A Study of the Fate of the Autochtonous Bacterial Flora of Still Mineral Waters by Analysis of Restriction Fragment Length Polymorphism of Genes Coding For rRNA

Abstract

From five French springs producing “still” waters, 36 water samples each were aseptically taken at three stages: at the source, just before bottling and upon bottling. Specimens were examined immediately after drawing and periodically during storage at ambient temperature for one year. Monitoring included (i) microscopic examination, combining acridine orange epifluorescence cell count with an INT stain; (ii) enumeration of colony forming units on R 2 A agar, incubated at 22 °C for 2 weeks. Colonies obtained on this medium were examined by a Gram stain, and for mobility, mode of attack on glucose, oxidase reaction, pigment production, in an API 20NE gallery, and restriction fragment length polymorphism profiles of RNA regions (“ribotyping”). In the pristine samples AODC counts varied between 10 3 and 10 4 ml -1 , with INT+ cells varying from 10 1 to 10 2 ml -1 , and colony counts from 1 to 10 ml -1 . Upon storage for one week AODC counts reached the order of 10 5 ml -1 , INT+ cells < 10 4 ml -1 and cfu's 10 3 -10 4 . These levels did not appreciably change upon further storage, except for INT+ counts which fluctuated somewhat, but never exceeded cfu numbers. Amongst 890 isolates from R 2 A-agar, originating from all specimens, only 18% could be identified as: Ps. fluorescens > Brevundimonas vesicularis > Sphingomonas paucimobilis > Comamonas testosteroni > C. acidovorans . From an occasional spring other non-fermentative Gram negative rods were isolated sporadically. The 890 isolates provided 378 different ribopatterns. Dependent on the spring, 44–114 different patterns were obtained per source. Dice coefficients of clustering varied from 20 to 95% similarity. Relative abundance of ribotypes in the different springs varied, some clusters being more specific for a given spring than others. Per source the incidence of repeatedly isolated types varied from 55–74%. Similarity of ribotypes recovered from the same source before and after bottling averaged 70%. These results suggest that a high percentage of closely related, if not indistinguishable, ribotypes of non-fermentative Gram negative rod-shaped bacteria occurring in pristine minerale waters is preserved throughout bottling and storage.