radiation sterilization. ... levels of radioresistance were already described for strains of M. radiotolerans (Green, 1992), survival ..... and Sterility Assurance Level.
Radiat. Phys.Chera.Vol. 52. Nos I -6, pp. 15 19, 1998
Pergamon PII: S0969-806X(98)0002A-3
RADIORESISTANCE
STUDIES
"~ 1998 ElsevierScienceLtd. All rights reserved Printed in Great Britain 0969-806X/98 $19.0o + 0.00
IN Methylobacterium spp.
F A T I M A N O G U E I R A I, M. L U I S A BOTELHO I, ROGI~RIO TENREIRO 2 ~Dept. Fisica, ITN, E.N. 10, P-2685 Sacav6m, Portugal; and 2 Centro de Gen6tica e Biologia Molecular, Universidade de Lisboa, Campo Grande, 1700 Lisboa, Portugal ABSTRACT
Methylobacterium extorquen'~ was isolated and was found as one of the most resistant microorganisms in the original bioburden of ophthalmic cotton dressings to be submitted to ~, radiation sterilization. Radiation survival curves were simultaneously performed in phosphate buffer and in test-pieces on two isolates, one obtained before irradiation (wild strain) and the other after irradiation at 20 kGy (rad strain), as well as on three type strains of Methylobacterium spp. (M. extorquensr, M, radiotoleransr and M. fujisawaenser). The radiation resistance was compared using Dv~u,s. To analyze the effect of non linearity on radioresistance other measures were applied, such as intercept point, fraction of surviving cells at a selected dose and area. The ranking of strains with these approaches showed to be different, pointing out the need of an integrated measure of radioresistance. Therefore, an index of relative survival (IRS) is proposed. KEYWORDS Radioresistance; gamma radiation; Methylobacterium; survival curves; Dva~ue. ~TRODUCTION G a m m a irradiation is an efficient process to reduce microbial levels in several materials, from food to health care products. However, both intrinsic microbial radioresistance and environmental factors must be taken into account when studying a process for reduction of microbial population. In fact, gramnegative bacilli are usually much more sensitive to 7 radiation than gram-positive specimens (Russell, 1993) and survival curves may be affected by the nature of the substrate (McCarthy, 1996). Pink-pigmented gram negative bacilli, further identified as Methylobacterium extorquens, showed to be one of the most radioresistant microorganisms isolated from ophthalmic cotton dressings. As high levels o f radioresistance were already described for strains of M. radiotolerans (Green, 1992), survival curves were performed to compare the isolated strains with type strains of Methylobacterium spp.. Although Dvalue is usually the comparative measure of the ability to recover from damaging events, its reliability becomes compromised when survival curves display shoulders, even short as were found for some of the above strains. To overcome this drawback, different approaches were applied, such as intercept point, fraction of survival cells at a selected dose, and area. MATERIALS AND METHODS
Microorganisms, media and growth conditions Type strains of Methylobacterium extorquens and M. radiotolerans were obtained from the National Collection of Industrial and Marine Bacteria (NCIMB). M. fujisawaense r was kindly provided by Peter N. Green (Torry Research Station, Aberdeen). Wild strain was isolated from the original bioburden of ophthalmic cotton dressings to be submitted to gamma radiation (Botelho, 1995) and further identified as M. extorquens by biochemical tests using API Systems (BioMerieux, France) and Biolog microplates (Biolog Inc., USA). Rad strain was isolated from membrane filters with colonies of wild strain, after irradiation at 20 kGy. To produce cell mass, bacterial cultures were incubated at 32 ° C for 6 to 7 days on glycerol peptone agar (Green, 1992). 15
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Ffitima Nogueira et al.
Preparation and irradiation o f samples
Active cultures were harvested and suspended on 5 ml of phosphate buffer (p.b.) to obtain approximately 108 cfu. Batches of test-pieces (t.p.) of approximately 108 cfu each, were prepared as described in Botelho et al (1988). Viable cell numbers were determined before irradiation by plate counting on peptone glycerol agar at 32 °C. Irradiation was carried out in gamma plant UTR at a dose rate of 1.47 kGy.h -j. Bacterial samples in p.b. and t.p. were irradiated simultaneously, at room temperature, with a dose range of 3 kGy up to 21 kGy, in increments of 3 kGy. The absorbed doses were measured using ceric-cerous dosimeters (Nordion Int., Inc.) and Red Perspex 4034 dosimeters (Harwell). After irradiation, viable ceils were determined as described above, with an extended incubation period of 14 days. Radioresistance measures
For each irradiation dose, the survival fraction (S) was estimated dividing the number of viable cells after irradiation (ND) by the initial viable cell number (No). Survival curves relating logS with irradiation dose in kGy were obtained for all strains in both substrates. D,a,ue was calculated according to the formula Dv~l,~ = II/bl, where b represents the slope estimated by linear regression of the overall survival curve. Linearity was assessed using an F test for r-square (r 2) and differences among Dv~l,~sassessed with multiple confidence intervals for b. In both tests, statistical significance was set at p
