterotoxins A and D. Six out of 64 quarter milk samples from three of 16 cows producing milk for dairy X tested positive for S. aureus. The isolates produced enterotoxins A and D, yielded genes encoding enterotoxins and D, and showed spa type t2953. S. aureus isolated from the nasal swab of the dairy owner harbored genes encoding enterotoxins C, G, H and I, and showed spa type t635. Our investigation revealed that the milk products produced in dairy X on June 7 were the source of the outbreak on June 8. The cows - not the dairy owner - the likely reservoir of the enterotoxin-producing S. aureus. From the risk assessment of the production process at the dairy, we hypothesize that staphylococcal toxin production took place during a 3-day period of storage of pasteurized milk prior to repasteurization for the production batch of 7.

With a positive predictive value of 100% and a negative predictive value of at least 99.9%, this combined HRM curve analysis is an ideal screening method for the TB laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.

Community-acquired pneumonia due to Pseudomonas aeruginosa in previously healthy individuals is a rare disease that is associated with high fatality. On 14 February 2010 a previously healthy 49-year-old woman presented to an emergency room with signs and symptoms of pneumonia, 2 days after returning from a spa holiday in a wellness hotel. Blood cultures and respiratory specimens grew P. aeruginosa. Despite adequate antimicrobial therapy, the patient died of septic multiorgan failure on day nine of hospitalization. On February 26, nine water samples were taken from the hotel facilities used by the patient: In the hot tub sample 37,000 colony-forming units of P. aeruginosa/100 ml were detected. Two of five individual colonies from the primary plate used for this hot tub water sample were found to be genetically closely related to the patients’ isolates. Results from PFGE, AFLP and MLST analysis allowed the two lung isolates gained at autopsy and the whirlpool bathtub isolates to be allocated into one cluster. The patient most likely acquired P. aeruginosa from the contaminated water in the hotel’s hot tub. The detection of P. aeruginosa in high numbers in a hot tub indicates massive biofilm formation in the bath circulation and severe deficiencies in hygienic maintenance. The increasing popularity of hot tubs in hotels and private homes demands increased awareness about potential health risks associated with deficient hygienic maintenance.

Fire blight is a destructive disease of apple and pear trees and other rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. Several molecular detection procedures described for this plant pathogen are either too time consuming, too insensitive or impractical when handling a large number of samples routinely. Here, we describe a modified protocol of the REDExtract-N-Amp TM Plant polymerase chain reaction kit for the detection of E. amylovora in planta and demonstrate that it provides simplicity, high sensitivity, speed and high throughput potential. A total of 951 samples collected in Austria were tested with the modified protocol and the standard protocol of our laboratory to validate the accuracy of this detection method.

In Austria, all laboratories are legally obligated to forward human and food/environmental L. monocytogenes isolates to the National Reference Laboratory/Center (NRL) for Listeria . Two invasive human isolates of L. monocytogenes serotype 1/2a of the same pulsed-field gel electrophoresis (PFGE) pattern, previously unknown in Austria, were cultured for the first time in January 2016. Five further human isolates, obtained from patients with invasive listeriosis between April 2016 and September 2017, showed this PFGE pattern. In Austria the NRL started to use whole-genome sequencing (WGS) based typing in 2016, using a core genome MLST (cgMLST) scheme developed by Ruppitsch et al. 2015, which contains 1701 target genes. Sequence data are submitted to a publicly available nomenclature server (Ridom GmbH, Münster, Germany) for allocation of the core genome complex type (CT). The seven invasive human isolates differed from each other with zero to two alleles and were allocated to CT1234 (declared as outbreak strain). Among the Austrian strain collection of about 6,000 cgMLST-characterized non-human isolates (i.e., food/environmental isolates) 90 isolates shared CT1234. Out of these, 83 isolates were traced back to one meat processing-company. They differed from the outbreak strain by up to seven alleles; one isolate originated from the company's industrial slicer. The remaining seven CT1234-isolates were obtained from food products of four other companies (five fish-products, one ready-to-eat dumpling and one deer-meat) and differed from the outbreak strain by six to eleven alleles. The outbreak described shows the considerable potential of WGS to identify the source of a listeriosis outbreak. Compared to PFGE analysis, WGS-based typing has higher discriminatory power, yields better data accuracy, and allows higher laboratory through-put at lower cost. Utilization of WGS-based typing results of human and food/ environmental L. monocytogenes isolates by appropriate public health analysts and epidemiologists is indispensable to support a successful outbreak investigation.

Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

ABSTRACTSalmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping ofSalmonella entericaisolates based on specific single-nucleotide polymorphisms in fragments offljB,gyrB, andycfQ. Simultaneous gene scanning offljB,gyrB, andycfQby high-resolution melting-curve analysis of 417Salmonellaisolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases fromSalmonella entericaserotype Enteritidis andSalmonella entericaserotype Dublin, in all cases fromSalmonella entericaserotype Ohio andSalmonella entericaserotype Rissen, fromSalmonella entericaserotype Mbandaka andSalmonella entericaserotype Kentucky, and fromSalmonella entericaserotype Bredeney,Salmonella entericaserotype Give, andSalmonella entericaserotype Schwarzengrund. To differentiate the most frequentSalmonellaserotype, Enteritidis, from someS. Dublin isolates, an additional single PCR assay was developed for specific identification ofS. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with anS. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39Salmonellaserotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhumanSalmonellaisolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.