The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS^® and DuPont Qualicon BAX^®-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C_T) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C_T values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10⁶ cells/sample or higher, which are levels not typically seen in ground beef.     

Alicyclobacillus acidoterrestris spores as a target for Cupuaçu (Theobroma grandiflorum) nectar thermal processing: kinetic parameters and experimental methods

The kinetic parameters of thermal inactivation of a spore former, Alicyclobacillus acidoterrestris, in a tropical fruit nectar [25% of Cupua?u (Theobroma grandiflorum) pulp and 15% sugar] were determined by the isothermal method (IM), under batch heating, and by the paired equivalent isothermal exposures (PEIE) method, under non-isothermal continuous conditions. The isothermal experiments were repeated three times, every 4 months, with the same spore suspension kept frozen between experiments. The aging of spores, under frozen storage, seemed to produce a notorious increase in the z-value from experiment to experiment: Experiment 1 (z = 7.8 +/- 2.6 degrees C, D(95 degrees C) = 5.29 +/- 0.96 min), Experiment 2 (z = 22 +/- 5 degrees C, D(95 degrees C) = 5.99 +/- 0.63 min), and Experiment 3 (z =29 +/- 10 degrees C, D(95 degrees C) = 3.82 +/- 0.48 min). The evaluation of the kinetic parameters by the PEIE method was carried out in parallel with Experiment 3, with the same aged spores, and the results (z = 31 +/- 6 degrees C, D(95 degrees C) = 5.5 +/- 1.2 min) were close to the ones obtained in this experiment. From this work, it seems that the PEIE method can also be applied to evaluate the reduction parameters of a spore-forming microorganism, and in a more realistic way, since the continuous system eliminates the errors caused by come-up and cool-down times (CUT and CDT) that are unavoidable in isothermal experiments. Therefore, when designing a thermal process for a continuous system, the PEIE method should be used, or the chances are that the process would be underdesigned, risking that the desired level of spore inactivation would not be achieved. An optimization of the thermal processing conditions was next performed for Cupua?u nectar, considering a 5D reduction in A. acidoterrestris spores. If a pasteurization process is considered, the conditions that ensure safety (9 min at 98 degrees C) only allow a 55% retention of ascorbic acid (AA). If sterilization is considered, 8 s at 115 degrees C will ensure a safe product and retain 98.5% of the original ascorbic acid. Therefore, if A. acidoterrestris is considered as the target microorganism, the nectar should undergo an aseptic high temperature short time principle (HTST) process to achieve a 5D reduction in this acidophilus spore former. However, if the hot-fill-and-hold pasteurization process is preferred, the product should be fortified with ascorbic acid.     

Characterization of O157:H7 and other Escherichia coli isolates recovered from cattle hides, feces, and carcasses

In a previous study, the seasonal prevalence was reported for stx+ Escherichia coli O157:H7 in feces and on hides and carcasses of cattle at processing. Overall, 1,697 O157:H7 isolates have now been characterized for the incidence of (i) eae(O157), hlyA, stx1, and stx2 in the recovered isolates and (ii) presumptive rough and presumptive nonmotile isolates. Seven O157:H7 isolates (0.4%) lacked stx genes, although they carried eae and hlyA. All but one of the isolates carried both eae and hlyA. Approximately two-thirds of the isolates (64% when one isolate per sample was considered) carried both stx1 and stx2. E. coli O157:H7 cells that harbored both stx1 and stx2 were more often recovered from hides in the fall (79% of the fall hide isolates) and winter (84% of the winter hide isolates) than in the spring (53%) and summer (59%). Isolates recovered from preevisceration carcasses showed a similar but not statistically significant trend. Twenty-three of the 25 O157:H7 isolates carrying stx1 but not stx2 were recovered during summer. Fifteen presumptive rough and 117 presumptive nonmotile stx+ O157:H7 isolates were recovered. Ten (67%) of the presumptive rough isolates were recovered during summer. Ninety-five of the presumptive nonmotile isolates (81%) were recovered during fall. Forty-eight percent of the false-positive isolates (175 of 363) tentatively identified as O157:H7 were O157+ H7- and lacked eae(O157), hlyA, and stx. These data suggest that in beef processing samples (i) there are minor seasonal variations in the prevalence of stx genes among E. coli O157:H7 isolates, (ii) presumptive rough and presumptive nonmotile stx+ O157:H7 isolates are present, (iii) E. coli O157:H7 isolates lacking stx genes may be rare, and (iv) O157+ H7- isolates lacking stx genes can result in many false-positive results.     

Some practical procedures for the solution of nonlinear finite element equations

Procedures for the solution of incremental finite element equations in practical nonlinear analysis are described and evaluated. The methods discussed are employed in static analysis and in dynamic analysis using implicit time integration. The solution procedures are implemented, and practical guidelines for their use are given.     

Minimum utility usage in heat exchanger network synthesis A transportation problem

This paper formulates the minimum utility calculation for a heat exchanger network synthesis as a “transportation problem” from linear programming, allowing one to develop an effective interactive computing aid for this problem. The approach is to linearize cooling/heating curves and partition the only at potential pinch points. In this formulation user imposed constraints are readily included, the latter permitting selected stream/stream matches disallowed in total or in part.By altering the formulation of the objective function, the paper also shows how to solve a minimum utility cost problem, where each utility is available at a single temperature level. A procedure is included to handle a utility which passes through a temperature change when being used.Extending the partitioning procedure permits the formulation to accommodate match dependent approach temperatures, an extension which is useful when heat transfer through a third fluid only is allowed for some matches.     

Synthesizing heat exchanger networks having restricted stream/stream matches using transportation problem formulations

This paper presents an algorithm which is readily computer implementable for the synthesis of heat exchanger networks. The algorithm permits the user to impose constraints disallowing in part or in total the matching of any of the stream pairs. Earlier work showed how to discover minimum utility use for these restricted match problems. Constraining utility use to any feasible limit desired, this paper shows how to discover networks requiring the fewest stream/stream matches and among those to discover networks of simply topology. If required, the algorithm will generate cyclic and/or split stream networks. The algorithm involves the formulation, solving and post processing of a series of linear “transportation” problems. Such problems are linear programming problems of special structure which can be solved extremely rapidly using existing computer codes.     

Partitioning trees: Matching,domination, and maximum diameter

A matching and a dominating set in a graph G are related in that they determine diameter-bounded subtree partitions of G. For a maximum matching and a minimum dominating set, the associate partitions have the fewest numbers of trees. The problem of determining a minimum dominating set in an arbitrary graph G is known to be NP-complete. In this paper we present a linear algorithm for partitioning an arbitrary tree into a minimum number of subtrees, each having a diameter at mostk, for a givenk.Research supported in part by the National Science Foundation under Grant ENG 7902960.Research supported in part by the National Science Foundation under Grant STI 7902960.     

The implementation of case statements in Pascal

This paper examines the probable implementations of the case statement in Pascal, and analyses their consequences. A particular implementation for the Burroughs B6700/7700 series illustrates the necessary investigations. The techniques, although not new, are brought together to serve as a stimulus to improving the implementation of case-statements in other compilers for Pascal, and to provide ideas for implementors. The architectural limitations of even high-level computers in implementing the case statement are briefly discussed.