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Susceptibility of isolates of Acinetobacter anitratus and Acinetobacter lwoffii to the bactericidal activity of normal human serum
FEMS Microbiology Immunology 89 (1992) 255-260 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00
255
FEMSIM 00213
Susceptibility of isolates of Acinetobacter anitratus and Acinetobacter lwoffii to the bactericidal activity of normal human serum Stanistaw Jankowski, Kryspina Grzybek-Hryncewicz, Malgorzata Fleischer and Maria Walczuk Department of Microbiology, Academy of Medic&e, Wroctaw, Poland Received 14 August 1991 Revision received 6 January 1992 Accepted 27 January 1992
Key words: Acinetobacter; Bactericidal action of serum; Complement
I. SUMMARY
2. INTRODUCTION
The bactericidal activity of normal human serum against the Gram-negative coccobacilli Acinetobacter anitratus and Acinetobacter lwoffii was studied; 12% and 84%, respectively, of the tested strains appeared to be sensitive. Thus, serum resistance may be an important factor contributing to the pathogenic potential of A. anitratus strains. Three types of bactericidal action were shown. In the first, the strains were killed when the alternative complement pathway was activated. In the second, some strains required both the classical and alternative pathways. In the third variant, the strains needed either the alternative or classical activiation pathway.
After 1980 a change in the spectrum of microbial factors causing hospital infections in patients with decreased immunity, e.g. those with cancer, was noticed. The number of Gram-positive bacteria, such as Streptococcus spp., coagulase-negative Staphylococcus and Mycobacterium as well as non-fermenting Gram-negative Acinetobacter strains, found in these infections increased markedly [1-3]. The number of coliforms and anaerobic bacteria decreased [2,4]. The change in these patients was caused by the use of a specific set of antibiotics. A. calcoaceticus ssp. anitratus and A. calcoaceticus ssp. lwoffii were frequently isolated from man [1,3]. Resistance to bactericidal factors in the host, e.g. to those in blood, may considerably influence the infectious activity of these bacteria. The bactericidal activity of serum is important for the protection of the higher organism against infection [5].
Correspondence to: S. Jankowski, Department of Microbiology, Academy of Medicine, Chatubifiskiego 4 str., P1-50368 Wroctaw, Poland.
256
In the present work the susceptibility of A. anitratus and A. lwoffii to the bactericidal action of normal human serum was tested.
3. M A T E R I A L S A N D M E T H O D S 3.1. Bacterial strains Twenty-five strains of A. anitratus or A. lwoffii isolated from man were used (Table 1). These strains were identified by the API 20 N E and Oxi Ferm Tube systems. 3.2. Serum Normal human serum (NHS) obtained from two healthy adult blood donors was pooled. The sera were stored at - 3 0 ° C in 1-ml portions for up to two months. These donors had not been treated with antibiotics in the period prior to blood donation. 3.3. Preparation of the serum The alternative pathway of complement activation was inhibited by incubating the serum for 20 rain at 50°C (NHS50°C) [6]. In such serum only the complement activated by the classical pathway exerts bactericidal action. To inhibit the classical pathway of complement activation, Ca 2+ ions were removed by E G T A from serum (NHS EGTAMg2+). EGTA-ethylene-glycol-bis-/3(aminoethyl e t h e r ) N , N '-tetraacetic acid solution was prepared according to
Fine et al. [7]. The final concentration of E G T A and MgCI~ was 10 raM. The bactericidal action of this preparation is dependent on the complement activated by the alternative pathway. The action of E G T A M g 2+ alone was investigated by introducing this factor to serum inactivated at 56°C ( N H S 5 6 ° C - E G T A M g 2+) and measuring the percentage survival of the bacteria. Inactivation of NHS was carried out at 56°C for 3(I rain (NHS56°C). 3.4. Complement assay The level of complement in the serum was determined by the method of Wedgwood and Janeway [8] and the results are given in units of complement Hs~ (50% haemolysis). 3.5. Bactericidal assay The bactericidal activity of serum was determined as described earlier [9]. The bacteria were harvested during early logarithmic growth. At an initial density of about 105 cells m l - 1 cells were added to a 50% NHS solution diluted with 0.1M NaC1. The mixture was incubated at 37°C and at time 0 and 0.5, 1, 2, 3 h, samples were taken, diluted and plated on nutrient agar. After incubation for 18 h at 37°C the number of colonies formed at t = 0 was taken as 100%. The strains which had a survival rate of not less than 50% after 3 h incubation in 50% serum were regarded as resistant.
