Salmonellosis outbreak due to Salmonella enteritidis phage type 14b resistant to nalidixic acid, Austria, September 2010.

We report on a salmonellosis-outbreak due to Salmonella Enteritidis phage type 14b resistant to nalidixic acid (S. Enteritidis PT14b Nx) among residents and employees of a student residence in Austria, September 2010. The outbreak was described and analysed by a retrospective cohort study, and microbiological environmental investigations were conducted to identify the outbreak source(s) and the reservoir of the outbreak strain. A total of 66 persons fulfilled the outbreak case definition including 14 laboratory-confirmed cases. Food specific cohort-analyses by day revealed that consumption of potato salad (RR: 1.65, 95%CI: 1.35­2.01, p=0.001) and a cheese-sausage cold plate (RR: 2.24, 95%CI: 1.29­3.88, p=0.002) on 14 September was associated with being an outbreak case. We hypothesised that cross-contamination with S. Enteritidis PT14b Nx positive eggs had occurred during preparation of the potato salad and cold plate as a result of preparing in parallel egg-containing breaded cutlets on 14 September. A traced laying hen holding in eastern Austria was identified as the sole source of the consumable eggs in the student residence. By applying the legally mandated sampling method for epidemiological-related laying hen farms (one pooled dust sample à 150g, two paired boot swabs cultured separately), the outbreak strain could not be detected. Our findings, that legally required sampling methods for laying hen farms failed to detect the causative pathogen in a laying hen holding, despite an epidemiological link, underline the request stated by the European Food Safety Authority Panel on Biological Hazards for a more sensitive sampling plan in epidemiologically-associated laying hen flocks.

Development of an oligonucleotide microarray method for Salmonella serotyping.

Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time-consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.

MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars.

Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.

Salmonella Enteritidis phage type 21 outbreak in Austria, 2005.

We report an outbreak of gastroenteritis due to Salmonella Enteritidis PT 21 associated with attending an annual traditional fair in a small Austrian village on 4 May 2005. The outbreak lasted from 4 to 8 May. Descriptive and analytical epidemiological investigations were conducted in order to determine the extent of the outbreak and to identify outbreak risk factors. Of the 115 persons who visited the fair, 85 persons fulfilled the criteria of an outbreak case (attack rate = 73.9%). Stool specimens from 52 patients, including two kitchen staff, were tested for salmonella, and 20 specimens were positive for Salmonella Enteritidis PT 21. The cohort study revealed mixed salad (which included potatoes) as the likely cause of the outbreak (RR: 10.4, 95%CI 2.8 - 39.1; P = < 0.001). The causative agent of the outbreak was cultured from the stock of eggs used at the fair and from all three drag swabs and one barn dust sample collected from the responsible egg laying flock. Molecular subtyping by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion showed that isolates from eggs, from the flock and from humans were indistinguishable. We hypothesise that cross contamination from eggs to boiled potatoes occurred in the kitchen area, where raw eggs were handled by village residents preparing a traditional Viennese egg dressing. Unrefrigerated storage of peeled potatoes may have favoured bacterial growth. Eggs from small rural flocks of laying hens kept in a traditional 'natural' way should not be assumed to be salmonella-free.

Salmonella Enteritidis phage type 21 outbreak in Austria, 2005.

We report an outbreak of gastroenteritis due to Salmonella Enteritidis PT 21 associated with attending an annual traditional fair in a small Austrian village on 4 May 2005. The outbreak lasted from 4 to 8 May. Descriptive and analytical epidemiological investigations were conducted in order to determine the extent of the outbreak and to identify outbreak risk factors. Of the 115 persons who visited the fair, 85 persons fulfilled the criteria of an outbreak case (attack rate=73.9%). Stool specimens from 52 patients, including two kitchen staff, were tested for salmonella, and 20 specimens were positive for Salmonella Enteritidis PT 21. The cohort study revealed mixed salad (which included potatoes) as the likely cause of the outbreak (RR: 10.4, 95%CI 2.8 - 39.1; P=<0.001). The causative agent of the outbreak was cultured from the stock of eggs used at the fair and from all three drag swabs and one barn dust sample collected from the responsible egg laying flock. Molecular subtyping by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion showed that isolates from eggs, from the flock and from humans were indistinguishable. We hypothesise that cross contamination from eggs to boiled potatoes occurred in the kitchen area, where raw eggs were handled by village residents preparing a traditional Viennese egg dressing. Unrefrigerated storage of peeled potatoes may have favoured bacterial growth. Eggs from small rural flocks of laying hens kept in a traditional 'natural' way should not be assumed to be salmonella-free.

