Multidisciplinary investigation of a multicountry outbreak of Salmonella Stanley infections associated with turkey meat in the European Union, August 2011 to January 2013.

Between August 2011 and January 2013, an outbreak of Salmonella enterica serovar Stanley (S. Stanley) infections affected 10 European Union (EU) countries, with a total of 710 cases recorded. Following an urgent inquiry in the Epidemic Intelligence Information System for food- and waterborne diseases (EPIS-FWD) on 29 June 2012, an international investigation was initiated including EU and national agencies for public health, veterinary health and food safety. Two of three local outbreak investigations undertaken by affected countries in 2012 identified turkey meat as a vehicle of infection. Furthermore, routine EU monitoring of animal sources showed that over 95% (n=298) of the 311 S. Stanley isolates reported from animal sampling in 2011 originated from the turkey food production chain. In 2004­10, none had this origin. Pulsed-field gel electrophoresis (PFGE) profile analysis of outbreak isolates and historical S. Stanley human isolates revealed that the outbreak isolates had a novel PFGE profile that emerged in Europe in 2011. An indistinguishable PFGE profile was identified in 346 of 464 human, food, feed, environmental and animal isolates from 16 EU countries: 102 of 112 non-human isolates tested were from the turkey production chain. On the basis of epidemiological and microbiological evidence, turkey meat was considered the primary source of human infection, following contamination early in the animal production chain.

Enumeration of bacteriophages in water by different laboratories of the European Union in two interlaboratory comparison studies.

As part of a European project on bacteriophages in bathing waters two interlaboratory comparison studies were carried out (May 1997 and March 1998). During these studies phage reference materials as well as naturally polluted standard samples were analysed in 16 European laboratories. Three groups of bacteriophages were tested using standardised methods: somatic coliphages, F-specific RNA-phages and phages of Bacteroides fragilis. Many of the participating laboratories applied one or more of the phage methods for the first time, after a one-week training session in a central laboratory. Nevertheless, the values of repeatability (r=1.35-1.38 calculated on log(10)-scale) and reproducibility (R=1.52-2.04 calculated on log(10)-scale) when analysing phage reference materials were close to the theoretical optimum for a Poisson distribution. When analysing the naturally polluted samples more variation in results within and between laboratories was found (r=1.63-2.34; R=3.10-5.72), in comparison with the results obtained with the pure phage reference materials.

Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe.

Somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. We tested fresh and marine bathing waters from North, South, East and West Europe expected to contain between 100 and 500 E. coli per 100 ml, although wider ranges were sometimes found. Bacteriophages were counted after concentration, since a preliminary study proved that this step was necessary to obtain positive counts. During monitoring, a first-line quality control with reference materials for bacteria and bacteriophages was performed by all the laboratories participating in the study. The same microbes were also counted in raw sewage samples from various areas in Europe, where the bacterial indicators and the three groups of bacteriophages were detected in roughly the same numbers. All groups of bacteriophages were detected in both fresh and marine bathing waters throughout Europe. Reliable and complete results from 147 samples showed that for log-transformed values, E. coli and bacteriophages were slightly correlated. However, the slope of the regression line changed according to E. coli concentration and the correlation diminished when this concentration was close to zero per 100 ml. The ratios between E. coli and phages in bathing waters differed significantly from those in sewage. The relative amounts of bacteriophages, mainly somatic coliphages and phages infecting Bact. fragilis RYC2056, increased in bathing waters with low E. coli concentration, especially in seawater samples containing < 100 E. coli per 100 ml. The relationship of bacteriophages with respect to enterococci paralleled that of bacteriophages with respect to E. coli. Somatic coliphages and bacteriophages infecting Bact. fragilis are useful to predict the presence of some pathogens with the same origin as present bacterial indicators but with higher survival rates.

Conservation of phage reference materials and water samples containing bacteriophages of enteric bacteria.

The survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA bacteriophages and phages infecting Bacteroides fragilis proposed as model micro-organisms for water quality control. Titres of phages of all groups either in pure culture phage suspensions or in naturally occurring phage suspensions were stable at (-70+/-10) degrees C and at (-20+/-5) degrees C when protected with glycerol. Moreover, phage analysis of stored suspensions demonstrated that their numbers were homogeneous, both between vials and within vials, and consequently they can be used as reference materials. Furthermore, changes in the storage temperature of the vials cause unpredictable changes in the numbers of bacteriophages. Consequently, phage reference materials and samples containing a quantitative number of phages must be maintained and dispatched at a constant temperature. Consequently, the results indicate that bacteriophages should be packed in dry ice during transport and storage. Finally, the number of phages in water samples stored at (5+/-3) degrees C in the dark does not decrease significantly during the first 72 h of storage. In addition, phage concentrates from natural samples obtained by adsorption-elution to cellulose nitrate filters and mixed with 10% glycerol were stable at least for 2 months at (-70+/-10) degrees C and at (-20+/-5) degrees C.

EU Drinking Water Directive reference methods for enumeration of total coliforms and Escherichia coli compared with alternative methods.

AIMS: The reference methods for enumeration of total coliforms and Escherichia coli as stated in the European Drinking Water Directive were compared with alternative methods. METHODS AND RESULTS: Laboratories used the reference method on Lactose TTC agar (LTTC), the Colilert/18 system, Laurysulphate Agar (LSA), Chromocult Coliform Agar and the E. coli Direct Plating (DP) method. They enumerated more total coliforms on LTTC than on LSA. CONCLUSIONS: LTTC is suitable for analysis of very clean water samples only, due to heavy background growth. Colilert/18 is a good alternative but it enumerates a broader group of total coliforms, resulting in higher counts. The DP method appeared to be the best choice for enumeration of E. coli because Colilert/18 produces lower counts and false-negative results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the limitations of the EU reference method on LTTC due to lack of selectivity and suggests alternative methods for the enumeration of total coliforms and E. coli.

Optimisation of the ISO-method on enumeration of somatic coliphages (draft ISO 10705-2).

As part of the EU project "Bacteriophages in Bathing Waters" (January 1996-June 1999) research was carried out to optimise the method for detection and enumeration of somatic coliphages in water as described in ISO/CD 10705-2 of August 1995. It was concluded that this draft ISO standard needed to be amended in certain aspects. For determining the viable count of the host culture WG5 Escherichia coli, a membrane filtration technique should be used instead of spread plate technique as the latter gives lower and less reproducible results. A freshly prepared inoculum culture of host strain WG5 should be used instead of a frozen inoculum culture as freezing of the inoculum culture is found to negatively influence the phage counts. The double agar layer method (DAL) is preferred to the single agar layer method (SAL) for performing the phage analysis as the DAL method gives higher phage counts than the SAL method.