Table 1 Source of A. lwoffii and A. anitrams strains Source
A. lwoffii strain
A. anitratus strain
Blood Sputum Bronchial secretion Urine Pharynx Wound Pus Vagina Bile Nose Cerebrospinal fluid
971,972, 84, 580, 54, 688, 71,937, 2, 798, 36, 766, 982 980
39, 807, 815, 267, 827 273 800, 841
978 832, 834, 952 693 733, 734 920, 965
768, 794
40, 704, 851,869 823, 833, 838, 844, 859, 902 824, 840, 842, 887, 926 810
884
257 4. R E S U L T S 2 Susceptibility of A. anitratus a n d A. lwoffii strains to N H S is r e p r e s e n t e d in T a b l e s 2 a n d 3. A. anitratus strains (22 of 25) were resistant to the bactericidal action of NHS. T h e s e resistant strains grew in the N H S after 3 h i n c u b a t i o n . T h e n u m ber of c o l o n y - f o r m i n g u n i t s in some strains increased 2 0 - 2 8 times. It can be seen from T a b l e 3 that most strains of A. lwoffii were susceptible to the bactericidal action of N H S ; after 30 m i n i n c u b a t i o n with the serum, the n u m b e r of colony-forming units was c o n s i d e r a b l y reduced. Five strains were resistant, the p e r c e n t a g e survival was from 56.5 to 109,5 of the initial c o n c e n t r a t i o n of bacteria. T h e s e strains, with the exception of No. 937, m u l t i p l i e d in N H S after i n c u b a tion for 3 h (Fig. 1). In f u r t h e r studies the m e c h a n i s m of the bactericidal action of c o m p l e m e n t in N H S on Acinetobacter strains was investigated. By t h e r m a l inactivation, the action of c o m p l e m e n t activated by the alternative pathway was e l i m i n a t e d , p r e p a r a t i o n NHS50°C. Strains of A. lwoffii chosen r a n d o m l y (Nos. 54, 84, 693) were susceptible to N H S b u t resistant to N H S 5 0 ° C (Fig. 2a). A. anitratus strain 851 was resistant to NHS50°C. T h e susceptibility of A. anitratus strain
stroin
/.0/
o
aa 0
>!00C
o
20~
97~3
~
~
_
.
,
0.5
,
1 Time
~
,
2 [
768
,
937
3
hours]
Fig. 1. Susceptibility of A. lwoffii to the bactericidal activityof normal human serum after 3 h incubation.
838 was not c h a n g e d by t h e r m a l inactivation of s e r u m (Fig. 2b). T h e s e e x p e r i m e n t s showed that the sera devoid of c o m p l e m e n t activated by the alternative pathway lose their bactericidal properties against 4 out of 5 strains of Acinetobacter.
Table 2 Susceptibility of A. anitratus strains to the bactericidal action of normal serum ~' after 3 h incubation Strain No.
Percent of colonyforming units (_+S.D.) b
Strain No.
Percent of colonyforming units (_+S.D.)
40 851 838 39 273 810 840 842 926 869 902 824 887
0.1 -+ 2.0 22.1+ 2.2 37.5_+ 3.2 117.5 + 14.0 133.3 +_15.7 184.3 _+16.2 245.6 + 15.9 329.5 + 45.1 407.0 _+29.1 637.2 _+19.9 944.6 _+31.2 954.5 _+30.9 1082.8 _+39.7
800 841 815 823 844 704 884 827 267 833 807 859
1084.8 + 40.2 1 107.6_+49.7 1 1t6.5+_51.0 1 159.3± 42.3 1230.9 _+38.2 1320.7+ 62.7 1398.1 _+50.2 1355.2 -+49.7 1747.5_+52.3 2000.9 +_60.9 2 220.2 _+62.2 2828,8 + 59.8
~' Level of complement - - CHs0 ml = 91. b The percentage of colony-forming units was calculated from 3 experiments taking into account standard deviation + S.D.
