Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-04-16
2001-02-13
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S176000, C435S178000, C435S180000, C435S243000, C435S252100, C435S252500, C435S822000, C435S832000, C435S839000
Reexamination Certificate
active
06187555
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to microbial spores treated with various additives in order to alter sensitivity to sterilants.
Biological indicators (BIs) have been used to test and/or determine the effectiveness of sterilization processes. Typically, biological indicators containing microbial spores are exposed to a selected sterilant or sterilizing process and then the survival of the exposed spores is determined by placing the exposed spores in an environment capable of sustaining germination and outgrowth of spores. Microbial spores are typically more resistant to sterilization processes than most types of microorganisms and it is assumed that a sterilization process that will kill microbial spores also will kill any contaminating microorganisms.
Traditional BIs based on growth could not be used to measure directly a sterilization assurance level as low as about 10
−1
and generally required incubation periods of at least two days and up to seven days before the effectiveness of the sterilization process could be assessed. The development of linear reaction velocity (LRV) technology has facilitated a sensitive method for the rapid determination of sterilization effectiveness over a wide range of frequency of survival from 10
9
to as low as 10
−16
viable spores per unit. As spores germinate, they absorb water and lose the capability of scattering light in spore-containing suspensions. This property allows the germination process to be followed spectrophotometrically as a decrease in light absorption or a decrease in light scattering. To determine spore germination rates, i.e., the decrease in absorbance per unit time, germination kinetics curves can be created by plotting the absorbance at 480 nm (“Abs
480
”) of a germinating spore suspension as a function of time. After spore germination is initiated, there is a lag period where little or no change in absorbance is observed. When a detectable percentage of the spores begin to germinate, a decrease in the Abs
480
of the spore suspension is observed. The decrease in the Abs
480
of the spore suspension is recorded until a majority of the spores germinate. The observed rate is affected by both the number of spores germinating and the time needed for a spore to complete germination. Thus, the more synchronous the spore germination of a given spore population, the higher the germination rate.
The LRV is the maximum spore germination rate for a particular population of spores in a particular germination medium. The LRV is computed from the descending linear portion of the germination kinetics curve that follows the lag period. The LRV is presented as the absolute value of the slope of the descending linear portion of the germination kinetics curve. Generally, LRV is expressed in units Abs
480
/min. Depending on the condition of the spores and the type of germination medium used, the lag period may vary and thus the time interval representing the descending linear portion may vary. After exposing spores to a sterilant, LRVs correlate with the survival of viable spores or cells in a linear relationship. The lower the LRV, the lower the probability of non-sterile units being present in a given biological load that was subjected to a sterilization process. See, WO 95/21936, filed Feb. 15, 1995.
It has been observed that death of microorganisms within a population due to an external factor, such as heat or gas sterilants, is described best using first order kinetics, since the decrease in the number of such organisms is logarithmic. See, for example, Pflug, I. J. and R. G. Holcomb, “Principles of the thermal destruction of microorganisms”, In
Disinfection, Sterilization, and Preservation,
Fourth Edition, S. S. Block, ed., Lea and Febiger, (1991), pp. 83-128. Thus, the number of organisms surviving per unit after increasingly longer exposure to a sterilant or killing treatment may be determined using the following linear regression equation (equation 1) and then plotting the calculated data on semilog graph paper.
log
N=−U/D+
log
N
0
(eq. 1)
U is equal to the number of minutes of sterilant exposure. N
0
is equal to the number of spores or cells per unit at the beginning of the sterilization process. N is equal to the number of microorganisms remaining per unit after sterilant exposure for a given time, U. D is a decimal reduction time (specifically, minutes required to kill one log of spores or cells), which is a constant for a given set of conditions and a given batch or crop of spores or cells. Thus, D is the negative reciprocal of the slope of a straight-line death curve.
Read-out reliability (ROR) is defined as the ratio of the number of positive BIs after two days of growth compared to the number of positive BIs after 7 days of growth. For a read-out of sterilization results earlier than 7 days to be valid, an ROR of at least 97% is required. Shortening readout time is highly desirable and would enhance the effectiveness of the BI assay.
Different methods of sterilization require spores with defined levels of resistance, which likewise give rise to different D values. Not all organisms can achieve the required D values for a particular sterilization method. For example, spores from
Bacillus subtilis
are best suited for ethylene oxide (EtO) sterilization, whereas spores from
Bacillus stearothermophilus
are best suited for high temperature steam sterilization. For other sterilization methods such as hydrogen peroxide plasma, the most suitable organism has not yet been identified. It would be useful if spore resistance to a certain sterilization method could be altered to allow use of an organism when sterilization conditions change or when it is more economical to produce spores from a particular organism whose native resistance to a particular sterilization process may not be optimal.
SUMMARY OF THE INVENTION
The present invention relates to microbial spores that include one or more additives specifically bound to sterilant-sensitive sites in the spores in an amount effective for altering the sensitivity of the spores to sterilants. As used herein, an additive is a substance added to microbial spores for the purpose of altering one or more native characteristics or properties of the spores. The spores can be prepared with various resistances, i.e. various D-values, to sterilization processes, and can provide measurable LRV responses even when a sterilization assurance level of 10
−16
is desired. Additives that enhance spore sensitivity can be combined with additives that decrease spore sensitivity to produce biological indicators with properties tailored to a specific sterilization method.
The invention features microbial spores that include an additive specifically bound to sterilant-sensitive sites in the spores. The additive increases the sensitivity of the spores to sterilants. As used herein, “sterilant-sensitive sites” refers to those sites within or on the spore that are necessary specifically for one or more of spore survival, germination, and outgrowth following exposure of the spores to a sterilant. Oligosaccharides, for example trehalose, raffinose, melibiose and maltose, increase the sensitivity of the spores to a sterilant when bound to sterilant-sensitive sites in the spores and can increase read-out reliability and shorten read-out time of a biological indicator. Trehalose is a particularly useful oligosaccharide for increasing spore sensitivity.
The invention also features microbial spores that include a dipeptide specifically bound to sterilant-sensitive sites in the spores. The dipeptide alters the sensitivity of the spores to sterilants and can be stereospecifically bound to the sterilant-sensitive sites. A dipeptide bound to the sterilant-sensitive sites can decrease spore sensitivity, increase the LRV of the spores and can increase the read-out reliability of a biological indicator. Read-out time can be shortened to less than two days.
Sterilants that are useful in the invention include steam, ethylene oxide, radiation, heat, sodi
3M Innovative Properties Company
Naff David M.
Rogers James A.
Ware Deborah K.
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