Fachbereich Veterinärmedizin



    Versuche zum Nachweis von Shigellen in Lebensmitteln (1987)

    Rutsch, Corinna (WE 8)
    Berlin, Freie Univ., 1987 — 138 Seiten
    Institut für Lebensmittelsicherheit und -hygiene

    Königsweg 69
    14163 Berlin
    Tel.+49 30 838 62550 Fax.+49 30 838 46029

    Abstract / Zusammenfassung

    In the present study, a method to improve the cultural detection of Shigella in foods has been developed and verified.In the initial orienting experiments, 7 solid selective media and 7 liquid enrichment media recommended in literature were compared. From the solid media, Mac Conkey Agar (MC) and Hektoen-Enteric Agar (H) to which 10 µg Novobiocin (N) had been added proved to be particularly suitable. From the liquid enrichment media, buffered peptone water (1%) to which also 10 µg N/ml had been added (PW + N) was chosen.Examination of the N sensitivity of various gram-negative bacteria revealed that the strains of Sh. sonnei and Sh. flexneri 2a used were hardly affected in their growth by the N dose mentioned while Sh. boydi proved to be sensitive. Some other Enterobacteriaceae species were inhibited with varying intensity. This effect was most intensive in Proteus. In contrast, Pseudomonas strains whose simultaneous presence with Enterobacteriaceae can often be expected and some of which had also been examined remained unaffected.In further orienting experiments using artificially contaminated materials such as animal feeds, minced pork and pork liver, the most favourable incubation period and temperature were determined. Results were best when inoculation took place after 4 and 6 hours of incubation in a water bathshaker at 37 °C. After 2 hours of incubation, Shigella growth was still too low while incubation over 18 hours in the incubator at 37 °C resulted in an overgrowth of the Shigella colonies which had grown on the selective plates by the concomitant bacterial flora.Based on the results of this preliminary testing, the following method was employed in the ensuing studies. The enrichment medium PW + N is inoculated at a ratio of one part of sample material/9 parts of the medium. The mixture is incubated in a waterbath shaker at 37 °C over 6 hours. Subcultures are prepared on solid selective media by streaking one loopful of incubated enrichment medium on one MC and one H + N plate after 4 and 6 hours incubation each. The solid selective media were incubated at 37 °C over 18-24 hours. Colonies showing a growth typical of Shigella were checked by agglutination in diagnostic polyvalent Shigella antiserum or, where the Shigella species used for artificial contamination was known, in antiserum to Shigella sonnei or Shigella flexneri. For onward studies, it will be necessary to establish pure cultures of agglutinating colonies on subsequent checking by means of serological and biochemical examinations.To determine the reliability and the limitations of this method, experiments with mixed bacteria were performed. One or two strains of gram-negative bacteria were mixed with 1000 cells of Sh. sonnei or Sh. flexneri 2a at Shigella/accompanying species ratios of 1:10, 1:100, 1:1.000 and 1:10.000.It could be seen that the success of reisolation of Shigella depended on the N sensitivity and the level of the bacterial count of the accompanying flora. The method will reach its limits if the major part of the concomitant florais N resistant and superior to the number of Shigella organisms by more than two logs. Under such conditions, a detection of Shigella will be a quite irregular and unreliable occurrence.In additional studies, various foods (potato salad with home-made and industry-made mayonnaise, industry-made mayonnaise only, ice-cream and shrimps) were contaminated with varying numbers(<100 to 10000 cells/g) of Sh. sonnei and Sh. flexneri 2a. It was then tried to reisolate Shigella with the aid of the above-described method after varying periods of storage.All trials to reisolate Shigella from potato salad an different days were successful irrespective of whether the salad had been prepared with home-made or industry-made mayonnaise. Shigella with an initial count of 1/g could be reisolated even after 21 days of storage in a refrigerator.Lactobacillus plantarum was added to another batch of potato salad at a ratio of 10.