258
Table 3 Susceptibility of A. lwoffii strains to the bactericidal action of the NHS after 30 min incubation Strain No.
Percent of colony forming units ( +- S.D.)
Strain No.
Percent of colony forming units ( ± S.D.)
971 2 965 798 952 972 980 794 580 920 832 834 734
0.1 ± 1.7 0.2 ± 1.0 0.3± 1.4 0.5 ± 1.0 0.5_+ 1.2 1.5_+ 1.2 2.2 ± 2.7 2.8±2.1 3.8+_ 1.0 6.0± 1.9 6.1 4-_2.2 6.5 ± 2. I 6.8 ± 6.1
54 84 766 693 36 71 733 937 768 978 688 982
6.9+_ 6.7 7.5 + 5.2 17.3± 5.1 27.2 +_ 7.9 3 7 . l ± 6.2 42,8± 9.2 43.3 ± 8.9 56.5± 8.8 80.2+_ 16.1 105.3 ± 15.7 107.4 + 14.0 109.5 ± 2 I. 1
By removal of Ca 2+ ions from serum to eliminate the classical pathway of complement activation ( N H S - E G T A M g 2+) the bactericidal action of the serum against two strains of A. anitratus was not inhibited (Table 4). The bactericidal activity of this serum, against three strains of A. lwoffii initially sensitive to NHS, are presented in Table 5. Two of the strains (Nos. 54, 84) were susceptible to N H S - E G T A M g 2+. The strain 693 was resistant and multiplied after 3 h incubation
in the modified serum. The E G T A concentration used does not influence the survival of bacteria in NHS56°C for 3 h.
5. D I S C U S S I O N The strains of A. anitratus and A. lwoffii isolated from patients differed in susceptibility to NHS. A. anitratus strains ( 1 2 % ) a n d A. lwoffii
500
A 4OO
2001
30[
100
8 O
20E 5O
C
"8 100
3 Q5 Time
1 [hours]
3
0.5 Time
1 [hours]
Fig. 2. Bactericidal activity of serum with thermally inactivated alternative pathway of complement against A. lwoffii (A) and A. anitratus (B) strains. A: Strains A. lwoffii Nos. 54 and 84 were used. Bars represent the standard deviation of three experiments.
NHS (o); NHS 50°C ( × ). B: A. anitratus No. 851; NHS (o); NHS 50°C ( x ); A. anitratus No. 838" NHS ( • ); NHS 50°C ( • ).
259
Table 4 Bactericidal activity of human serum after removing Ca 2+ ions against A. anitratus strains Percent of colony-forming units _+S.D. Time of incubation (h)
Strain No.
Serum
1
3
838
NHS NHS E G T A M g 2+ NHS 56°C NHS 56°C E G T A M g 2+
82.6-+ 17.5 47.1 _+ 14.9 159.1 _+27.1 132.4 -+ 29.1
37.9-+ 9.7 20.3_+ 7.9 270.2 -+ 31.2 250.7 .+ 30.1
851
NHS NHS EGTAMg 2+ NHS 56°C NHS 56°C EGTAMg 2+
75.4_+ 19.9 64.0+ 15.1 170.7-+27.7 149.2.+ 15.1
20.6-+ 10.1 19.1 -+ 10.01 310.1 +31.1 270.7_+ 27.1
strains (84%) were susceptible. The low susceptibility of A. anitratus to the bactericidal action of NHS may be responsible for the occurrence of these strains as an etiological factor of bacteraemia and for their frequent isolation from other clinical sources [1]. Differences in susceptibility to NHS between A. anitratus and A. lwoffii are possibly associated with a variation in cell wall structure [10]. Surface proteins are one of the
main components of the outer membrane protecting bacteria from the action of complement [5]. In the present work, A. lwoffii strains showed a high degree of susceptibility to the bactericidal action of serum. This implies a possibility of rapid elimination of these bacteria after infection. The contribution of complement activated by the classical pathway implies the presence in the serum of antibodies against these bacteria. Absence of
Table 5 Bactericidal activity of human serum after removing Ca 2+ ions against A. lwoffii strains Strain No.
