Radiation resistance of clinical Acinetobacter spp.: a need for concern?

Radiation resistance of clinical Acinetobacter spp.: a need for concern?

Journal of Hospital Infection (1991) resistance of clinical Acinetobacter a need for concern? Radiation E. A. Christensen*, *Control 18, 85-92 ...

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of Hospital



resistance of clinical Acinetobacter a need for concern?


E. A. Christensen*, *Control

18, 85-92


P. Gerner-Smidtf-

and H. Kristensen*

of Diagnostic Bacteriology, and TDepartment Seruminstitut, Copenhagen, Denmark

Accepted for publication


4 March



Summary: As part of an epidemiological investigation of hospital infections caused by Acinetobacter spp. the radiation resistance of 15 clinical isolates and four reference strains was assessed. The radiation resistance (in D-6 values, viz. the dose necessary for reducing the initial number of colony forming units by a factor of 10’) was, in general, higher in the isolates of A. radioresistens than in the isolates of the A. calcoaceticus-A. baumannii complex and of A. Iwo@. However, the least resistant isolates of A. radioresistens had a D-6 value equal to or lower than the most resistant isolates of the other groups. The lowest D-6 values found were for two of the reference strains. The highest D-6 value was 35 kGv. Three isolates of A. iohnsonii could not survive-long enough in a dried preparation to make an assessment of the D-6 values possible. The radiation resistance of the 15 clinical isolates in the present study was higher than the resistance found in a study of similar isolates in 1970. Keywords:


spp.; radiation




Introduction Strains of the genus Acinetobacter are widespread in nature, and are common contaminants in clinical bacteriological cultures. They may also especially in hospitalized patients.‘,2 These cause serious infections, microorganisms multiply in water at ambient temperature and many strains can survive for months in the environment at ambient temperature and humidity. Acinetobacters with high radiation resistance compared to most other bacteria have been found in different environments, e.g. irradiated poultry,3 premises for manufacturing radiation sterilized medical devices,4 irradiated beef,’ irradiated cotton6 and soil.7 Recently the genus Acinetobacter has been restructured on the basis of Correspondence to: E. A. Christensen, DK-2300 Copenhagen, Denmark. OlY5-6701~91,'060085




$03 0~1~0 85





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et al.

genotypical and phenotypical data. ‘-lo High radiation resistance has been used as a characteristic in the description of a new species: A. radioresistens.” As a part of an epidemiological investigation of Acinetobacter strains causing nosocomial infections in a Danish hospital since 1982, the radiation resistances of 15 clinical isolates and four reference strains were assessed. Materials

and methods

Terminology For genotypically related strains the expression DNA group is used in the present study. The DNA group numbers are those of Tjernberg & Ursing.12 Named species are designated by their name and when possible also by DNA group number (Table 1). Strains which have only been identified phenotypically are designated by name only and strains identified phenotypically as belonging to the genotypically closely related DNA groups 1, 2, 3 and 13 are designated as strains of the A. calcoaceticus-A. baumannii complex.9~‘0 Bacterial isolates Nineteen isolates of Acinetobacter from six DNA groups were investigated (Table I). Of these 12 were recent Danish clinical isolates: two from an Table



I. 1989 Study-collection


50853-82 53893-82 6.5109-84 86981-84 283 286 56474-84 65219-84 65321-84 70819-85 291 353 M 163 M 50 M 195 ATCC 19606”‘” ATCC 1 7909“h NCTC 5866’rc ATCC 17903d


of Acinetobacter

Origin CSF CSF CSF CSF Dialysate Dialysate Dialysate Dialysate Dialysate Dialysate Sputum Sputum Sputum Urine Exudate Urine Gut Not known Not known

DNA group

D-6 value in kGy

2 13

18 15

ii 8

:: 12’ 13

:; 10 10 8 S.D.* S.D.* 15 13

:; 12 12

:; 35 18


SD6 * 16’ 6



“N.I., Not identified to any known DNA-group; *S.D., fast spontaneous decrease of cfu in dried preparation; “type-strain for A. baumannii (DNA-group 2); hype-strain for A. johnsonii (DNA-group 7); ‘type-strain for A. lwofii (DNA-group 8); “reference-strain for DNA-group 13; ‘DNA-group 12: A. radioresistens.



