COSTES INDIRECTOS

The ratio of extracellular and intracellular AK provides a rapid and reliable indicator of the health of bacterial cells in culture. Careful monitoring of samples before phage infection was required to ensure that the bacteria were

growing well. This was assessed by removing a sample from the bacterial culture and comparing the extracellular AK activity (ADP alone) with the total (measurable with chemical extractant + ADP). It was found that more than 95% of the total AK activity in actively growing cultures was intracellular, whereas dead or dying cells produced much higher extracellular AK. Table 7.1 shows the proportion of extracellular AK over the growth cycle of a culture of E.coli cells. Extracellular levels were relatively high immediately after sub­ culture, probably due to the presence of cell debris from the starter culture, which had been growing for 16 hours. The proportion of extracellular AK reduced as the cells began to grow, increasing again when stationary phase was reached. During the log phase of growth, absolute extracellular AK levels slowly increase in rough proportion with the total number of cells. This may be as a result o f cell death and autolysis of defective cells, or perhaps have been caused as a by product of cell division where the pinching off o f daughter cell membranes might entail a certain amount of leakage of cell contents. Care must be taken, when only the extracellular AK activity is measured, not to confuse spontaneously released AK form a dense bacterial population, with phage mediated lysis from a lower number of cells.

Time from sub-culture Number of cells (cfti / ml) Approximate place in growth cycle % Extracellular AK 0 4.8x10“' Lag phase 3.6

1.5 hours 9.5x10^ Early log 1.9

4 hours 2.8x10' Mid log 1.6

6 hours 5.3x10* Late log 1.4

22 hours 4.8x10' Stationary 13.0

Table 7.1: Comparison o f extracellular AK levels at different time points in a culture o f E.coli: Cells were grown in nutrient broth at 37®C with shaking. Sub culture at t=0 was from lOOpl o f an overnight broth culture into 10ml fresh medium.

In all experiments where it was possible to compare the total AK contents of uninfected and phage infected cells, there appeared to be a small initial

increase in AK in the infected cells. Possibly, to assist its own reproduction, the phage enhances the synthesis of AK as part o f a gearing up the host cell's machinery for energy metabolism. Alternatively, the selective inhibition of some the host cell's own metabolic processes might lead to more resources being available for the production of unaffected enzymes. The position switched as lysis neared when the uninfected cultures contained more AK, because these were able to undergo growth and division whilst the infected cells were dying.

Because o f the sensitivity of the AK assay, bacterial growth in apparently clear culture medium could be monitored, and the condition o f a culture determined within 15 minutes of sub-culture. It should be noted that once the culture began to appear cloudy (usually the first indication that it is growing), dilution was required to keep the bioluminescent signal within the dynamic range o f the luminometer.

7.7 Conclusions

The work presented here suggests that AK-phage assays could provide a useful technique for the rapid identification of bacterial contaminants. The results,

4 -1

with limits o f detection of fewer than 10 cells ml , compare favourably with those obtainable from immunoassays (Blackburn, 1993, 1994), which have typical detection limits of 10^ m f \ The technique would be qualitative in nature, due to its dependence on factors which affect the growth rate of the cell. For a BW detector, the main consideration will be the presence and nature of competing organisms in the sample. This is almost as difficult to characterise and quantify as the detection of the target organism itself. Additionally, since the time to lysis is ultimately dependent upon the growth rate of the host cells, there is little or no scope for reducing the assay time to below 20-40 minutes (depending upon the target organism). This is not fast enough to meet the requirements for a BW detector.

7.7.1 Alternative Applications

In addition to its use as an identification method for bacteria, it can be seen that the measurement o f total and extracellular AK levels would provide a valuable tool in the microbiology laboratory. Experience gained from this work has shown that the viability of old cell stocks, freshly sub-cultured into new media can be determined rapidly. This has saved many hours of waiting to see if the culture was growing before an experiment could be started.

Although not particularly relevant to a quantitative assay, the comparison of extracellular and total AK may be used to monitor the effects of biocides and antibiotics on bacterial cells. If cells are prevented from growing, by the effects of biostatic chemicals or antibiotics, total AK levels should not increase with time, in comparison with untreated controls. The effects o f lytic agents on cell populations would be manifested as a rapid increase in extracellular AK levels. It is therefore possible that this technique could be applied to assays for

determining the susceptibility of bacteria to such agents. The methods would be simple and rapid, taking no more than an hour. This would provide a considerable advantage over current, cell culture methods, where it can take several days to obtain a result.

7.8 Bibliography

BLACKBURN, C. D. W., CURTIS, L. M., HUMPHESON, L. & PETIT, S. B. (1994). Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the Detection of Salmonella in Foods. Letters in Applied Microbiology 19, 32- 36.

BLACKBURN, C. D. W. (1993). Rapid and Alternative Methods for the Detection of Salmonellas in Foods. Journal o f Applied Bacteriology 75, 190- 214

HOLT, J. G., KRIEG, N. R., SHEATH, P. H., STALEY, J. T. & WILLIAMS, S. T. (1994). Bergey's Manual o f Determinative Bacteriology. London:

Williams and Wilkins.

MADIGAN, M. T., MARTINKO, J. M. & PARKER, J. (1997). Brock Biology o f Microorganisms. London: Prentise Hall International Ltd.

SANDERS, M. F. (1994). A Rapid Bioluminescent Technique for the Detection and Identification of Listeria monocytogenes in the Presence of Listeria innocua. In Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects, eds. CAMPBELL, A. K., KRICKA, L. J. & STANLEY, P. E. pp. 454-457. Chichester: John Wiley and sons.

SCHLEGEL, H. G. (1993). General Microbiology. Cambridge University Press.