54
84
693
NT: not tested.
Serum
Percent of colony-forming units _+S.D. Time of incubation (h) 0.5
1
3
NHS NHS E G T A M g 2+ NHS 56°C NHS 56°C E G T A M g 2+
7.1+ 2.7 15.2+ 1.2 101.2 .+ 17.7
1.1_+ 1.0 3.2.+ 2.1 157.7 _+29.1
NT NT 285.1 -+ 30.9
97.2 -+ 27.2
119.7-+ 22.2
229.9 + 19.2
NHS N H S E G T A M g 2+ NHS 56°C NHS 56°C EGTAMg 2+
10.1.+ 2.1 19.2.+ 1.9 110.9 .+ 17.1
0.7+ 1.0 3.1.+ 1.0 170.2 _+21.2
NT NT 301.0 + 21.7
100.2-+ 18.9
159.2 + 21.1
278.9 ± 17.2
NHS NHS EGTAMg 2+ NHS 56°C NHS 56°C E G T A M g 2+
31.2-+ 3.2 98.2_+ 17.6 117.2 .+ 10.1
7.1-+ 1.2 110.9.+21.1 162.2 + 29.1
0.1-+ 1.0 210.2.+32.2 290.2 .+ 27.1
116,0 .+ 12.1
167.2 _+30.1
269.9 -+ 30.2
260
these antibodies excludes the possibility of bactericidal activity. Therefore, it seems that these bacteria do not stimulate sufficient levels of antibody to initiate complement binding. In consequence these bacteria may be dangerous in clinical infections mainly in patients with a lower degree of immunity. Lack of complement activation by the classical pathway caused by the absence of antibodies also creates the possibility of bacteraemia or other kinds of infection. Investigation of the mechanism of killing of Acinetobacter bacilli by complement has shown that it is complicated. Complement induced killing of some strains after activation by the alternative pathway. The bactericidal action of complement against other strains may be induced independently by its activation by the classical or alternative pathway. One strain, A. lwoffii 693, required the presence of both the alternative and classical pathways of complement activation for bactericidal process. The diversity of activity of bactericidal serum factors against Shigella was noticed previously [11,12]. The bactericidal action of complement, activated by the alternative pathway, against Acinetobacter strains is undoubtedly beneficial in human infection because it can also work in the absence of antibodies. The induction of the antibodies by Acinetobacter strains is, from the epidemiological point of view, limited. These bacteria occur in the external environment and may be a component of the normal flora. The influence of these bacteria on the immunological system is weak and does not lead to the stimulation of humoral immunity. Nevertheless, the mechanism of non-specific defence by the action of complement activated by the alternative pathway can destroy the majority of strains of A. anitratus before they can penetrate the tissues and thus prevent development of disease.
REFERENCES [1] Beck-Sague, C.M., Jarvis, W.R., Brook, J.H., Culver, D.H., Potts, A., Gay, E., Shotts, B.W., Hill, B., Anderson, R.L. and Weinsteein, M,P, 11990) Epidemic bacteremia due to Acinetobacter baumanii in five intensive care units. Am. J. Epidemiol. 132, 723 733. [2] Kiem T.E. (1989) Bacteremia and fungemia in the immuno-compromised patient. Eur. J. Clin. Microbiol. Infect. Dis. 8, 832-837. [3] Traub, W.H. and Sper, M. 11989) Antimicrobial drug susceptibility of clinical isolates of Acinetobacter species CA. baumanii. A. haemolyticus, genospecies 3 and genospecies 6). Antimicrobial. Agents Chemother. 33, 1617-1619. [4] Reuben, A.G., Musher, D.M., Hamill, R.J. and Broucke, I. (1989) Polymicrobial bacteremia: clinical and microbiologic patterns, Rev. Infect. Dis. 11, 161-183. [5] Taylor, P.W. (1988) Bacterial resistance to ct)mplement. In: Virulence Mechanisms of Bacterial Pathogens (Roth, J.A., Ed.), Am. Soc. Microbiol., Washington. 