Biodiversity of anti-listerial microbial cheese ripening consortia and monitoring of a recombinant Yersinia enterocolitica reporter strain on soft cheese

First, the temporal stability and diversity of the bacterial species composition as well as the anti-listerial potential of two different, complex and undefined microbial consortia from red-smear soft cheeses were investigated. Samples from two food producers (R and K) were collected two times each at six months intervals and a total of 400 bacterial isolates were identified by Fourier-transform infrared (FT-IR) spectroscopy and 16S rDNA sequence analysis. Coryneform bacteria represented the majority of the isolates, with certain species being predominant. In addition, Marinolactobacillus psychrotolerans, Halomonas venusta, Halomonas variabilis, Halomonas sp. (between 106 and 107 cfu per gram smear) and unknown Gram positive bacteria (between 107 and 108 cfu per gram smear) are described for the first time in this environment. The species composition of consortium R was quite stable over a period of six months, but consortium K revealed less diversity of coryneform species as well as less stability. While consortium R had a stable, extraordinary high anti listerial potential in situ, the anti-listerial activity of consortium K was lower and decreased with time. The cause for the anti-listerial activity of the two consortia remained unknown, but is not due to the secretion of soluble, inhibitory substances by the individual components of the consortium. Furthermore, the effect of the ripening temperature on the biodiversity and the anti-listerial activity in situ of undefined red smear cheese ripening consortia was analyzed. Undefined microbial surface ripening consortia of mature retail red smear cheeses produced in dairies R and K were derived twice (I and II). Each of the four consortia (R I, R II, K I, K II) was used for inoculation of soft cheeses, which were ripened at two temperature regimes (13°C and 16/12°C). The bacterial composition of the consortia was analyzed in the smear waters and the corresponding surface floras derived from mature cheeses at the expiry date. A total of 1200 isolates, was identified by FT- IR spectroscopy, partial 16S rDNA sequence analysis of some isolates and physiological methods. Coryneform bacteria represented 78% of the isolates. As far as we are aware, this is the first report on the presence of Vagococcus carniphilus, Psychrobacter spp. and Marinobacter sp. in smear cheese surface floras. Furthermore, the two coryneform bacteria Brachybacterium tyrofermentans and Leucobacter komagatae are described for the first time on German red smear cheeses. The bacterial composition of consortia R I and R II differed significantly. At a ripening temperature of 13°C, in both consortia, the proportions of coryneforms increased from about 82% in the smear water to 96% and even 100% of the isolates in the corresponding surface floras derived at the expiry date. At 16/12°C, their proportions remained about the same level. With respect to the species level, significant flora successions occurred with both temperature regimes. In case of consortia K I and K II the coryneform content decreased with both temperature regimes compared to the initial content in smear water. In K II at 16/12°C, the coryneforms decreased and, instead, Marinolactobacilli dominated, which had not been detected in the smear water of K II. Despite the structural differences of K I and K II in the smear water the trend of reduction of A. nicotianane alongside with an increase of B. linens was similar in both consortia at the 16/12°C regime. While in the case of R I and R II the anti-listerial activity was not affected by the different ripening temperatures, with consortia K I and K II this feature appeared to be dependent on the ripening temperature regime. As a step towards real time and in situ monitoring of pathogens in cold stored soft cheeses with a real product status, bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promotorless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens. Their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10°C by quantifying bioluminescence using a photon counting intensified charge-coupled device (ICCD) camera. The detection limit on cheese was 200 cfu/cm2. Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde into the food matrix in order to gain quantitative, reproducible bioluminescence data.