Most of the previous research on the release o f process micro-organisms has focussed
on the routes used by the organisms to escape from a unit operation (Hambleton et al,
1991) and the effect o f exposure to these released organisms (Winkler and Parke, 1992). However, little attention has been paid to the survival o f released organisms within the
environment external to a process. Colwell et al, 1995 has shown how a significant
proportion of water home viable cells may exist in a non-culturable state (Colwell et al,
1995), and that these viable but non-culturable cells can retain their pathogenicity
(Colwell et al, 1990).
One advantage of the QPCR reaction is its ability to estimate any release without concern for the viable state of the cells. With this in mind a series o f semi-quantitative experiments were performed to test whether it would be possible to detect any release that had occurred over the time course of a fermentation and for how long after the end of the fermentation the organism might still be detected in the local environment.
H .l Detection of Standard DNA Samples.
A sample from an E.coli RV308 pHKY531 fermentation (Section 2.1.2.3.1) was diluted
to a concentration of 2 x 10^ cells/mL as determined by a visual count under a microscope (Section 2.1.4.1). Samples (50 pL) of serial dilutions o f the cell suspension containing between 2x10^ and 200 cells/mL were plated onto a 1cm diameter area of phenol-formaldehyde resin (Formica) benchtop. After the elapsed time a sample was recovered by scraping the contaminated benchtop with the end of a sterile cotton wool bud wetted in phosphate buffer. The bud was then shaken with 0.2mL of phosphate buffer, from which a lOmL sample was used for a QPCR assay (Section 2.2).
After two weeks, E.coli was detected in all but the most dilute sample (Table H .l), and
this was derived from the patch where only 1 0 organisms were plated onto the bench.
The measurements taken after two weeks are only qualitative due to the degradation of the internal standard used in the PCR assay.
Appendix H
The sensitivity o f the QPCR assay, in the format that we use it, has a lower limit of about 250 molecules o f the target DNA per sample (Noble et al, 1997). Assuming some 40 to 50 plasmids per cell (Bradley, 1999) this is the equivalent of about 5 cells, or 100 on the swab taken from the bench.
After longer periods (4 to 8 weeks) the response is more variable, and there may be
none at all from a patch which later responds positively (Table H .l). Nothing was detected at later times. For these samples the number of cells collected was roughly estimated. The trend in the estimates generally follows the number of cells applied to the patches on the bench, but the actual values could not be used to determine the
surface concentration of the E.coli cells. In a few cases the results were at the limit of
detection and there is a poor correlation between the number o f organisms applied to the bench and the number recovered. This is hardly surprising given the very simple method used to convey the cells from the surface into the tubes for the QPCR assay. However the nature of the response does suggest that, with care, a genuine correlation could be established, perhaps as good as the one which exists when the cells are
sampled from aerosols (Noble et al, 1997; Noble et al, 1999).
Table H .l Detection of DNA from standardised contamination o f surfaces with E.coli
pHKY531.
Cells detected/enumerated
Cells plated onto surface
after 2 weeks after 4 weeks After 6 weeks after 8 weeks
1 0® detected 34x10" 6 6x1 0" 790 1 0" detected 6 .2x1 0" 3.3x10" Nd 1 0^ detected Nd 9.5x10" - 1 0 0 1 0^ detected 4.5x10" 280 Nd 1 0" detected Nd nd Nd 1 0" detected 790 - 1 0 0 Nd 1 0" detected Nd nd Nd 1 0 nd Nd nd Nd
Appendix H
H.2 Detection of DNA in the Process Environment.
The same swabbing procedure was used to test surfaces on and surrounding several
fermenters at various time intervals after an E.coli RV308 pHKY531 (see Section
2.1.2.3.1) fermentation. The sample was recovered by scraping the contaminated area of the processing environment with the end of a cocktail stick wetted in phosphate buffer. The end of the stick was then shaken with 0.2mL of phosphate buffer, from which a 10
pL sample was used for a QPCR assay (Section 2.2). Cells were detected up to 6 weeks
after the fermentation, but were not found on a 2L fermenter after 9 months (Table H.2). The highest level of contamination was detected on and near to the sampling valve. This suggests that release was partly caused by aerosols formed during the sampling process.
During the 6 -week period the fermenters had been used for the culture of other
organisms and had passed through several cleaning and sterilisation cycles. Moreover, the floor of the fermentation hall had been antiseptically scrubbed in the interim period, indicating that the contamination is remarkably persistent. Once present its removal owes more to natural decay than to hygiene in the process environment. However their detection with the QPCR does not mean that the cells are viable; only the target DNA need be present for the assay to respond positively.
The level of the contamination is difficult to determine from the data, but it would be consistent with the loss of about 50 pL of fermentation broth at harvest containing perhaps 2 x 10^^ cells mL'^ spread across an area of 30cm square (10^ cells on the 1cm diameter disk in Table H .l). This is not an unreasonable quantity to find deposited underneath the sampling valve of a large fermenter where aerosols accumulate.
These results have a number of implications on the potential health effects on plant operators. The main hazards arising from a release o f a micro-organism will be due to inhalation, ingestion and skin contact (Norris, 1994). Most common is the allergic response to an organism or product. Workers may also suffer other toxic effects from unprotected exposure to micro-organisms, products or bi-products. This is particularly true for people working with antibiotics. Although these health effects can be avoided through suitable protective clothing and training, the results presented here suggest that
Appendix H
the hazard although probably much reduced after the operation has ceased is not reduced to zero and precautions still should be taken during periods o f down time.
Table H.2 The Detection o f E.coli pHKY531 DNA in the processing environment.
Vessel Sample Position Sample Time Cells collected
75 L sample valve 1 month nd
75 L surface below sample valve 1 month 670
75 L floor 30 cm from sample valve 1 month 3300
450 L sample valve 1 month 9500
450 L surface below sample valve 1 month 280
450 L transfer port 1 month nd
450 L floor 30 cm from transfer port 1 month 6000
450 L sample valve 1 month nd
250 L floor below sample valve 6 weeks 2400
250 L floor below sample valve 6 weeks 480
250 L top plate 6 weeks 250
2L support table 9 months nd
2L top plate 9 months nd