107-1211. [6] Edinger, D., Bello, E. and Mates, A. 11977) The heterocytotoxicity of human serum. I. Activatkm of the alternative complement pathway by heterologus target cells. Cell lmmunol, 29, 174-186. [7] Fine, D.P., Marney, S.R., Colley, D.C., Sergent, J.S. and Des Prez, R.M. 11972) C3 shunt activation in human serum chelated with EGTA. J, Immunol, 1119, 807-809. [8] Wedgwood, R.J. and Janewy, C.A. (1953) Technique for complement titration in human sera. Pediatrics 11, 569572. [9] Jankowski, S. and Doroszkiewicz, W. (1990) Prelimina~' studies of the effect of ozone on the bactericidal properties of complement. Complement lnflamm. 7, 57-62. [10] Boevet, P.J.M., Jeanjean, S., Vieu, J.V. and Dijkshoorn, L. (199/)) Species, biotype and bacteriophage type determinations compared with protein prophiles for typing Acinetobacter strains. J. Clin. Microbiol. 28, 170-176. [11] Mokracka-Latajka, G., Grzybek-Hryncewicz, K. and Jankowski, S. (1990) Sensitivity of Shigella strains to bactericidal activity of human serum. 1. Participation of two independently active mechanisms in bactericidal actkm against Shigella sonnei phase If. Acta Microbiol. Pol. 39. 111-16. [12] Mokracka-Latajka, G., Grzybeck-Hryncewiez, K. and Jankowski, S. (1990) Sensitivity of Shigella strains to the bactericidal activity of human serum. Ill. The diversity of activity variants of bactericidal serum factors against Shigella fh'xneri serotypes. Acta Microbiol. Pol, 39, 121127.
255
FEMSIM 00213
Susceptibility of isolates of Acinetobacter anitratus and Acinetobacter lwoffii to the bactericidal activity of normal human serum Stanistaw Jankowski, Kryspina Grzybek-Hryncewicz, Malgorzata Fleischer and Maria Walczuk Department of Microbiology, Academy of Medic&e, Wroctaw, Poland Received 14 August 1991 Revision received 6 January 1992 Accepted 27 January 1992
Key words: Acinetobacter; Bactericidal action of serum; Complement
I. SUMMARY
2. INTRODUCTION
The bactericidal activity of normal human serum against the Gram-negative coccobacilli Acinetobacter anitratus and Acinetobacter lwoffii was studied; 12% and 84%, respectively, of the tested strains appeared to be sensitive. Thus, serum resistance may be an important factor contributing to the pathogenic potential of A. anitratus strains. Three types of bactericidal action were shown. In the first, the strains were killed when the alternative complement pathway was activated. In the second, some strains required both the classical and alternative pathways. In the third variant, the strains needed either the alternative or classical activiation pathway.
After 1980 a change in the spectrum of microbial factors causing hospital infections in patients with decreased immunity, e.g. those with cancer, was noticed. The number of Gram-positive bacteria, such as Streptococcus spp., coagulase-negative Staphylococcus and Mycobacterium as well as non-fermenting Gram-negative Acinetobacter strains, found in these infections increased markedly [1-3]. The number of coliforms and anaerobic bacteria decreased [2,4]. The change in these patients was caused by the use of a specific set of antibiotics. A. calcoaceticus ssp. anitratus and A. calcoaceticus ssp. lwoffii were frequently isolated from man [1,3]. Resistance to bactericidal factors in the host, e.g. to those in blood, may considerably influence the infectious activity of these bacteria. The bactericidal activity of serum is important for the protection of the higher organism against infection [5].
Correspondence to: S. Jankowski, Department of Microbiology, Academy of Medicine, Chatubifiskiego 4 str., P1-50368 Wroctaw, Poland.
256
In the present work the susceptibility of A. anitratus and A. lwoffii to the bactericidal action of normal human serum was tested.