Occurrence of Salmonella Enteritidis phage type 29 in Austria: an opportunity to assess the relevance of chicken meat as source of human Salmonella infections.

Assuming that the various phage types of Salmonella Enteritidis (S. Enteritidis) are largely equally virulent, the importance of certain foods as sources of infection for human salmonellosis can be deduced from differences in the distribution of phage types in human and non-human samples. In 2002, S. Enteritidis phage type 29 (PT29) was first isolated from non-human test samples in Austria. S. Enteritidis PT29 accounted for 44 (27.7%) of 159 S. Enteritidis strains, derived from veterinary samples of chicken (e.g. meat, giblets) or chicken habitations (e.g. swabs from the coop and excrement). At the food retail level (chicken meat, chicken liver), five (13.1%) of 38 S. Enteritidis isolates were PT29. The proportion of S. Enteritidis PT29 in human samples was much lower. Only 0.4% (30 human primary isolates) of all S. Enteritidis isolates in the year 2002, and 0.33% (23 human primary isolates) of all human S. Enteritidis strains in 2003 were PT29. In our opinion, the discrepancy between the high prevalence of S. Enteritidis PT29 in broilers and chicken meat and the low number of PT29 cases in humans indicates that chicken meat of Austrian origin is currently only a minor source of human S. Enteritidis infections.

Occurrence of Salmonella Enteritidis phage type 29 in Austria : an opportunity to assess the relevance of chicken meat as source of human salmonella infections.

Assuming that the various phage types of Salmonella Enteritidis (S. Enteritidis) are largely equally virulent, the importance of certain foods as sources of infection for human salmonellosis can be deduced from differences in the distribution of phage types in human and non-human samples. In 2002, S. Enteritidis phage type 29 (PT29) was first isolated from non-human test samples in Austria. S. Enteritidis PT29 accounted for 44 (27.7%) of 159 S. Enteritidis strains, derived from veterinary samples of chicken (e.g. meat, giblets) or chicken habitations (e.g. swabs from the coop and excrement). At the food retail level (chicken meat, chicken liver), five (13.1%) of 38 S. Enteritidis isolates were PT29. The proportion of S. Enteritidis PT29 in human samples was much lower. Only 0.4% (30 human primary isolates) of all S. Enteritidis isolates in the year 2002, and 0.33% (23 human primary isolates) of all human S. Enteritidis strains in 2003 were PT29. In our opinion, the discrepancy between the high prevalence of S. Enteritidis PT29 in broilers and chicken meat and the low number of PT29 cases in humans indicates that chicken meat of Austrian origin is currently only a minor source of human S. Enteritidis infections.

A regional outbreak of S. Enteritidis phage type 5, traced back to the flocks of an egg producer, Austria.

In the spring and summer of 2002, the Nationale Referenzzentrale für Salmonellen (National Reference Centre for Salmonella - NRCS) in Austria noticed a cluster of human Salmonella enterica subsp. enterica ser. Enteritidis phage type 5 (S. Enteritidis PT5) infections in two neighbouring districts of Austria. Another small outbreak of S. Enteritidis PT5 infections that occurred in the same region in 1999 had been traced back to the flocks of a local egg producer (approximately 6 000 hens). Attention was therefore again directed at this farm. The results of voluntary bacteriological examinations from the farm and further epidemiological investigations identified the same egg producer as the source of the second outbreak. The 70 human isolates of S. Enteritidis PT5 ascertained in 2002 represented a minority of all infections. It is realistic to estimate that several hundred infections occurred in the course of the 2002 outbreak. The farmer had not vaccinated new flocks against Salmonella since August 2001. It is likely that the change in vaccination policy resulted in the reappearance of the S. Enteritidis PT5 infections. By the end of September 2002 the farmer had stopped selling untreated table eggs. In October 2002 only one isolate of S. Enteritidis PT5 was ascertained in the region.