A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooled D values (decimal reduction times) at 63 and 66°C were 15.0 ؎ 2.8 s (95% confidence interval) and 5.9 ؎ 0.7 s (95% confidence interval), respectively. The mean extrapolated D 72°C was <2.03 s. This was equivalent to a >7 log 10 kill at 72°C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log 10

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis Clo, H1, and 133 grown on galactose. The lactose phosphoenolpyruvatedependent phosphotransferase system and phospho-p-galactosidase activities were also absent in Lac-derivatives of strains H1 and 133 and were low (possibly absent) in C1o Lac-. In all three Lac-derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac-derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac' derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lacmutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6phosphate pathway.Lactic streptococci (Streptococcus cremoris and S. lactis) transport lactose via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (26, 37). The lactose phosphate formed is hydrolyzed by phospho-f3galactosidase (16), giving D-glucose and D-galactose 6-phosphate (Gal-6P) (37). The latter intermediate is further metabolized to triose phosphates by the three enzymes (see Fig. 2) of the D-tagatose 6-phosphate (Tag-6P) pathway(3).In Staphylococcus aureus, the Tag-6P pathway is required for utilization of galactose as well as lactose (4). Lactic streptococci, however, have the enzymatic potential to metabolize galactose via two initially separate routes, namely, the Tag-6P pathway and the galactose 1phosphate (Gal-1P) pathway (Leloir pathway) (3). Either one or both pathways appear to operate, depending on the strain and the exogenous galactose concentration (36). With most S. lactis and S. cremoris strains, galactose fermentation is heterolactic (36) whereas lactose fermentation is homolactic (33).The instability of lactose metabolism in lactic streptococci was noted by earlier workers (14, 15, 40), and the frequency of lactose-negative (Lac-) variants was increased by curing agents (24,25,27). Subsequent work (for a review, see reference 10) has supported the early suggestion (25) that lactose metabolism is plasmid linked. The lactose-PTS and phospho-,B-galactosidase have been associated with the lactose plasmid in strains of S. lactis and S. cremoris (1,20,21,26,32), and the lactose plasmid influences the metabolism of galactose by S. lactis (21). However, the three enzymes involved in the metabolism of Gal-6P (see Fig. 2) have not been assayed in Lac-variants. A deficie...

The sequence and genetic organization was determined of the 2508 bp lactococcal portion of pFX2, which was derived from a cryptic Lactococcus lactis subsp. lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA and repB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Up-stream of the ori site, and in a region which was non-essential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.

This is the first study to report kinetic data on the survival of a range of significant milk-borne pathogens under commercial-type pasteurization conditions. The most heat-resistant strain of each of the milk-borne pathogens Staphylococcus aureus, Yersinia enterocolitica, pathogenic Escherichia coli, Cronobacter sakazakii (formerly known as Enterobacter sakazakii), Listeria monocytogenes, and Salmonella was selected to obtain the worst-case scenario in heat inactivation trials using a pilot-plant-scale pasteurizer. Initially, approximately 30 of each species were screened using a submerged coil unit. Then, UHT milk was inoculated with the most heat-resistant pathogens at ~10(7)/mL and heat treated in a pilot-plant-scale pasteurizer under commercial-type conditions of turbulent flow for 15s over a temperature range from 56 to 66°C and at 72°C. Survivors were enumerated on nonselective media chosen for the highest efficiency of plating of heat-damaged bacteria of each of the chosen strains. The mean log(10) reductions and temperatures of inactivation of the 6 pathogens during a 15-s treatment were Staph. aureus >6.7 at 66.5°C, Y. enterocolitica >6.8 at 62.5°C, pathogenic E. coli >6.8 at 65°C, C. sakazakii >6.7 at 67.5°C, L. monocytogenes >6.9 at 65.5°C, and Salmonella ser. Typhimurium >6.9 at 61.5°C. The kinetic data from these experiments will be used by the New Zealand Ministry of Agriculture and Forestry to populate the quantitative risk assessment model being developed to investigate the risks to New Zealand consumers from pasteurized, compared with nonpasteurized, milk and milk products.

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