3. M A T E R I A L S A N D M E T H O D S 3.1. Bacterial strains Twenty-five strains of A. anitratus or A. lwoffii isolated from man were used (Table 1). These strains were identified by the API 20 N E and Oxi Ferm Tube systems. 3.2. Serum Normal human serum (NHS) obtained from two healthy adult blood donors was pooled. The sera were stored at - 3 0 ° C in 1-ml portions for up to two months. These donors had not been treated with antibiotics in the period prior to blood donation. 3.3. Preparation of the serum The alternative pathway of complement activation was inhibited by incubating the serum for 20 rain at 50°C (NHS50°C) [6]. In such serum only the complement activated by the classical pathway exerts bactericidal action. To inhibit the classical pathway of complement activation, Ca 2+ ions were removed by E G T A from serum (NHS EGTAMg2+). EGTA-ethylene-glycol-bis-/3(aminoethyl e t h e r ) N , N '-tetraacetic acid solution was prepared according to
Fine et al. [7]. The final concentration of E G T A and MgCI~ was 10 raM. The bactericidal action of this preparation is dependent on the complement activated by the alternative pathway. The action of E G T A M g 2+ alone was investigated by introducing this factor to serum inactivated at 56°C ( N H S 5 6 ° C - E G T A M g 2+) and measuring the percentage survival of the bacteria. Inactivation of NHS was carried out at 56°C for 3(I rain (NHS56°C). 3.4. Complement assay The level of complement in the serum was determined by the method of Wedgwood and Janeway [8] and the results are given in units of complement Hs~ (50% haemolysis). 3.5. Bactericidal assay The bactericidal activity of serum was determined as described earlier [9]. The bacteria were harvested during early logarithmic growth. At an initial density of about 105 cells m l - 1 cells were added to a 50% NHS solution diluted with 0.1M NaC1. The mixture was incubated at 37°C and at time 0 and 0.5, 1, 2, 3 h, samples were taken, diluted and plated on nutrient agar. After incubation for 18 h at 37°C the number of colonies formed at t = 0 was taken as 100%. The strains which had a survival rate of not less than 50% after 3 h incubation in 50% serum were regarded as resistant.
Table 1 Source of A. lwoffii and A. anitrams strains Source
A. lwoffii strain
A. anitratus strain
Blood Sputum Bronchial secretion Urine Pharynx Wound Pus Vagina Bile Nose Cerebrospinal fluid
971,972, 84, 580, 54, 688, 71,937, 2, 798, 36, 766, 982 980
39, 807, 815, 267, 827 273 800, 841
978 832, 834, 952 693 733, 734 920, 965
768, 794
40, 704, 851,869 823, 833, 838, 844, 859, 902 824, 840, 842, 887, 926 810
884
257 4. R E S U L T S 2 Susceptibility of A. anitratus a n d A. lwoffii strains to N H S is r e p r e s e n t e d in T a b l e s 2 a n d 3. A. anitratus strains (22 of 25) were resistant to the bactericidal action of NHS. T h e s e resistant strains grew in the N H S after 3 h i n c u b a t i o n . T h e n u m ber of c o l o n y - f o r m i n g u n i t s in some strains increased 2 0 - 2 8 times. It can be seen from T a b l e 3 that most strains of A. lwoffii were susceptible to the bactericidal action of N H S ; after 30 m i n i n c u b a t i o n with the serum, the n u m b e r of colony-forming units was c o n s i d e r a b l y reduced. Five strains were resistant, the p e r c e n t a g e survival was from 56.5 to 109,5 of the initial c o n c e n t r a t i o n of bacteria. T h e s e strains, with the exception of No. 937, m u l t i p l i e d in N H S after i n c u b a tion for 3 h (Fig. 1). In f u r t h e r studies the m e c h a n i s m of the bactericidal action of c o m p l e m e n t in N H S on Acinetobacter strains was investigated. By t h e r m a l inactivation, the action of c o m p l e m e n t activated by the alternative pathway was e l i m i n a t e d , p r e p a r a t i o n NHS50°C. Strains of A. lwoffii chosen r a n d o m l y (Nos. 54, 84, 693) were susceptible to N H S b u t resistant to N H S 5 0 ° C (Fig. 2a). A. anitratus strain 851 was resistant to NHS50°C. T h e susceptibility of A. anitratus strain
stroin
/.0/
o
aa 0
>!00C
o
20~
97~3
~
~
_
.
,
0.5
,
1 Time
~
,
2 [
768
,
937
3
hours]
Fig. 1. Susceptibility of A. lwoffii to the bactericidal activityof normal human serum after 3 h incubation.