of Acinetobacter



epidemic of ventriculitis described earlier,13 two that were endemic in an isolates from patients suffering intensive care unit,14 and eight unrelated from ventriculostomy related ventriculitis or dialysis-associated peritonitis. Three were Swedish clinical isolates of A. radioresistens (group 12) (M163, MS0 and M195). Four were reference strains: ATCC 19606T A. baumannii 51866~ A. Zwofii (group 2), ATCC 17909T A. johnsonii (group 7), NCTC (group S), and ATCC 17903, reference strain for DNA group 13.‘* The clinical isolates were grouped genotypically and phenotypically in an earlier investigation.” Strain 56474-84 could not be identified genotypically to any known DNA group, but the strain was phenotypically similar to strains of A. lwofii. Radiation resistance The radiation resistance of the isolates was characterized by dose-response relationships in semilogarithmic presentation. Pure cultures of each isolate cultivated 2-4 days at 36°C on TGY agar plates (Bacto-tryptone 0.5%, yeast extract Difco 0.3%, glucose O-l%, agar l*S”/,) were scraped off the agar surfaces. The layer of culture of each isolate was suspended in 3 ml of serum broth (beef broth with horse serum 5%, glucose O*l%, horse haemoglobin 0.05%). Droplets of 0.02 ml of the suspensions were dried on plastic foil at ambient temperature and humidity and enclosed in sealed plastic envelopes. This method for assessment of the radiation resistance of strains of bacteria has been used with minor modifications since the early 1960~.‘~ The number of colony forming units (cfu) per droplet was determined c. 24 h after the droplets had been distributed on plastic foil. The counts were repeated after storage of the test pieces for 3 weeks at ambient temperature. Groups of two test pieces were irradiated with varying doses in a laboratory 6oCo facility,” the doses being increased until an inactivation factor of c. lo6 was achieved. These series of irradiations with incremental doses were performed within the first 4 days after the preparation of test pieces and were repeated after approximately three weeks. At least two batches of test pieces of each isolate were examined. The resistance was assessed in dried laboratory preparation only. All counts were evaluated by a method for simplified statistics for small numbers of observations.” The doses necessary for reducing the initial numbers of cfu by a factor of lo6 (D-6) were read on semilogarithmic dose-response curves, the 95% confidence intervals for the D-6 values being within f 15 %. Results

The D-6 values for 16 of the Acinetobacter isolates were found to be between 6 and 35 kGy (Figure 1). The isolates of A. radioresistens (group 12) had D-6 values between 15 and 35 kGy. The clinical isolates of the groups most frequently causing hospital infections, A. baumannii (group 2) and

E. A. Christensen

et al.

Figure 1. Dose-response relationships for nine different group number of the isolates are shown on the graphs.



Acinetobacter. The DNA

DNA-group 13,9 had D-6 values between 13 and 18 kGy. However, the reference strains for A. baumannii (group 2) and for group 13 had D-6 values of only 6 kGy. The isolates of A. Zwofii (group 8) had D-6 values between 10 and 16 kGy (Table I). It was impossible to assess the D-6 values for three isolates of A. johnsonii (group 7) because of a low storage stability of the test pieces manifested by a first initial count below 104, despite the droplets being dried from suspensions of between lo6 and 10’ cfu per 0.01 ml. Further decrease in the number of cfu per test piece was found at re-examination a few days later. Of the 16 isolates for which dose-response curves were achieved four belonging to the A. calcoaceticus-A. buumunnii complex had linear curves (two group 2 and two group 13). The other 12 isolates had non-linear curves with the slopes of the inactivation curves increasing with increasing radiation doses (Fig. 1). Discussion

The three Swedish isolates and one of the Danish isolates were A. rudioresistens (group 12). The high D-6 values for these four isolates were expected as the description of this species includes high radiation resistance.” However, the relatively high radiation resistance of some of the A. Iwo@ (group 8) isolates and of isolates belonging to the A.