838 was not c h a n g e d by t h e r m a l inactivation of s e r u m (Fig. 2b). T h e s e e x p e r i m e n t s showed that the sera devoid of c o m p l e m e n t activated by the alternative pathway lose their bactericidal properties against 4 out of 5 strains of Acinetobacter.
Table 2 Susceptibility of A. anitratus strains to the bactericidal action of normal serum ~' after 3 h incubation Strain No.
Percent of colonyforming units (_+S.D.) b
Strain No.
Percent of colonyforming units (_+S.D.)
40 851 838 39 273 810 840 842 926 869 902 824 887
0.1 -+ 2.0 22.1+ 2.2 37.5_+ 3.2 117.5 + 14.0 133.3 +_15.7 184.3 _+16.2 245.6 + 15.9 329.5 + 45.1 407.0 _+29.1 637.2 _+19.9 944.6 _+31.2 954.5 _+30.9 1082.8 _+39.7
800 841 815 823 844 704 884 827 267 833 807 859
1084.8 + 40.2 1 107.6_+49.7 1 1t6.5+_51.0 1 159.3± 42.3 1230.9 _+38.2 1320.7+ 62.7 1398.1 _+50.2 1355.2 -+49.7 1747.5_+52.3 2000.9 +_60.9 2 220.2 _+62.2 2828,8 + 59.8
~' Level of complement - - CHs0 ml = 91. b The percentage of colony-forming units was calculated from 3 experiments taking into account standard deviation + S.D.
258
Table 3 Susceptibility of A. lwoffii strains to the bactericidal action of the NHS after 30 min incubation Strain No.
Percent of colony forming units ( +- S.D.)
Strain No.
Percent of colony forming units ( ± S.D.)
971 2 965 798 952 972 980 794 580 920 832 834 734
0.1 ± 1.7 0.2 ± 1.0 0.3± 1.4 0.5 ± 1.0 0.5_+ 1.2 1.5_+ 1.2 2.2 ± 2.7 2.8±2.1 3.8+_ 1.0 6.0± 1.9 6.1 4-_2.2 6.5 ± 2. I 6.8 ± 6.1
54 84 766 693 36 71 733 937 768 978 688 982
6.9+_ 6.7 7.5 + 5.2 17.3± 5.1 27.2 +_ 7.9 3 7 . l ± 6.2 42,8± 9.2 43.3 ± 8.9 56.5± 8.8 80.2+_ 16.1 105.3 ± 15.7 107.4 + 14.0 109.5 ± 2 I. 1
By removal of Ca 2+ ions from serum to eliminate the classical pathway of complement activation ( N H S - E G T A M g 2+) the bactericidal action of the serum against two strains of A. anitratus was not inhibited (Table 4). The bactericidal activity of this serum, against three strains of A. lwoffii initially sensitive to NHS, are presented in Table 5. Two of the strains (Nos. 54, 84) were susceptible to N H S - E G T A M g 2+. The strain 693 was resistant and multiplied after 3 h incubation
in the modified serum. The E G T A concentration used does not influence the survival of bacteria in NHS56°C for 3 h.
5. D I S C U S S I O N The strains of A. anitratus and A. lwoffii isolated from patients differed in susceptibility to NHS. A. anitratus strains ( 1 2 % ) a n d A. lwoffii
500
A 4OO
2001
30[
100
8 O
20E 5O
C
"8 100
3 Q5 Time
1 [hours]
3
0.5 Time
1 [hours]
Fig. 2. Bactericidal activity of serum with thermally inactivated alternative pathway of complement against A. lwoffii (A) and A. anitratus (B) strains. A: Strains A. lwoffii Nos. 54 and 84 were used. Bars represent the standard deviation of three experiments.
NHS (o); NHS 50°C ( × ). B: A. anitratus No. 851; NHS (o); NHS 50°C ( x ); A. anitratus No. 838" NHS ( • ); NHS 50°C ( • ).
259
Table 4 Bactericidal activity of human serum after removing Ca 2+ ions against A. anitratus strains Percent of colony-forming units _+S.D. Time of incubation (h)
Strain No.