of Acinetobacter



calcoaceticus-A. baumannii complex (group 2 and 13) was unexpected. Twenty years ago a number of isolates of Gram-negative bacteria with unusually high radiation resistance were collected from samples of dust from premises at a Danish factory manufacturing medical devices sterilized by irradiation. A total of 14 isolates were all classified as Acinetobacter (formerly Bacterium anitratum). These isolates had D-6 values between 17 and 38 kGy (at that time assessed by the same method as used in the present study). For comparison an assessment was carried out of the radiation resistance of a collection of 37 Acinetobacter isolates without any known connection to an environment exposed to ionizing radiation.4 These isolates were obtained from the Department of Diagnostic Bacteriology, Statens Seruminstitut. The 37 isolates had D-6 values between 2 and 10 kGy, i.e. all had a lower resistance than the isolates from the factory environment. In the present study the D-6 values for the clinical isolates were 8 to 35 kGy (Figure 2). Several of the isolates from the study in 1970 have been kept in the freeze-dried state, and were recently characterized phenotypically. Eight isolates from the factory environment were all A. radioresistens. The majority of the 1970 isolates from the Department of Diagnostic Bacteriology were probably clinical isolates (information about the origin has been lost). Seventeen of these isolates have been re-examined: nine were A. lwofii and four belonged to the A. calcoaceticus-A. baumannii complex. Two of the 17 isolates were A. haemolyticus, one was A. junii, and one belonged to the unnamed DXA-group 10, three species that were not represented in the present study. The change in resistance observed by assessment of dose-response curves for acinetobacters collected from clinical samples in the same part of Europe, with about 20 years between the sampling periods, cannot be explained solely by the occurrence of A. radioresistens among the clinical isolates in the present study. Also, two of the four A. Zwofii isolates, and all four isolates of the A. calcoaceticus-A. baumannii complex, had higher D-6 values than were found for the same species in 1970. Three of these six isolates had D-6 values comparable to the D-6 value for the least resistant isolates of A. radioresistens. Repeated irradiation and subsequent growth can for some acinetobacters cause a doubling or more of the D-6 values.4 This phenomenon could explain the change in radiation resistance of clinical Acinetobacter isolates from the 1960s to the 198Os, provided that a connection to gamma- or electron-irradiated of UV-radiation for products, or to use decontamination, could be demonstrated. We are studying experimentally induced increase of radiation resistance of strains of A. lwofii (group 8) and of the A. calcoaceticus-A. baumannii complex (group 2 and 13), and possible links between radiation resistance and other selecting factors in the hospital environment are being investigated.

E. A. Christensen D-6 0 1





in kGy

20 I

30 I

20 D-6


et al.


40 I


in kGy

Figure 2. D-6 values in two collections of Acinetobacter isolates from 1970 and 1989, respectively. One square=one isolate. Open squares: Isolates from Department of Diagnostic Bacteriology. Black squares: Isolates from a factory producing radiation sterilized medical devices in 1970. Crossed squares: Culture collection strains.

Many different disposable medical devices have been used in the management of the patients from whom the isolates in the present study originated and several of these devices were sterilized by irradiation. However, when the present study was initiated it was not possible to focus on any specific irradiated device as a possible source for resistant bacteria. Neither could any source for intense UV-radiation be identified in the relevant environment. The number of patients with reduced defences against infections has been greater in the 1980s than it was in the 1960s. However, it is not clear why this should favour acinetobacters with relatively high radiation resistance and not acinetobacters with low resistance. Is the change in radiation



of Acinetobacter



resistance favoured by, or due to exposure to ionizing radiation, or is the change caused by other factors, and the increased radiation resistance only by chance connected to these factors? As mentioned earlier acinetobacters with high radiation resistance have been found in different irradiated products.3,5s6 The reported D-values are high enough to indicate that the probability for survival of some Acinetobacter strains, when occurring in a mixed population of microorganisms, would be rather high even if these products (poultry, beef, cotton) had been irradiated with the dose of 25 kGy which is commonly used for sterilization of medical products. It has been suggested’* that doses lower than 25 kGy could be used for sterilization of medical devices, provided that approved rules for Good Manufacturing Practice (GMP) were followed and a validation of the sterilization of the specific product was performed. The experimental evidence for the validity of the suggested validation procedures has so far been scarce.” Some of the A. radioresistens isolates in the present investigation gave characteristics indicating that special attention should be paid to medical devices sterilized by irradiation with reference to the use of such devices in intensive care units. Experience shows that A. radioresistens (DNA-group 12) can survive in production premiseq4 the radiation resistance of some isolates is very high (Table I); the dose-response relationships are often non-linear (Figure 1) and finally, the inactivation factors achieved at low doses are therefore comparatively smaller than for organisms with linear dose-response curves. A potential risk for unintended selection of bacteria with high radiation resistance should be recognized in connection to the use of ionizing radiation for decontamination in the food industry and for reducing microbial contamination in raw-products in the pharmaceutical industry. The authors wish to thank Rise National Laboratory, Roskilde, Denmark, and in particular the Accelerator Department and Arne Miller, for making the laboratory “‘Co source available and for maintaining the radiation facility.