Serum
1
3
838
NHS NHS E G T A M g 2+ NHS 56°C NHS 56°C E G T A M g 2+
82.6-+ 17.5 47.1 _+ 14.9 159.1 _+27.1 132.4 -+ 29.1
37.9-+ 9.7 20.3_+ 7.9 270.2 -+ 31.2 250.7 .+ 30.1
851
NHS NHS EGTAMg 2+ NHS 56°C NHS 56°C EGTAMg 2+
75.4_+ 19.9 64.0+ 15.1 170.7-+27.7 149.2.+ 15.1
20.6-+ 10.1 19.1 -+ 10.01 310.1 +31.1 270.7_+ 27.1
strains (84%) were susceptible. The low susceptibility of A. anitratus to the bactericidal action of NHS may be responsible for the occurrence of these strains as an etiological factor of bacteraemia and for their frequent isolation from other clinical sources [1]. Differences in susceptibility to NHS between A. anitratus and A. lwoffii are possibly associated with a variation in cell wall structure [10]. Surface proteins are one of the
main components of the outer membrane protecting bacteria from the action of complement [5]. In the present work, A. lwoffii strains showed a high degree of susceptibility to the bactericidal action of serum. This implies a possibility of rapid elimination of these bacteria after infection. The contribution of complement activated by the classical pathway implies the presence in the serum of antibodies against these bacteria. Absence of
Table 5 Bactericidal activity of human serum after removing Ca 2+ ions against A. lwoffii strains Strain No.
54
84
693
NT: not tested.
Serum
Percent of colony-forming units _+S.D. Time of incubation (h) 0.5
1
3
NHS NHS E G T A M g 2+ NHS 56°C NHS 56°C E G T A M g 2+
7.1+ 2.7 15.2+ 1.2 101.2 .+ 17.7
1.1_+ 1.0 3.2.+ 2.1 157.7 _+29.1
NT NT 285.1 -+ 30.9
97.2 -+ 27.2
119.7-+ 22.2
229.9 + 19.2
NHS N H S E G T A M g 2+ NHS 56°C NHS 56°C EGTAMg 2+
10.1.+ 2.1 19.2.+ 1.9 110.9 .+ 17.1
0.7+ 1.0 3.1.+ 1.0 170.2 _+21.2
NT NT 301.0 + 21.7
100.2-+ 18.9
159.2 + 21.1
278.9 ± 17.2
NHS NHS EGTAMg 2+ NHS 56°C NHS 56°C E G T A M g 2+
31.2-+ 3.2 98.2_+ 17.6 117.2 .+ 10.1
7.1-+ 1.2 110.9.+21.1 162.2 + 29.1
0.1-+ 1.0 210.2.+32.2 290.2 .+ 27.1
116,0 .+ 12.1
167.2 _+30.1
269.9 -+ 30.2
260
these antibodies excludes the possibility of bactericidal activity. Therefore, it seems that these bacteria do not stimulate sufficient levels of antibody to initiate complement binding. In consequence these bacteria may be dangerous in clinical infections mainly in patients with a lower degree of immunity. Lack of complement activation by the classical pathway caused by the absence of antibodies also creates the possibility of bacteraemia or other kinds of infection. Investigation of the mechanism of killing of Acinetobacter bacilli by complement has shown that it is complicated. Complement induced killing of some strains after activation by the alternative pathway. The bactericidal action of complement against other strains may be induced independently by its activation by the classical or alternative pathway. One strain, A. lwoffii 693, required the presence of both the alternative and classical pathways of complement activation for bactericidal process. The diversity of activity of bactericidal serum factors against Shigella was noticed previously [11,12]. The bactericidal action of complement, activated by the alternative pathway, against Acinetobacter strains is undoubtedly beneficial in human infection because it can also work in the absence of antibodies. The induction of the antibodies by Acinetobacter strains is, from the epidemiological point of view, limited. These bacteria occur in the external environment and may be a component of the normal flora. The influence of these bacteria on the immunological system is weak and does not lead to the stimulation of humoral immunity. Nevertheless, the mechanism of non-specific defence by the action of complement activated by the alternative pathway can destroy the majority of strains of A. anitratus before they can penetrate the tissues and thus prevent development of disease.
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