References 1. Clew RH, Moellering RC, Kunz LJ. Infections with Acinetobacter calcoaceticus (Herellea vaginicola): Clinical and laboratory studies. Medicine 1977; 56: 79-97. 2. Holton J. A report of a further hospital outbreak caused by a multi-resistant Acinetobacter anitratus. J Hasp Infect 1982; 3: 305-309. 3. Thornley MJ, Ingram M, Barnes EM. The effects of antibiotics and irradiation on the Pseudomonas-Achromobacter flora of chilled poultry. J Appl Bacterial 1960; 23: 487498. 4. Christensen EA. Radiation-induced mutants with increased resistance against ionizing radiation. In: Experiences in radiation sterilization of medical products. Technical Report IAEA-159, International Atomic Energy Agency, Vienna 1974: 29-34. 5. Welch AB, Maxcy RB. Characterization of radiation-resistant vegetative bacteria in beef. Appl Microbial 1975; 30: 242-250. 6. Kairiyama E, Nishimura Y, Iizuka H. Radioresistance of an Acinetobacter species. J Gen Appl Microbial 1979; 25: 401406.


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7. Nishimura Y, Kairiyama E, Shimadzu M, Iizuka H. Characterization of a radiation resistant Acinetobacter. Z Allg Mikrobiol 1981; 21: 125-l 30. 8. Bouvet PJM, Grimont PAD. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nav., Acinetobacter haemolyticus sp. nov., Acinetobacter of johnsonii sp. nov., and Acinetobacter junii sp. nov. and amended descriptions Acinetobacter calcoaceticus and Acinetobacter CwoJii. Int J Syst Bacterial 1986; 36: 228-240. 9. Tjernberg I. Taxonomic studies of the genus Acinetobacter (Thesis). Grafic Systems AB, Malmo, Sweden 1990: 49. 10. Gerner-Smidt P, Tjernberg F, nrsing J. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbial 1991; 29: 277-282. 11. Nishimura Y, Ino T, Iizuka H. Acinetobacter radioresistens sp. nov. isolated from cotton and soil. IntJ Syst Bacterial 1988; 38: 209-211. 12. Tjernberg I, Ursing J. Clinical strains of Acinetobacter classified by DNA-DNA hybridization. APMIS 1989; 97: 59.5-605. 13. Gerner-Smidt P, Hansen L, Knudsen A, Siboni K, Soegaard I. Epidemic spread of Acinetobacter calcoaceticus in a neurosurgical department analyzed by electronic data processing. J Hasp Infect 1985; 6: 166-l 74. 14. Gerner-Smidt P. Endemic occurrence of Acinetobacter calcoaceticus biovar anitratus in an intensive care unit. J Hosp Infect 1987; 10: 265-272. 15. Christensen EA, Holm NW. Inactivation of dried bacteria and bacterial spores by means of ionizing radiation. Acta Path Microbial Stand 1964; 60: 253-264. 16. Bjergbakke E, Larsen E. The 10000 Ci 6oCo facility and the 3000 Ci 6oCo gamma cell, Accelerator department, Rise. Riss Report M-2651. Rise National Laboratory, Roskilde, Denmark 1973: 14. 17. Dean RB, Dixon WJ. Simplified statistics for small numbers of observations. Anal Chem 1951; 23: 636638. 18. Association for Advancement of Medical Instrumentation. Process Control Guidelinesfor Gamma Radiation Sterilization of Medical Devices. AAMI, 1901 North Fort Myer Drive, Suite 603, Arlington, Virginia, USA 1984: 24. 19. Doolan PT, Halls NA, Tallentire A. Sub-process irradiation of naturally contaminated hypodermic needles. Radiat Phys Chem 1988; 31: 699-703.